Russian Contributions to Invertebrate Behavior

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Author: Charles I Abramson | Yuri Burmistrov | Zhanna Shuranova

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Russian Contributions to Invertebrate Behavior

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Russian Contributions to Invertebrate Behavior Edited by

Charles I. Abramson Zhanna P. Shuranova Yuri M. Burmistrov

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Westport, Connecticut

London

Library of Congress Cataloging-in-Publication Data Russian contributions to invertebrate behavior / edited by Charles I. Abramson, Zhanna P. Shuranova, Yuri M. Burmistrov. p. cm. Includes bibliographical references and index. ISBN 0-275-94525-1 (alk. paper) 1. Invertebrates—Behavior. 2. Learning in animals. I. Abramson, Charles I. II. Shuranova, Zh. P. (Zhanna Petrovna) III. Burmistrov, IU. M. (IUrii Mikhailovich) QL364.2.R87 1996 592'.051—dc20 95-40578 British Library Cataloguing in Publication Data is available. Copyright © 1996 by Charles I. Abramson, Zhanna P. Shuranova, and Yuri M. Burmistrov All rights reserved. No portion of this book may be reproduced, by any process or technique, without the express written consent of the publisher. Library of Congress Catalog Card Number: 95-40578 ISBN: 0-275-94525-1 First published in 1996 Praeger Publishers, 88 Post Road West, Westport, CT 06881 An imprint of Greenwood Publishing Group, Inc. Printed in the United States of America

@r The paper used in this book complies with the Permanent Paper Standard issued by the National Information Standards Organization (Z39.48-1984). 10

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Contents

Figures

vii

Preface

ix

1. Introduction Charles I. Abramson

1

2. History of Invertebrate Behavioral Studies in Russia Zhanna P. Shuranova

5

3. Memory and Morphogenesis in Planaria and Beetle Inna M. Sheiman and Kharlampi P. Tiras

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4. Innate and Acquired Behavior of Mollusks Pavel M. Balaban and Igor I. Stepanov

77

5. Individual Features in Invertebrate Behavior: Crustacea Yuri M. Burmistrov and Zhanna P. Shuranova

111

6. Learning, Memory, and Motivation in Ants Galina P. Udalova and Anna Ja. Karas

145

1. Local Orientation and Learning in Insects Vladimir M. Kartsev

177

VI

CONTENTS

Appendix A: Directory of Russian Scientists Engaged in Invertebrate Research

213

Appendix B: Bibliography of Invertebrate Articles Appearing in Neuroscience and Behavioral Physiology

217

Index

227

Addresses of the Editors and Contributors

229

Figures

Figure 2.1: V. A. Vagner (Courtesy of B. V. Lukin, Priroda, 1987, issue 1, p. 50).

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Figure 2.2: N. N. Ladygina-Kots (From Ladygina-Kots 1923)

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Figure 2.3: L. A. Orbeli (Courtesy of Yu. Burmistrov).

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Figure 2.4: L. V. Krushinsky (Courtesy of N. Krushinskaya).

19

Figure 3.1: Conditioning of planarians with light as the conditioned stimulus. For Day 9, only the conditioned stimulus was changed to vibration.

46

Figure 4.1: Experimental setup for behavioral experiments. The animal is fixed by its shell to a holder (H), but in such a manner that it can move freely on a plastic ball (B). I: indifferent carbon electrode, T: electrically driven tapper, S: electrode for manual application of electric shock to the skin.

81

Figure 4.2: Performance of snails in a food capture task. Curve 1 represents the time needed to capture a piece of cabbage. Curve 2 represents the time needed to capture a piece of carrot.

84

Vlll

FIGURES

Figure 4.3: Pneumostome closure responses in the snail during paired presentation of watermelon odor (filled dot) and moderately intense noxious stimulus (triangle, upper trace) across training trials. The upward shift in the trace represents pneumostome closure. The unfilled dot represents the presentation of carrot. Carrot odor was not paired with the noxious stimulus.

92

Figure 4.4: The dynamics of food avoidance learning in Helix fitted to the mathematical model. 1: conditioned stimulus—carrot, differentiated stimulus—cabbage; 2: conditioned stimulus—cabbage, no differentiated stimulus; 3: conditioned stimulus—carrot, no differentiated stimulus.

95

Figure 4.5: An application of the model to the instrumental conditioning of left tentacle withdrawal reflex in Helix after 1, 2, 3, or 4 training sessions.

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Figure 4.6: The influence of blood, sampled from donors after their first left tentacle withdrawal reflex learning session, on the learning of naive recipients in the snail Helix. Curve 1: the learning curve of the donors' first session. Curve 2: the learning curve of the recipients' first session. Curve 3: the learning curve of the recipients' second session.

99

Figure 4.7: The influence of blood, sampled from donors after their fourth left tentacle withdrawal reflex learning session, on the learning of naive recipients in the snail Helix. Curve 1: the learning curve of the donors' first session. Curve 2: the learning curve of the recipients' first session. Curve 3: the learning curve of the recipients' second session.

1000

Figure 4.8: Average frequency of bar contacts. Pooled results.

103

FIGURES

IX

Figure 6.1: The scheme of the experimental apparatus: I: the earthenware pot with ant nest. II: the tray. Ill: the screen. IV: the arena. V: the maze. 1: uprights. 2 entrance to nest. 3: the central bridge. 4: the side bridges. 5: the starting zone. 6: removable bridges. O: entrance to maze. A and G: target areas. B and V: symmetrical areas: T and P exits: K,L,E,C,D and Y,Q,J,U: other sections of right and left halves of maze, respectively.

155

Figure 7.1: Discriminative abilities in bees. 1: shape discrimination, 2: color discrimination, 3: shape of figures, 4: size of figures, 5: shape and size simultaneously.

187

Figure 7.2: Performance of two groups of bees discriminating stimuli that lead to food (curve 1) or to the nest entrance (curve 2), over the course of 80 visits.

190

Figure 7.3: Four possible trajectories around the perimeter of the experimental table containing four flower-like feeders; a and c were preferred flight paths, and b and d were rarely chosen.

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Preface

Analysis of associative and nonassociative behavior of invertebrates is one of the current approaches to understanding the neuronal and chemical bases of learning and memory. Associative and/or nonassociative learning has been demonstrated in all of the major classes of invertebrates, and underlying cellular substrates have been identified in several cases, largely in molluscs. The overwhelming majority of what American researchers know about invertebrate behavior, however, comes from Western laboratories. A large amount of the Russian invertebrate behavior literature remains untranslated and not readily available to the Western researcher. This book addresses and is a necessary step in correcting this imbalance. I first became interested in Russian studies of invertebrate behavior from reading the literature reviews of Gregory Razran (1901-1973). For nearly fifty years, Razran provided Western readers with surveys of the Russian literature on "higher nervous activity" or, as it is known in the West, learning. This work culminated in his 1971 book, Mind in Evolution: An East-West Synthesis (Boston: Houghton Mifflin), which reviewed over one thousand Russian studies. Razran's survey of the Russian work prepared me to find a vast and vibrant literature on invertebrate behavior. I was not disappointed. In a book describing, for example, the behavior of microorganisms, Tushmalova [1986, Functional Analysis of Acquired Behavior of Lower Invertebrates (in Russian), Moscow: Moscow University] cites 128 Russian studies and Sheiman [1984, The Regulators of Morphogenesis and their Adaptive Function (in Russian), Moskva: Nauka] cites 58 in her discussion of planarian and beetle behavior. In two books written by Shuranova and Burmistrov [1988, Neurophysiology of the Crayfish (in Russian), Moskva: Nauka; 1991, Progress in Crustacean Neurophysiology (in Russian), Moskva: Viniti], 123 Russian citations are presented on various aspects of crusta-

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PREFACE

ceans; and Balaban and Zakharov [1992, Learning and Development: Common Basis of Two Phenomena (in Russian), Moskva: Nauka] present 99 Russian citations relevant to the behavior of the snail. During my visits to Russia I had the opportunity to explore several research laboratories and speak with investigators engaged in invertebrate research, the work of some of whom is presented here. The continuation of invertebrate behavior work, however, is in jeopardy. It is obvious to any visitor to Russia that the growth of democracy is going to be a slow and painful process. The effect of this process is nowhere more evident than in the Russian scientific infrastructure. The current conditions, with an average salary of $30 a month, the cost of an airline ticket equivalent to at least two years salary, a shortage of even the most basic laboratory supplies, competition to obtain extramural funding with little or no grant writing skills, immigration of senior scientists to the West, a severe shortage of graduate students—for, after all, who but the most dedicated would risk spending a lifetime studying invertebrates for $30 a month—will in the short run confine the study of invertebrate behavior to a few relatively well funded laboratories studying only a few species. Without assistance from Western scientists engaged in the study of invertebrate behavior, the end of invertebrate research in Russia is rapidly approaching. In the course of conversations, Zhanna Shuranova, Yuri Burmistrov, and I came upon the idea that one way to provide such assistance is to make Russian work on invertebrate behavior known in the West. In this way collaborations in the form of, for instance, student and faculty exchanges, research and grant writing, and invitations to submit review articles and book chapters might be stimulated. Moreover, it would facilitate answers to fundamental questions in behavior by minimizing duplication and by fine-tuning research efforts. It is obvious from reading Western reviews of the invertebrate behavior literature that the Russian work is seldom cited. For example, out of a combined total of 700 references, only the 1927 work of Pavlov was mentioned in two major reviews of invertebrate behavior (Farley and Alkon 1985, Ann. Rev. Psy. 36: 419494; Carew and Sahley 1986, Ann. Rev. Neuros. 9: 435-487). In a recent review of invertebrate learning, no Russian work was cited in 257 references (Krasne and Glanzman 1995, Ann. Rev. Psy. 46: 585-624) even though a special issue of Neuroscience and Behavioral Physiology (1994, vol. 24) was dedicated to the neurobiology of the snail. The same situation is evident in books on invertebrate learning and behavior. For instance, in the thirteen chapters comprising Corning, Dyal, and Willows' three volume set on invertebrate learning there are no Russian contributors (Invertebrate Learning: Vol: 1-3, 1973-1975, New York: Plenum); and there is only 1 Russian contributor out of 103 in a recent encyclopedic treatment of learning and memory (Squire 1992, Encyclopedia of Learning and Memory, New York: Macmillan). One factor in the lack of Russian citations in Western journals and books is that the Russians themselves do not do a good job of making their invertebrate research known to Western readers. In a journal devoted to translations of selected Russian work a survey of Neuroscience and Behavioral Physiology between 1972 and

PREFACE

Xlll

1994, only 129 of 1661 papers pertained to invertebrates. The notable exception is a 1994 issue (vol. 24), which presents twenty papers dealing with snail behavior and physiology. As I mentioned above, this issue was not cited in a recent review of the literature. With this book the editors and contributors hope to increase the awareness of Russian contributions to invertebrate behavior. Russian Contributions to Invertebrate Behavior is an edited volume describing Russian experiments on invertebrate behavior, especially learning. Each chapter, written by a leading Russian scientist, provides a review of his or her subject area. Subject areas include a historical introduction to the role of vertebrate and invertebrate behavior in Russian science, the behavior of crustaceans, the orientation and learning of insects, the behavior and morphogenesis of invertebrates, and the role of motivation in learning and memory of ants. The audience for this book ranges from students and instructors of invertebrate behavior to any professional interested in knowing the contributions made by Russian scientists to the important field of invertebrate behavior. In addition to reviews of Russian work, additional features are appendices that provide the names and addresses of Russian scientists engaged in invertebrate work and a bibliography of invertebrate articles appearing in Neuroscience and Behavioral Physiology. It is hoped that students and professionals interested in invertebrate behavior can use the information to forge and to facilitate collaborative efforts between Russian and Western scientists. It is the sincere wish of the editors and authors that this book will serve the scientific community as a reference tool and, more important, will stimulate contacts between Russian scientists and Western researchers doing related work. We would like to thank Dolores A. Buckbee, Bryan Bruno, and Jim Hellwege for their help in the final stages of this project. Thanks are also due to Ann Newman, Liz Murphy, Linda Robinson, and Laura Clark from Praeger Publishing for making this book possible. Charles I. Abramson Departments of Psychology and Zoology Oklahoma State University


Russian Contributions to Invertebrate Behavior


Chapter One Introduction Charles I. Abramson

Among the many advances made possible by the study of invertebrates are an increase in knowledge of behavior, a greater appreciation of the plasticity inherent in stereotypic behavior, and the use of training methods to assist bees in the pollination of crops. In addition, researchers who have taken advantage of the invertebrate nervous system have made significant inroads in understanding the cellular mechanisms of behavior. As I have said elsewhere (Abramson 1994), for those of us who seek basic principles of behavior, invertebrates offer several important advantages. For example, the varieties of invertebrate nervous systems created in the course of evolution provide an excellent opportunity to begin to understand ourselves. When we understand the workings of the single cell of a protozoan, the regeneration of ganglia in a planarian, the retention of a memory in a beetle as it progresses from a larvae to an adult, the physiological basis of learning in a snail, or the individual behavior of a crayfish, we are that much closer to unravelling the mysteries of our own brains and behavior. In addition to what they can tell us about ourselves, invertebrates are useful to test the generality of both behavioral theories of learning and any proposed underlying physiological or biochemical mechanisms. Invertebrate nervous systems are, on the whole, more amenable to physiological and biochemical manipulations than vertebrate nervous systems. Moreover, because the behavior of invertebrates is generally less complex and more reflexive than our own, the genetic analysis of physiological mechanisms is readily amenable to experimental manipulations. The fruit fly, Drosophila, and the nematode Caenorhabditis elegans have become the invertebrates of choice in the effort to identify genetic components in behavioral traits. If we find, for instance, in a vertebrate that a particular type of learning depends on the presence of a cerebral cortex, a specific

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RUSSIAN CONTRIBUTIONS TO INVERTEBRATE BEHAVIOR

type of neural organization, or a certain level of cognitive development, an invertebrate can reveal whether such features are always required. We may find, for instance, that an invertebrate can learn the same task as a vertebrate but that the underlying mechanisms or solutions are different. Invertebrates are also used to answer questions such as, When does learning first appear in the animal kingdom? Is it possible to trace the evolutionary development of a particular type of behavior? Do invertebrates and vertebrates behave the same way, under the same conditions? Can invertebrates be used as a bioassay to detect pollutants? The intriguing nature of invertebrate behavior, the accessibility of the nervous system, and the use of genetic manipulations combine to make invertebrates an attractive group to answer questions about learning and memory. This attractiveness is multiplied by the sheer number and diversity of invertebrates, which make it easy to select behavior and physiology uniquely suited to a particular experimental design. For instance, if one is interested in the effect of regeneration of the nervous system on retention of a learned response, planarians make fine subjects. If one is interested in how conditions inside a cell influence learning, one approach is to use aneural single cells of such protozoans as Stentor. Russian Contributions to Invertebrate Behavior is an introduction to the invertebrate work performed by Russian scientists. The major emphasis is on studies of learning. In this book the editors and contributors have brought together contemporary Russian experimental data on the behavior of various invertebrates, including crustaceans, insects, and molluscs. The book should be useful for those interested in acquiring a working knowledge of the behavioral techniques, data, issues, and history of Russian studies of invertebrate behavior. It is also of interest to those studying the history of Russian behavioral science. The book is designed primarily to stimulate Western researchers in contacting and forming collaborations with Russian colleagues. It can stand alone, as a valuable resource of the Russian literature, or can be used as a supplemental text for courses in animal behavior and physiological psychology. The book consists of Seven chapters and Two appendices. Chapters Two through Seven begin with a history of Russian work in a particular area and highlights the investigators research. Each chapter contains a reference section citing related Russian articles. Much of the data discussed in the chapters was not previously available, or at least not readily available, to the Western researcher. Chapter Two provides a historical overview of invertebrate behavioral studies in Russian. Here you will see the contributions made by Russian scientists at the turn of the century and read about the downfall of Russian behavioral science due to the October Revolution, the resulting civil war, and the Communist leadership. You will learn about V. A. Vagner and the rise of the Russian ethological movement and about other Russian scientists, such as L. A. Orbeli and N. N. Ladygina-Kots, who contributed to the comparative analysis of behavior in Russia, often at great personal risk. Chapter Three presents a discussion of memory and morphogenesis using planarians and beetles as model animals. Experiments are presented that attempt to

INTRODUCTION

3

uncover the underlying mechanisms of the survival of memories following regeneration and metamorphosis. An experimental technique is presented for the training of planarians that eliminates the problems inherent in the conditioning procedures popular in the West during the 1950s and 1960s. Of interest also is a technique for quantifying the process of regeneration. Chapter Four is an excellent introduction to the type of simple systems work performed in Russia. The authors discuss a wide range of behavior in the snail Helix and discuss various training techniques and the influence of developmental stage on learning. A mathematical model is presented that accounts for performance under a range of training situations. A series of experiments that particularly captures the imagination comes from a transfer of training study in which the blood from a trained snail is injected into a naive snail. This work has much in common with that in Chapter Three on the transfer of learning in planarians following regeneration. Chapter Five provides a discussion of Russian studies of crustacean behavior. Of particular interest here is the value placed on the study of individual behavior when observed over a long period of time. The authors believe that long term observation under various conditions, coupled with physiological data, indicate that crayfish can be used to create an invertebrate model of the emotional process. A strong case is also made that the crayfish Procambarus cubensis, because of its size and ease of housing, makes an excellent subject for research. The behavior of this species is discussed in detail. Chapters Six and Seven outline several investigations on the learning and memory of ants and of bees and wasps, respectively. The learning of ants is studied under various levels of motivation; the data obtained in a complex maze situation indicate that the behavior of ants is more flexible than one might expect from Western data. The learning of bees and wasps is also studied under various conditions of motivation and under cognitive-like experiments based on problems of logic. The experiments with bees and wasps provide an excellent example of the comparative approach to the study of behavior and provide the surprising conclusion that bees and wasps can solve logical tasks; wasps are more flexible in their ability to do so than honey bees. The remaining sections of the book consist of two appendices. Appendix A presents the names and addresses of some Russian workers in the area of invertebrate learning to enable readers to contact scientists whose work excites them. Appendix B is a bibliography of every article using invertebrates (behavior, physiology, and/or a combination of both) that appeared in the journal Neuroscience and Behavioral Physiology until 1995. Other translated materials are also listed. SOME DIFFERENCES BETWEEN RUSSIAN AND WESTERN STUDIES OF INVERTEBRATE LEARNING AND BEHAVIOR In reading these chapters, one is struck by the similarities between Western and

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Russian studies of invertebrate behavior. As their Western counterparts, Russian scientists have exploited the invertebrate nervous system for what it can reveal about the underlying mechanisms of behavior; and, as in the West, invertebrates are recognized in Russia as important in research and teaching (Abramson 1990, 1994). There are also important differences. Russian studies of invertebrate learning, by and large, minimize the differences between instrumental/operant conditioning and classical conditioning. This stems from the Pavlovian tradition, which stressed instrumental conditioning as a special case of classical conditioning. In the West the distinction is stressed, although at the level of vertebrates the practice seems to be fading. A second difference is that the Russians employ an evolutionary perspective in their research much more than is done in the West. In the 1950s, for instance, both Schneirla and Beach noted the lack of an evolutionary perspective in comparative psychology. At least as it applies to invertebrates this problem still remains (Abramson 1994). As Chapter Two will show, there were a number of Russian theorists who routinely considered invertebrate data often at great personal risk. Related to the importance of having an evolutionary perspective is the need to conduct comparative analysis. In my opinion the West certainly lags behind the Russians. As Kartsev's chapter on learning in honey bees and wasps indicates he routinely compares the performance of several invertebrates, as do Sheiman and Tiras and others described in this book. The Sheiman and Tiras contribution is also interesting in that they continue to use the planarian in studies of learning and memory—an animal long ago forgotten in invertebrate learning research in the West. It should also be noted that there is a trend in the Russian invertebrate literature to consider cognitive capabilities in higher invertebrates such as Decapods and in insects such as ants and bees and wasps. Such research has been going on since the 1950s. I remember as a student thinking how silly it was to impart cognitive abilities to invertebrates. Yet now, in the West, for some, it seems silly not to. There also seems to be a greater Russian interest in the behavior of invertebrates particularly in the study of individual differences. As the chapters point out, a number of research institutions are devoted to the study of invertebrates and how they can be used as bioassays and, in the case of ants, "forest defense." In the West much invertebrate research focuses on their nervous systems. The above descriptions of Russian and Western perspectives come, of course, from a personal perspective. In the following pages, readers can determine for themselves the extent of the similarities and differences.

REFERENCES Abramson, C. I. 1990. Invertebrate Learning: A Laboratory Manual and Source Washington, D.C.: American Psychological Association. . 1994. A Primer of Invertebrate Learning: The Behavioral Perspective ington, D.C.: American Psychological Association.

Chapter Two History of Invertebrate Behavioral Studies in Russia Zhanna P. Shuranova

This chapter is dedicated to the memory of B. S. Kuzin, A. A. Lyubishchev, B. N. Veprintsev, and many other talented Soviet biologists who enjoyed doing research but could not do very much, for reasons that are so far from science.

Systematic investigations of invertebrate behavior in Russia began at the end of the nineteenth century, primarily because of the efforts of V. A. Vagner (Wagner) (Vagner 1896). It is well known that the twentieth century has been an extremely difficult time in Russian history. Therefore, it is impossible to discuss the status of any specialized scientific field (such as invertebrate behavioral studies) if we ignore the general picture of social life and the status of science and scientists in Russia (Aleksandrov 1992). The aim of this chapter is to analyze the main tendencies of invertebrate behavioral investigations in Russia from the beginning of this century. This time period can be divided arbitrarily into several subperiods. The first period continued from the end of the nineteenth century up to the beginning of World War I. Russian science during this period was a respectable member of the international scientific community, the equal of other highly developed Western countries. There was, for instance, an established tradition of sending young scientists abroad to further their education. Such famous Russian physiologists as V. M. Bekhterev, N. E. Vedensky, A. A. Ukhtomsky, I. S. Beritov, and L. A. Orbeli spent time working in laboratories abroad. I. P. Pavlov also spent time abroad as a student, spending two years (1884-1886) in Germany, where he worked in Heidenhain's and Ludwig's laboratories (Gureeva and Chebysheva 1969). Throughout his life Pavlov remembered these German physiologists and considered them among his teachers (see, for example, his speech after Heidenhain's death, October 23, 1897: Pavlov 1952, 6:108). As for Vagner, he had a rather long journey abroad sponsored by the St.

6


Petersburg Society of Natural Sciences in 1889. He visited the zoological station in Naples and the Russian zoological station in Villafranca and spent time in Paris, Haage, Bruxelles, and Cairo (Lukin 1987). It is clear that both Vagner and Pavlov and other biologists of that time knew the chief European languages well. They could read scientific papers in German, French, and English; and they had no problem communicating with their foreign colleagues, often publishing their work in foreign journals (see, for example, both Pavlov's and Vagner's list of publications: Pavlov 1954; Vagner 1907). Thus, Russian scientists of the day were well informed about all scientific events, whether be by printed matter or from personal participation in international conferences (Yaroshevsky 1985). All this seems very simple and self-evident. However, this "normal" lifestyle began to deteriorate in 1914, when the world war resulted in the suspension of international scientific exchange. This situation was estimated at that time as "abnormal" and "temporary" (see, for instance, Ladygina-Kots 1923); but it lasted in Russia for many decades, fueled in part by the October Revolution in 1917. The period of the revolution itself and the resulting civil war was extremely hard on scientists, as it was for the average citizen. Life was a daily struggle; perhaps worse for many scientists, it interfered with their experimental and theoretical work. Though most naturalists had antimonarchist views and became loyal to the new power, often collaborating with it (see, for example, A. A. Ukhtomsky [Merkulov 1960; Pomper 1970; Shlupikova 1968], who held the title of "Prince" and whose ancestors were related to Rurik), the conditions for living were so hard that some scientists died from hunger (see short notes in the magazine Priroda in the years 1920-1921). In 1921, life began to be easier because of the "new economic policy" (NEP) of the Soviet government and Lenin. During the 1920s there was a short "Renaissance" in various areas of Russian cultural and scientific life (Shnol 1990). New scientific institutions were constructed, and there was a renewed interest in publishing scientific literature. There was unusual progress in printing books devoted to many biological problems. As the main reason was the intention to educate millions of nonliterate people, most of these printed materials were popular and inexpensive. Among them were books by Russian biologists and translations of interesting books by foreign writers (Sobol 1925). It should be noted that the books and magazines created during this period were to remain the primary reading material until the 1950s and 1960s. Some of these materials are still of interest today. The second time period spanned the beginning of the 1930s to about the 1950s. This period seemed to be harder on science and scientists than the preceding period. The main reason was that such scientific leaders as Bekhterev, Pavlov, Vagner, and Ukhtomsky died, with the leadership of Russian science being passed along to their pupils. Many of these new leaders were selected not according to scientific achievement but rather according to social and political activity, particularly as it related to the Communist party. Highly capable scientists with a deep interest in research found it difficult to maintain scientific careers. Some of

HISTORY OF INVERTEBRATE BEHAVIORAL STUDIES IN RUSSIA

7

these scientists were repressed by being confined to gulags. This was the fate of physiologists E. M. Kreps (Pavlov's pupil) and V. M. Merkulov (Ukhtomsky's pupil), the biochemist A. A. Baev (Engelgardt's pupil), and entomologist B. S. Kuzin. The head of Russian genetics, academician N. N. Vavilov; E. Bauer, the author of the brilliant book Theoretical Biology; the practical entomologist N. A. Alekseenko; and the talented scientific historian E. M. Vermel (among many other scientists) died in these camps and prisons. The great zoologist and geneticist N. K. Koltsov, who "fought" for many years against T. D. Lysenko, died at home in 1939. The main reason for his death was considered by many to be the frustration and sorrow of not being able to carry on his research. It is interesting to note that, though he was not repressed formally, books written by Koltsov were removed from libraries. His books still could not be obtained through official channels such as interlibrary loan as recently as the late 1970s. This time period was known as "the midnight of the century," (from the title of the book written in 1937 by the famous Russian-French writer V. Serge (V. Kibalchich). The period known as the midnight of the century was dominated by World War II ("the Great National War"). Many scientists died in the bloodshed, and scientific life all but stopped. There were attempts to move some scientific institutions away from the fighting. The physiological and medical institutions of Leningrad, for instance, were moved to Kazan and Middle Asia. Scientists who wished to stay in Leningrad (such as Pavlov's collaborator M. K. Petrova; the chief of the ornithological laboratory in Koltushi, A. N. Promptov; and the academician A. A. Ukhtomsky) tried to continue their scientific investigations and hold traditional scientific meetings. However, this heroic activity could hardly be successful in the face of the continued battle for survival. A. A. Ukhtomsky, for instance, died during the blockade of Leningrad, in the winter of 1942. Perhaps the most tragic event of this time as it relates to science is the so-called Vaskhnil Session (1948) of the Ail-Union Agricultural Academy, organized initially by T. D. Lysenko. The goal of this famous session appeared to be the destruction of true genetic investigations in Russia. The Vaskhnil Session spawned the "daughter" session of the "big" and "small" academies (Academy of Sciences and Medical Academy) devoted to Pavlovian "uchenije" (teaching). These daughter sessions were especially important for scientists studying behavior. The goal of these sessions appeared to be the same as that of the 1948 session: the destruction of true behavioral investigations in Russia. It is clear now that these two so-called scientific meetings were inspired directly by the Central Committee of the Communist party and personally by I. V. Stalin (Yaroshevsky 1991a, 1991b). After these Sessions (whose critical analysis has only just begun; see for example Leibson 1990; Grigorian and Roitbak 1991), L. A. Orbeli was relieved from the leadership of both the Biological Section of the USSR Academy of Science (in 1948) and the physiological institutes, which he had controlled after Pavlov's death. Some of Orbeli's collaborators lost their jobs in Leningrad; others had to change their topics. At this last session many scientists were criticized (I. S. Beritov, P. K. Anokhin, L. S. Stern, and A. D. Speransky, to name but a few), but

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the main coup was directed against Orbeli (Leibson 1990). Even now it is extremely difficult emotionally for many Russian scientists to reread the materials of that session. In fact, even in 1948 it was difficult to believe in the sincerity of many of those speakers. Thus it seems reasonable not to analyze seriously their arguments directed against their colleagues and teachers. This period of the "repressed science" ended in principle after Stalin's death in March 1953, when all life in the USSR began to change rather quickly. His death begins the third period, which continues to the present. However, the situation in science (at least in biology) seemed to remain unchanged for many subsequent years. It is important to note, though it seems unbelievable, especially for the Western reader, that the "Sessions" did not solve any scientific problems. The purpose of these sessions was to change the leadership in neurophysiology and related scientific fields. During many years and decades following these sessions the main scientific leadership positions were in the hands of those who "won the battle," inspired by nonscientific motives. For instance, the main "speakers" of these sessions attained high office: K. M. Bykov and A. Ivanov-Smolensky soon became the director and vice-director of the leading Institutes; L. G. Voronin and E. Sh. Ajrapetjants became the heads of the Departments of Higher Nervous Activity in Moscow and Leningrad Universities respectively. They held these positions mostly up to their deaths. Later their places were occupied by their pupils. It may be interesting to know that even in 1973 (twenty years after Stalin's death!) L. G. Leibson, who wrote a book about his teacher L. A. Orbeli, could not describe what actually happened at these sessions because the facts were unknown to ordinary scientists. The behind the scenes injustices of the Pavlovian sessions came to light only because of the influence of "perestrojka" in 1985 (Leibson 1990). However, Leibson's book is only the first bird signalling the spring. Critical analyses of these sessions on Russian science are now being performed (Krementsov 1991; Umrikhin 1991). Thus one may say that after Stalin's death the conditions for scientific work in Russia changed but the "repressive atmosphere" (at least in some biological sciences) remained for many years. The situation changed rather drastically only in the last five or so years, when scientists became much more free in their behavior. Ironically, few scientists can take advantage of this newfound freedom because of the worsening Russian economic situation. HISTORY OF BEHAVIORAL INVESTIGATIONS IN RUSSIA One may divide the behavioral studies performed in Russia during this century into at least three periods. The first period lasted from the beginning of the century up to the middle 1920s when the two main approaches to behavior, the psychological and the physiological, were equally respected. This sentiment is reflected well in the following quotation from a book by Ladygina-Kots: It [the book] is edited at the time of progressive development of the young science zoo-


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psychology when some scientists try to limit behavioral manifestations putting the animal in arbitrary, narrow experimental conditions (the American school), the others "talk" with animals by means of knocks and look for human capabilities in them (German school of Krallists"), the third glue the tag of "reflex" to all the psychical events (from the lowest to the highest) and look for the animal as for the automatic device (Russian reflexologists), the fourth (anatomo-physiologists) hope to understand the psychical life by means of skillful sophisticated surgical technique. (Ladygina-Kots 1923:9) During the second interval, lasting from the mid 1920s up to the end of the 1950s, there was a gradual and complete elimination of the psychological approach to the study of behavior. Moreover, at that time investigations that used physiological techniques differing from those favored by Pavlov and his school were eliminated. For example, at the end of the 1930s V. M. Borovsky (Borovsky 1936) had to stop his scientific work; and after the "daughter" Session in 1950 (the so-called Pavlovian Session) many research programs not fitting the traditional Pavlovian views were closed. In the third period, which began approximately at the end of the 1950s and the beginning of the 1960s, we see a gradual restoration of psychology and comparative psychology. During this period, which continues probably up to the present, we see a shift in the primary interest of Russian scientists to research and theoretical problems posed by Western science. Simultaneously, there appears to be a loss of interest in traditional questions of conditioned reflexes. This change is rather unexpected, considering the declarations of the Pavlovian Sessions. Another remarkable feature of our time is the revival of behavioral investigations using nonphysiological measures. Such research is based on ethological ideas that came to this country from abroad and not from the Russian investigations. It is a testament to the effectiveness of the Pavlovian Sessions and other Russian scientific catastrophes that the vast majority of Russian students have no collective recognition of the rich Russian tradition in ethology or "zoopsychology" started by Vagner. Let us look at these periods of behavioral research in more detail. "Objective Biopsychology" and the Theory of Conditioned Reflexes The development of "objective biopsychology" in Russia is connected entirely with V. A. Vagner (1849-1934). Today his name and his views are almost unknown to Russian behavioral scientists (Vagner 1900a, 1900b, 1910a, 1910b, 1913, 1914b, 1923). It seems that recently he is known better abroad than at home, because of the publications of Joravsky (1989) and especially of N. Krementsov (1992). Even here, however, the information about Vagner is incomplete. In this section I will present some information relating to Vagner's personality (Fabri 1969; Kim 1966; Roginsky 1940a; Strelchenko 1975). As a student Vagner entered both the Faculty of Law and the Faculty of Physics and Mathematics of Moscow University. Among his teachers were S. A. Usov,

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Ya. A. Borsenkov, and A. P. Bogdanov, who were pupils of the famous Russian zoologist K. F. Rulje. In his third year at the university he began to study seriously the anatomy and taxonomy of spiders. Figure 2.1 shows a picture of Vagner during this time period. While investigating the fauna of the Black Sea in 1882 he met with the famous biologists 1.1. Mechnikov and A. O. Kovalevsky. At this time he turned his attention toward the investigation of blood content in different sea invertebrates. Clearly, from the very beginning of his university education he had a deep interest in invertebrates. Vagner's thesis was devoted to an analysis of spider taxonomy in which he based his classification mainly on their sex organs (Vagner 1890). He proposed that behavioral features were good taxonomic indices, whose value was just as important as morphological indicators. His use of interspecies variability of behavioral features was heralded as a new method in comparative morphology. He also attempted to apply the scientific method to zoopsychology, which he called "objective biopsychology." For Vagner, biopsychology was the study of rules and tendencies in the evolution of behavior, using field observations and experiments under natural conditions. His main ideas, together with thorough, critical reviews of psychological and biological literature, are represented in his book "Biological Bases of Comparative Psychology: Biopsychology, printed in 1910 (vol. 1) and 1913 (vol. 2). The original data used in these books were obtained by Vagner earlier. Vagner's views concerning the evolution of behavior and the specificity of invertebrate behavior will be treated later in this chapter. One may state here, however, that Vagner provided a strong foundation for a naturalistic approach to behavior that is very similar to what is known in the West as ethology. The aims of biopsychology and its methods coincide with the program of "classical" ethological research (Krementsov 1992). It is obvious that his ideas were very popular at the beginning of this century. They influenced, for instance, such biologists as Kholodkovsky (1897), Shimkevich (1897), and Severtsev (1922). Vagner was well known, too, for his social activity. He lectured for many years on biology and comparative psychology at Moscow University. Later (from 1905) he was the director of the so-called Commercial College in St. Petersburg. In 1912 he realized his dream of creating a new popular magazine, Priroda. He discussed the idea of forming such a magazine with the writer A. P. Chekhov in the 1890s and became (with L. V. Pisarzhevsky) its first editor. It is interesting to note that the first issue of this magazine, which is still in circulation, has at the masthead a picture of the "assiduous bee" that Vagner displayed on his calling card. For ten years he was the editor of the famous series New ideas in biology; he also founded periodicals in the field of pedagogics. The physiological approach to behavioral research in this century was represented in Russia first of all by I. P. Pavlov (1849-1936) and V. M. Bekhterev (1857-1927). The names and works of both these famous scientists are well known in this country and abroad. Many papers and books have been written about their lives and scientific activity. Their works (especially those of Pavlov) were


Figure 2.1 V. A. Vagner (Courtesy of B. V. Lukin, Priroda, 1987, issue 1, p. 50).

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translated into other languages and re-edited many times in Russian. Pavlov and Vagner were contemporaries. They worked for a time in the same city (St. Petersburg) and in the same scientific field. Both had common teachers and common views and were strong personalities. As so happens in science, despite sharing many characteristics, they were competitors rather then friends. They worked independently and moved in different circles. As is well known, Pavlov had both university and medical education. He had surgical skill, which he first applied to the study of the heart and the blood vessels. Later he used these skills to investigate the physiology of the digestive system. For this research he was awarded the Nobel Prize in 1904. However, at the end of the last century his interests shifted to the event called by him the "conditioned reflex." For the rest of his life Pavlov, together with many pupils, studied this wonderful phenomenon, mostly in dogs, by means of a technique he developed for this purpose (Pavlov 1923, 1951, 1954). V. M. Bekhterev, a psychiatrist and neurologist by education, worked in clinics and studied for many years the functional anatomy of the human central nervous system. Throughout his life Bekhterev maintained a deep interest in human psychology (Bekhterev 1907/1912). About the same time as Pavlov, Bekhterev also discovered the conditioned reflex, though Bekhterev called the phenomenon the "combination reflex." Rather than study the salivary reaction as Pavlov did, he preferred to study the movements of humans. He criticized Pavlov's technique but used much of Pavlov's data to confirm his own views. In the beginning of their research in this new field, Pavlov and Bekhterev believed that these phenomena belonged to the domain of psychology. However, later they changed their minds, realizing that all the "psychical" events can be studied by means of "conditioned" or "combination" reflexes. Thus there arose such terms as "higher nervous activity," introduced by Pavlov, and "reflexology," used by Bekhterev. Both of these "teachings" developed in the beginning of this century. Now they seem rather similar. At the turn of the century, however, they appeared contradictory, and their fate was different. "Victory" of Pavlovian Views: But at What Cost? I have tried to show that in the beginning of the century two big physiological schools in Russia were extremely active in the study of behavior. It seems that psychological views were not as popular even at that time. This probably reflects the general tendency of the development of natural sciences in the last century and the status of psychology itself at the end of the nineteenth century. However, as the Ladygina-Kots quote indicates, different approaches to the study of behavior and were available and given scientific expression in journals and other forums. This situation changed rather soon. In the 1920s the main way to study behavior became the method of conditioned reflexes (CR). Moreover, the theory of CR served as the foundation for not only physiology but psychology as well. Soviet


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psychologists viewed Pavlov's teaching as the basis for the establishment of a truly scientific approach to human psychical functions (Yaroshevsky 1985). As for Bekhterev, he was also extremely active during these years. Twelve institutions that he founded were eventually consolidated by Bekhterev into the State Psycho-Neurological Academy (Mojiseeva 1988). The academy had two scientific departments, clinical and experimental. It also included several additional institutions serving practical purposes, such as a school for retarded children. Bekhterev generated many ideas of great importance concerning human research in general and especially in children. He did not, however, have a systematic scientific program of research (Yaroshevsky 1985). He died suddenly in 1927 after visiting Stalin in his capacity as a psychiatrist. For many years there was a rumor that Bekhterev was poisoned (Guberman 1977). There are different opinions as to what happened to him. It is obvious that Bekhterev's name was forgotten soon after. Vagner, whose career was severely hurt by World War I and the civil war (Lukin 1987), was mainly occupied in the 1920s with pedagogical work (Ya. M. Pressman, personal communication) and with the organization of the Museum for the Evolution of the Nervous System and Comparative Psychology, which was based on the Anatomical Museum of the Bekhterev Institute of Brain Research. The exposition proposed by Vagner represented the correlation between nervous structure and function on one hand and behavioral and psychic activity on the other hand. The museum contained exhibitions of conditioned reflexes in fish and dogs, habits in ants, maze experiments with rats, and so on (Roginsky 1940b). Throughout these years, up to his death in 1934, Vagner wrote much but could publish rather little. His main book published during this time was The Essays in Comparative Psychology, which was based on the third volume of his Biopsychology (which was to be edited in 1915 but was "lost"). The essays were published in the form of ten thin booklets of low quality (especially if one compares them with the first two volumes) in 1925-1929. These volumes as well as other published work were never re-edited. Among his last published books is a very small and rare volume entitled Some Ways to Observe Animals (1926). His Comparative Psychology written at the end of his life, could not be published despite his and his wife's efforts (Lukin 1987). In Krementsov's opinion (1992), the main reason for the loss of Vagner's popularity after the October Revolution is the obvious discrepancy between his views and Marxist ideology. One may also add, the popularity and political power of Pavlov's views. There are no data about personal contacts between Pavlov and Vagner or about Pavlov's comments concerning Vagner's work. However, some of Pavlov's students (Yu. P. Frolov, G. P. Zeleny) "fought" with Vagner from the 1910s and for the next several years. These students of Pavlov denied the importance of behavioral research in itself and proclaimed the physiological approach to behavior as the only reasonable approach (Frolov 1925; Zeleny 1913). Vagner's reaction to this attack has been expressed several times (see, for instance, Vagner 1914a, 1914b). In Vagner's opinion, the physiological approach is insufficient, because

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higher mental capabilities are outside the methods used by the physiological school and because the reflex theory cannot explain the existence or formation of instincts. His conclusion was that the physiological school has major problems dealing with the facts of comparative psychology. The weakness of the physiological school becomes clear when they try to resolve what Vagner considered the main task of comparative psychology: to discover the rules governing the evolution of psychical events. Without this knowledge, according to Vagner, it is impossible to understand human psychology, just as it is impossible to understand human anatomy without an understanding of comparative anatomy. The criticisms that Vagner leveled at the physiological approach have been ignored since shortly after the October Revolution. At the end of the 1930s Vagner's contribution to Russian science was all but forgotten. He had few students; the best known were G. Z. Roginsky, who worked primarily with monkeys (Roginsky 1947), and B. I. Khotin, who studied imitation from an evolutionary perspective (Khotin 1990). Unfortunately Khotin could not work during the war (he was a soldier) and died soon after the war. With his death the comparative psychological work in the USSR also seemed to die. Around the period of the October Revolution, several famous scientists in addition to Vagner worked in the field of comparative psychology. D. N. Kashkarov, who is known as the founder of ecological zoology in the USSR, lectured on comparative psychology at Moscow University. The materials from these lectures were used for writing a book that remains useful even today (Kashkarov 1928). Another important figure was V. M. Borovsky, who was the head of the zoopsychological laboratory in the Moscow Psychological Institute. His topic was not far from Vagner's, but his approach was purely physiological (Borovsky 1936). He proposed, for instance, that instinctive actions are the result of unbalanced physiological rhythms in the organism. Moreover, he rejected the term "instinct" for its mystical and religious connotations. Although he had many more students than Vagner (for example, Akimov, Kobozjeva, Tikh, Skrjebitsky, Bibikova), like Vagner's, his contributions are all but forgotten. Another great personality of this time was N. N. Ladygina-Kots. She was the wife of the famous zoologist A. P. Kots, who worked for many years at the Moscow Zoo (Kots 1917) and was the founder of the wonderful Zoological Museum named for Darwin. In addition to her scientific training she was also a student of the famous Russian painter A. M. Vatagin, who is known most of all for his drawings of wild and domestic animals. She studied in detail the elaboration of new habits and sensory capabilities of monkeys, using methods she devised for the purpose. In Figure 2.2 she is seen holding one of her subjects. Ladygina-Kots proposed techniques for studying monkey behavior based on many hours of observation of the animal's natural habits under conditions of maximum freedom (Ladygina-Kots 1928, 1935, 1958, 1960). She criticized not only the purely physiological approach, by which some fragmentary reaction is studied, often under conditions of restraint, but also the approach of some psychologists of that time.


Figure 2.2 N. N. Ladygina-Kots (From Ladygina-Kots 1923).

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In the author's opinion, one may force the silent animal to "open" his mouth only if the common language between the experimenter and the animal will be . . . specific for each animal (and only for it). If we stay face to face with Nature itself, before the live animal we should first of all not dictate to it our conditions and our demands but to try to sense it from the interior, to listen for it, to watch it and to find the best way to make contact with it; then it should "open" itself and "reveal" itself extremely easily and completely. (Ladygina-Kots 1923:9) Ladygina-Kots was certainly a unique scientist and not typical of Russian behavioral scientists of that time. The great bulk of young scientists who had an interest in animal behavior were involved by necessity with the views expressed by Pavlov and his school. After Pavlov's death in 1936, the main person responsible for the development of higher nervous activity became L. A. Orbeli (1882-1958). Orbeli was a student of Pavlov and a renowned scientist in theoretical and applied physiology. Orbeli is presented in Figure 2.3. His interests were rather broad. They included higher nervous activity, evolutionary physiology, trophic functions of the nervous system, kidney functions, and problems associated with pain (Leibson 1973; Svidersky, Veselkin, and Natochin 1988). Soon after he become the head of Pavlov's laboratories, he proposed a broad and innovative program for investigating "the teaching of conditioned reflexes." It is important to note that, for Orbeli, the application of the evolutionary, or comparative, approach to different functional systems, including problems associated with the central nervous system, was the correct approach. He wrote on many occasions that such an approach had been advocated by Pavlov himself at the end of his life. However, it now seems clear that it was mainly Orbeli's initiative to investigate the interaction between inborn and acquired behavior across taxons. In his behavioral experiments, he decided to use three lines of the evolutionary process: the insects, in whose behavior instinctive reactions predominate; the birds, which have a good representation of inborn and acquired behaviors; and the mammals, in which conditioned behavior attained its maximum expression. In his work with mammals the dog was often the animal of choice, but monkeys were also employed (Orbeli 1949). The comparative investigations of behavior took place primarily in Koltushi. Koltushi is a village not far from Leningrad where by special decision of the Soviet government, Pavlov founded one of his laboratories. Later it was reorganized into the Institute of Evolutionary Physiology and Pathology of the Higher Nervous Activity (see Zakharzhevsky and Andreeva 1984). Though most studies were performed at the institute on dogs, the ornithological laboratory and the laboratory of insect biology were also in operation. In some behavioral experiments Drosophila was used (Masing 1947). The major findings of the investigations performed in Koltushi during this time period are represented in a book devoted to the tenth anniversary of Pavlov's death but published some years later. In this book appear both a list of scientific publications during 1936-1946 and a list of theses defended in the institute. It offers an


Figure 2.3 L. A. Orbeli (Courtesy of Yu. Burmistrov).

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interesting glimpse into scientific life during the war years. For the purposes of this chapter we will focus later on the work from the laboratory of insect biology headed by S. I. Malyshev (Malyshev 1949). As for general approaches to studying behavior, one should first mention A. N. Promptov (1898-1948). Promptov's views are the combination of the two main Russian approaches to behavior, the naturalistic (Vagner) and the physiological (Pavlov). Throughout his life, especially after leaving the Faculty of Physics and Mathematics of Moscow University, Promptov studied the behavior of birds (Promptov 1947). His main interest was in natural behavior. After 1940 he was invited by Orbeli to Koltushi, where he established a laboratory for the study of birds. One should note that during the war (and the famous blockade of Leningrad) he stayed behind in Koltushi so that he could protect his birds. Moreover, during this extremely hard time he had enough courage, strength, and will to continue his experiments (Lukina 1956). His main topic was the interaction of inborn and acquired behavior. For instance, he thoroughly investigated, using field and laboratory techniques, the orientation reaction to visual and auditory stimuli in such birds as skylarks, meadow pipits, northern nightingales and blue throats. In a book written during his last years (published in 1956, after his death) Promptov provided a description of the "biocomplex," a central theme of his research program. It may be described as "the set of reactions specific for every biological situation which has phylogenetically adaptive value and which is determined by the functional activity of the nervous and humoral systems" (Promptov 1956:110). It is similar to Vagner's idea of the "complex instinct." Rather close to Promptov's position was that of another scientist who was invited by Orbeli to Koltushi: L. V. Krushinsky (1911-1984). Throughout his life Krushinsky was associated with Moscow University (Semiokhina 1991). Like Promptov, he was a true naturalist who loved observing animals in their natural habitat. He was an expert in dog behavior and training, skills that were put to use during the war years. Later, using Promptov's idea about the role of excitability in the nervous system as a factor in the modification of animal behavior, he and his collaborators produced a rat that was extremely sensitive to sound. The line of such hyperexcitable rats (named KM, which means Krushinsky-Molodkina line) still remains unique in world practice and is used in many laboratories in this country and abroad. Figure 2.4 presents a picture of Krushinsky. When Krushinsky worked in Koltushi, his primary research was performing a behavioral-genetic analysis of learning in dogs (Krushinsky 1991). During thistime he wrote a very interesting paper entitled "Some integrative stages in theformation of behavior" (Krushinsky 1948). He argued that it was impossible to separate inborn from acquired behaviors and proposed the concept of "unitary responses." Such responses recognize the unity of both behavioral forms and the interrelations between them. He believed that these unitary responses combine to form a single adaptive action. He described the ways of forming these reactions and their possible involvement in behavioral evolution. Later, as a professor at the Department of Higher Nervous Activity in Moscow


Figure 2.4 L. V. Krushinsky (Courtesy of N. Krushinskaya).

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University, Krushinsky investigated "elementary intellectual activity" in animals. He proposed techniques for studying this activity across developmental stages and across the phylogenetic scale. In his opinion, Intellectual activity is a kind of higher nervous activity; due to this, it is possible to solve— without some special learning—many tasks based on certain logical connections between the components. By means of intellectual activity one may detect the rules which connect the events and things in the environment. Certainly intellectual activity is a behavioral form, due to which the animal can react highly adaptively to rapidly changing environmental conditions. (Krushinsky 1977:207) Krushinsky and his collaborators investigated behavioral reactions of different animals when faced with a common set of problems. The results of these experiments indicated that monkeys were the best problem solvers, closely followed by dolphins. Following the dolphins were the carnivorous mammals, and much further behind were hares and rats. Good results were obtained also with birds, especially with the crow and its relatives. In contrast, "lower" vertebrates such as turtles and lizards could solve only the simplest kind of extrapolation task and fish could solve them only after extensive training. Thus Krushinsky showed that great differences are found between animals belonging to low and high stages of phylogenesis if one compares their capabilities to detect some simple natural rules and how to use them. It seems evident from the above-mentioned facts that the investigations headed by L. A. Orbeli, which took place in Koltushi during the 1940s had good prospects for continued success. The experiments showed the importance, indeed the necessity, of using a wide range of species in behavioral experiments and examining various types of behavior, not just higher nervous activity. Unfortunately this fruitful period was rather short, limited first of all by the Great National War and by the previously mentioned Pavlovian Sessions of 1948 and 1950. A different approach to Pavlov's study of higher nervous activity is connected with the name of the famous Georgian scientist I. S. Beritashvili (Beritov, in Russian transcription) (1885-1974). He was N. E. Vedensky's student at St. Petersburg University, where he specialized in neuromuscular physiology and was very skillful in electrophysiological techniques of that time. It is interesting to note that Pavlov did not approve of all this "wire" physiology. In 1915 Beritashvili obtained a job in the famous Novorosijsky University (Odessa), where he began to study conditioned reflexes in different animals. Though it was the time of World War I, which was followed by the October Revolution and civil war, the years spent in Odessa were some of the most fruitful in his life (Roitbak 1991). In 1919 he obtained work in the Georgian capital of Tbilisi, where he remained until his death. He began teaching in Tbilisi University and wrote books in various fields of neurophysiology. He had an encyclopedic knowledge of neurophysiology and was a popular person in scientific and social circles. Moreover, he was a brilliant experimenter who routinely developed new techniques. He also had the


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very rare ability of being able to generate novel scientific ideas. Being an expert in neuromorphology, he tried to connect structure and function. He was one of the first to approach a research problem from the physiological, morphological and biochemical perspectives (Roitbak and Bakuradze 1988). In addition to his work in neurophysiology Beritashvili simultaneously had a deep and lifelong interest in the study of animal behavior. He began replicating Pavlov's experiments with conditioned reflexes in dogs and soon understood their limitations. Like Vagner, Beritashvili believed that the behavior of the whole animal must be studied under conditions in which the animal is free to move about. He developed some specific techniques that allowed him to establish, among other things, the essential differences between reflexive and complex behavior, the ontogenetic and phylogenetic development of specific types of behavior, and the dependence of behavior on context. Though he was a "pure" physiologist and did much in the field of neurophysiology, his main approach to behavior was neuropsychological. He proposed the concept of "psycho-nervous" activity, which he considered to be a higher form of individual behavior than either reflexive or instinctive behavior. As opposed to conditioned reflexes, the reactions of the psycho-nervous type are formed rapidly. They arise due to remembering some events (that is, due to the psycho-nervous representation, or image, of the event) that the animal experienced only once (Beritov 1947). Later he investigated the mechanisms of spatial orientation of animals across species. At the end of his life he was completely occupied with the problem of memory. He divided the memory of vertebrates into three types: imaginative, emotional, and conditioned. He proposed that highly organized vertebrates have some elements of intellectual activity that permit goal-directed behavior. It had been noted previously that Beritov was one of the main figures condemned during the Pavlovian Session of 1950. In fact, the critique of him began some years earlier, after the publication of his small book entitled The Main Forms of Nervous and Psycho-Nervous Activity (Beritov 1947). It is necessary to pay attention to his extremely courageous behavior at that time. To gain some appreciation of this period in Russian scientific history, it is instructive to read a volume of his own material (most of which was completely unknown) published after his death (Beritashvili 1984). In this book are many papers written by him in response to accusations directed against him by some fellow neurophysiologists (for instance, Vatsuro and Voronin) and philosophers during the period before and after the Pavlovian Session of 1950. It was during this session that he was removed from the post of director of the Physiological Institute, Georgian Academy of Sciences. The Current Stage of Behavioral Investigations The beginning of this period seems to be at the end of the 1950s. As is known that one may see the great only from a certain distance, it is not easy to describe

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the most essential features of the time span to which we belong. From the historical point of view this period may be characterized as the time of "coming full circle." The pretensions of many neurophysiologists who believe that there is only one way of conducting behavioral research was expressed extremely well at the Pavlovian Session of 1950 and in the first few years after it. Such an attitude gradually disappeared as the Communist party loosened its grip on science and access to Western scientific literature improved. After approximately a thirty year absence, the teaching of comparative psychology was reinstated at the Psychological Faculty (opened at the beginning of the 1960s) in Moscow University. The restoration of the lectures on comparative psychology is the achievement of K. E. Fabri, who lectured at Moscow University for many years and wrote a book based upon these lectures (Fabri 1976). On the other hand, due mainly to the investigators in the field of vertebrate zoology (which had excellent traditions in this country), there was a revival of behavioral research based mostly on ethological concepts. Some results of their activity are represented in the proceedings of the All-Union Conferences on Animal Behavior organized by the Institute for Evolutionary Morphology and Animal Ecology and held at Moscow University in 1972, 1976, and 1984. In the preface to the proceedings of the last conference, one of its leaders, E. N. Panov, noted that there were big changes in the development of behavioral investigations during these twelve years. For instance, the number of participants increased from 146 to more than 600, and the topics of the reports became much broader. Reading the papers of these conferences one can clearly see the flowering of new approaches and new combinations of existing approaches to the study of behavior. It is obvious, however, that it is only the beginning in the development of this perspective. As for the achievements of the physiological school of Pavlov, in the narrow sense, it is not easy to estimate them. On the one hand, hundreds of papers have been written, especially in the 1950s, about the great victories of the teachings of Pavlov. The tradition of describing the investigations of conditioned reflexes exclusively in "pink light" is slowly fading (see, for example, Simonov 1991; Suvorov and Andreeva 1990). In my opinion Russians are not ready for a true critical analysis of the achievements and mistakes brought upon Russian behavioral science by the incidents reported here. It seems such an analysis is easier to conduct abroad (see, for example, Joravsky 1989). To conduct such an analysis properly, however, several generations of behavioral scientists should pass. This includes not only the old generation (whose life was more or less destroyed during these years of the "great jumps" and "big terror") but also people of young and middle age whose minds were damaged by the type of educational system that dominated in this country for so many decades. BEHAVIORAL STUDIES OF INVERTEBRATES The history of behavioral investigations of the main invertebrate groups will be


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presented in the chapters that follow. This section will offer some general considerations and provide a short review of studies omitted by other authors. My additional purpose is to illustrate (by examples taken from behavioral studies of invertebrates) the great influence of nonscientific factors on scientific work in this country. General Remarks It is well known that there were many great names in the last century in the field of invertebrate zoology in Russia (Strelkov 1967). First of all are the names of A. O. Kovalevsky and I. I. Mechnikov. Many brilliant investigations describing the natural history and morphological features of various invertebrates were performed at the end of the nineteenth and in the beginning of the twentieth century. These investigations were performed in universities throughout Russia, including those, for example, in St. Petersburg, Moscow, Kazan, Kijev, and Odessa. After the October Revolution there were a number of famous Russian invertebrate zoologists, including K. I. Skrjabin (the founder of helminthology in this country), E. N. Pavlovsky (who worked in many fields but is known primarily for his work with mosquitoes as disease vectors), V. A. Dogel (a specialist in ecological parasitology and comparative anatomy of invertebrates, whose manual Invertebrate Zoology first appeared in the 1920s and was re-edited until the 1980s, who for many years lectured at Leningrad University), V. N. Beklemishev (who lectured on comparative invertebrate anatomy at Moscow University), N. A. Livanov (who worked at Kazan University and had a special interest in the evolution of the nervous system of invertebrates), and L. A. Zenkevich (the specialist in sea hydrobiology who studied the evolution of movement in different invertebrate taxons), to name but a few. From just this short list it is easy to conclude that there was a rather high level of invertebrate research performed by Soviet zoologists. As for comparative physiological investigations of invertebrates, two main groups—one headed by L. A. Orbeli (in Leningrad) and the other headed by Kh. S. Koshtojants (in Moscow)—should be noted. Orbeli's students who were interested in evolutionary problems worked in the Institute of Evolutionary Physiology and Biochemistry, founded in 1956. Kh. S. Koshtojants (1900-1961), who was the head of the Department of Physiology at Moscow University, created the Moscow school of comparative physiology, which differed from Leningrad's mainly in its biological direction (Sakharov and Turpayev 1988). Koshtojants tried to incorporate physiology into the biological sciences. He had some personal experience in conducting invertebrate research from his work in G. Jordan's laboratory. He retained interest in these animals throughout his work at Moscow University and encouraged many of his students to use different invertebrates in their experimental research. The results of this research, together with an exhaustive review of the literature, were presented in his two-volume book, The Comparative Physiology (the second volume was devoted to the comparative physiology of the nervous system) (Koshtojants 1957). One

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may note that many students of Koshtojants retained an interest in evolutionary problems and invertebrate research, for example, T. M. Turpajev, who for many years was the director of the Institute for Developmental Biology; D. A. Sakharov, the great specialist in mollusc neurobiology; and J. Salanki, the director of the ADD Balaton Limnological Research Institute (Hungry), and president of the International Society for Invertebrate Neurobiology. However, the main interests of these scientists were rather far from behavior itself. As for behavioral research on invertebrates, it has been previously noted that for many years in this country there existed only one "acceptable" way of studying behavior, which, of course, was the method proposed by Pavlov. Unfortunately, he had no obvious interest in invertebrate behavioral studies. Though he once worked with the clam Anodonta, it was during his visit to Heidenhain's laboratory in 1884. The history of this work described by Pavlov himself is rather interesting (Pavlov 1885). He wrote in the introduction that he could not fulfill the work proposed by Heidenhain because not much was known about the organization of the nervous system as it related to the control of "leg" movements. Pavlov decided to investigate neural organization of motor control in the clam. This line of research was in fact related to his ongoing interest in the excitatory and inhibitory control of peripheral structures. Work on this problem was still of interest to Koshtojants' students (Koshtojants 1957). It seems, however, that it had no deep influence on Pavlov's investigations of behavior. It is well known that for Pavlov the conditioned reflex was the universal event in the animal world. However, this belief remained to be proved. It is obvious from the book of Majorov (Majorov 1954), which describes in chronological order all work performed in Pavlov's laboratories, that the first investigation of invertebrate behavior was made in 1924 by Kreps (Kreps 1925 and see below). Only then was the following problem considered: Is it possible to elaborate the conditioned reflex in animals without a cerebral cortex? (Majorov 1954). To judge the level of Pavlov's interest in invertebrates, it is helpful to look at his complete works including the "Pavlovskije sredy," three books of notes that were written during Pavlov's laboratory sessions held every Wednesday (Pavlov 1954). It was noted above that the evolutionary approach to studying behavior was generated by Orbeli. One of his students, L. G. Voronin (who was one of the "winners" at the Session of 1950) became the head of the new department founded at Moscow University after this session (the Department of the Physiology of the Higher Nervous Activity). Under his leadership, many investigations were done on the comparative physiology of conditioned reflexes, and some of them used invertebrates (see below). The main results of these studies and Voronin's view concerning this problem were represented in his lectures, which were edited several times (Voronin 1957, 1977, 1984). The comparative physiological approach to behavior and the study of the evolution of conditioned reflexes took place at the Rostov-on-Don University and were performed by A. B. Kogan. Kogan wrote a manual entitled The Bases of the Physiology of Higher Nervous Activity (first edition 1959; second edition 1988), in


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which he considered the problem of the evolution of higher nervous activity from the Protozoa on up. Moreover, his experimental course at the university was organized in such a way that students used various invertebrates for the study of behavior (Kogan and Shchitov 1954). The practical works in Kogan's course on physiology of the nervous system were divided into two groups: "lower" and "higher" nervous activity. In the first group, there was a description of such tasks as hydra reflexes, the reflectory movements of the isolated snail "leg," the reflectory coordination of worm movements, the role of the head ganglia in worm movement, the reflexive behavior of antennas in the cockroach, the reflexes in the isolated ant head, and circling movements of the crayfish after unilateral deletion of the cerebral ganglion. In the second part of this course there were experiments directed toward the study of the evolution of conditioned reflexes: conditioned orientation of searching movements in hydra (see below); conditioned reflexes to sound in Ascidia (see below); conditioned reflex to skin and muscle stimulation in worms; the extinction of natural defense reactions in the snail; the transition of the "negative" response to illumination into a "positive" one in the beetle Tenebrio molitor; and the elaboration of color discrimination in the cockroach. Considered below will be work done at Rostov University on the conditioned reflexes of Daphnia. Unfortunately, no data are available about other works in the field of invertebrate behavior that were made by this group of investigators. Following these general remarks is a description of some papers concerning behavioral investigations in invertebrates that were omitted in subsequent chapters and are important from a historical point of view. The first to be represented is a group of organisms considered by many not to be animals. There is, however, a long tradition of describing the nervous functions and behavioral organization in unicellular organisms, the Protozoa. Protozoa In the beginning of this century many countries, including Russia, expressed great interest in unicellular organisms. S. I. Metalnikov, who worked for many years with infusorians, published in the prestigious journal Izvestiya Imperatorskoj Akademii Nauk (Metalnikov 1915) a remarkable paper "Reflex as a creative act" which was later criticized by some scientists up until the 1950s (Koshtojants 1957; Lobashov 1951). In this paper, he discussed some general problems concerning reflexive reactions. In his opinion, even in infusorians the reflex reactions to different extrinsic influences are extremely variable. The variability of reflexes can easily be observed, "especially in unicellular organisms. In this case we can see every reaction in its entity, in all its appearances. The whole animal—the single cell—is before our eyes under the microscope, and we can discriminate all the peculiarities of every response" (Metalnikov 1915:1803). The variability observed in the reflexive behavior of protozoans suggested to Metalinikov the presence of individual behavior acquired through experience.

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Certainly this position of the author could not be accepted by many behavioral scientists of that time (and especially of the time that followed). The position expressed in that paper was criticized as an "idealistic" one. Different from this "idealistic" paper is another remarkable investigation on Protozoa which seems to be forgotten in Russian literature. This work was a "pioneer" in several respects. I refer to a paper of N. N. Plavilshchikov (18921962), who later became the famous entomologist who contributed much to the Zoological Museum at Moscow University. The paper, which was published in 1928 (Plavilstchikov 1928) in one of the journals arising at that time (see above), had the modest title, "Some observations on the irritability phenomena in infusorians." The purpose of the experiments was to elaborate the conditioned reflex in the colonial infusorians Carchesium lachmanni. Touching the colony produced a contraction, which was used as the unconditioned stimulus (US). The conditioned stimulus (CS) was a one minute illumination with red or blue light. Both the conditioned and the unconditioned response were estimated visually by means of a rating scale. After 150-200 CS-US pairings the infusorians began to contract in response to the CS alone. At this stage the CS was presented on test trials in the absence of the US. The conditioned reflex was retained for several days and could be extinguished. Plavilstchikov's experiment was one of the first on classical conditioning in Protozoa. A second purpose of his study was to see what will happen after the transplantation of trained organisms into a colony of untrained organisms. Part of the "learned" colony was transferred to a "naive" colony. The experiments began one or two days after the successful surgery. First, the effects of the CS and US on the "learned" organisms were tested. After 7-22 trials, the "learned" organisms were removed (together with a little part of a "host" colony), and the responses of the "host" colony to the same stimuli were compared with the responses of "controls" (colonies that were simply damaged). The main result was that the host colony had the same reactions to the light that were elaborated in the trained colony. "This second part of the experiment, in my opinion, is especially important because it indicates the possibility of a special kind of induction' of intracellular transmission of capability for a conditioned response" (Plavilstchikov 1928:24). Later, the author noted that he obtained similar results in experiments with worms. As for the mechanism of this phenomenon, he tried to explain it by means of the "irritation concept" of P. Lazarev and by the "biological rays" of A. Gourvich, which were popular at that time in Russia. It is evident that this work opened several lines of investigation. First of all it introduced a new technique for studying associative processes in Protozoa. Second, it indicated the possibility of memory transfer in these organisms (it is interesting to note that the problem of memory transfer seemed rather important in those years). Third, the author's explanation of his data indicated the high level of biological thought in this country during the 1920s; it was later destroyed when such biological branches as embryology, biophysics, and genetics were repressed. It would certainly be of interest to repeat these experiments today.


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Coelenterata A short paper of Zubkov and Polikarpov (1951), which was rather popular in its time, seems now to have interest only from a historical point of view. The main purpose of this work (as noted by the authors) was to discredit Beritov, who had suggested that animals with "primitive" nervous systems had only inborn or innate behavioral reactions. The experiments were performed on hydra. The animal was placed in a watch-glass filled with water. It was discovered that the incidents of the hydra's searching movements inside the glass depended on the animal's position relative to the previous water level. Changing the water level influenced these movements, but the movements remained for a time in the same form as before. The authors believed that this fact (keeping the movements characteristic of one situation an hour or more after a new water level was introduced) indicated the elaboration of a conditioned reflex in hydra. "This experimental fact gives the refutation of the metaphysical concept of the academician I. S. Beritov concerning the supposed lack of the acquired nervous activity at the early stages of nervous system development" (1951:302). The above mentioned paper seems strange in its purpose and in its results. A more detailed investigation made several years later (Chaylakhian 1957) did not support the conclusion of Zubkov and Polikarpov. He tried to form a classically conditioned reflex in hydras by means of combining illumination with electrical stimulation through the water. After special consideration of their responses to CS, US, and their combination, L. M. Chaylakhian concluded that "the phenomena which from the first sight seem to indicate the presence of contemporary connections in . . . Coelenterata after more detailed physiological analysis can be explained with more simple physiological processes" such as the rise of excitability, accommodation, and the like (Chaylakhian 1957:773). He noted too that the data obtained in Zubkov's and Polikarpov's experiments can also be interpreted this way. Ascidians As noted above, the first work on invertebrates done in Pavlov's laboratory belonged to E. M. Kreps, who is known as a prominent comparative physiologist and biochemist and as Director of the Institute of the Evolutionary Physiology and Biochemistry (1960-1975). At the time of that work, however, he was a student of the Military Medical Academy. He participated also in the work of Pavlov. In the summer of 1921 he visited the Biological Station near Murmansk, which was founded in the last century and belonged to the Society of Natural Scientists in Petrograd (the name of St. Petersburg in the first years after the October Revolution). Kreps convinced Pavlov to request this society to organize at the Murmansk Station, a laboratory for studying higher nervous activity of the primitive ("the lower") animals. This request was fulfilled, and Kreps began to investigate the behavioral reactions of animals abundant in the White Sea that

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could be kept easily in the laboratory. These included Ciona intestinalis, Pyura aurantia, Styela rustica, and Phallusia obliqua, who belong to a very strange taxon, Ascidia. Included in this taxon are very primitive animals with sedentary lives, which nevertheless are relatives, not of the invertebrates, but of Chordata. The results of this thorough investigation were reported at the meeting in Pavlov's laboratory ("Wednesday" meetings) and approved by Pavlov, who recommended them for publication (however, Pavlov never referred to them). Kreps began with a detailed study of the ascidian response to a single mechanical stimulus (a drop of water striking from above). The strength of the stimulation could be easily controlled by adjusting the height at which the drop was released. With this method it was possible to adjust the stimulation so that there was a contraction of only the syphon or, alternatively, a contraction of the whole body. Then he investigated these responses to repetitive mechanical stimuli in intact animals and in animals with ablated ganglion. He also studied the influence of environmental factors such as temperature and tried to elaborate the conditioned reflex to illumination and sound. It appeared that ascidians could produce only two kinds of movements: (1) contraction of the body or the syphon (defined as "defense reaction") caused by some extrinsic stimuli and (2) contraction of the syphon ("throw out reaction") in response to some intrinsic agents (such as chemicals introduced into the syphon). Weak artificial light was ineffective, and strong illumination resulted in a defensive reaction. Sound itself caused no reaction; after pairing with the mechanical stimuli, "it became the excitor of the defense reaction." It is evident that the author was very cautious in speaking about conditioning in ascidians. He was not sure that he could discriminate between the conditioned reflex in ascidians and the rise of excitability due to repetitive stimulation. "It is not easy to draw the distinct line between the conditioned response and the simple rise of the excitability in primitive animals with a very limited range of reactions" (Kreps 1925:226). It should be noted that Kreps maintained this point of view throughout the remainder of his life, though some authors tried to conclude on the basis of this work that even in ascidians the conditioned reflexes have been demonstrated unequivocally (Lobashov 1951). Thus the first work devoted to conditioned reflexes in invertebrates in Pavlov's laboratory was undoubtedly performed at the personal initiative of E. M. Kreps (Kreps 1989). He worked at the Murmansk Biological Station for ten years. However, it was his first and last work with invertebrate behavior. Very soon his interests shifted into comparative investigation of different functional systems, using biochemical techniques. It also seems that the behavioral investigation of these interesting animals has never been sustained in this country. Lower Crustaceans Work performed at the beginning of the 1950s at Rostov University concerning the hereditary fixation of the conditioned reflexes in lower animals (Kogan and Semenovykh, 1955) is representative of work performed at that particular time


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period in Russia and points nicely to the fact that the inheritability of conditioned reflexes was a popular research topic. The history of Pavlov's views on this problem and his experimental approach have been described many times before (see, for instance, Voronin 1957). Kogan and Semenovykh (1955) thought that the main factor hindering the experimental proof of the heredity of conditioned reflexes was the technical difficulty of producing so many generations of vertebrate material. They selected as their research animal a lower crustacean that is very common in small freshwater ponds: Daphnia. Daphnia needs only 2-3 weeks for parthenogenetic reproduction. In nature these animals prefer illuminated places where they search for food. In the laboratory, food was withheld during the daytime. Instead, they were fed only at night. Once a week the light-dark preference was estimated from the number of animals located in the illuminated and darkened area of the tank. The offspring were given a light-dark pretest to determine their preference. After this pretest, they experienced the same procedure as the "parents." By the seventh and eighth generations there was a significant increase in the "positive reaction to darkness," and by the fourteenth to seventeenth generations most of the animals preferred darkness. The authors concluded that "the conditioned feeding reflex to darkness which has been elaborated during many generations became the unconditioned one" (Kogan and Semenovykh 1955:111). This conclusion was criticized several times in the 1960s. As L. V. Krushinsky noted, It is absolutely clear that in this work there was no estimation of the individual survival and individual rate of reproduction as dependent on the time needed for the elaboration of the conditioned reflex to the darkness in a single animal. However, the individual differences in the adaptation to unusual conditions of feeding should be reflected obviously in the survival and the reproduction rate. Thus the selection process will be inevitable. (Krushinsky 1968:122) The authors proclaimed in their preface, however, that they paid special attention to eliminate the effect of natural selection. It is not easy to understand now why this opportunity to elaborate on hereditary conditioned reflexes seemed so pleasing to many scientists. Evidently the individual experience—even it would be hereditary—cannot influence the whole population and should be extinct. On the other hand, there are some questions concerning the above-described conditioned reflex in itself. Strictly speaking, it is difficult to compare it with the conditioned reflexes in mammalia, because of its schedule (first of all, there was no individual modification of behavior caused by the combination of CS and US) and to the incompleteness of such data in the paper. Insecta I would like to end this brief account of the works that have used rather "exotic"

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animals for behavioral research by referring to the investigation of the most "popular" invertebrates, which will be considered in the following chapters. The reason for this approach is twofold: First, these works have obvious interest from a historical point of view. Second, they are forgotten in this country and probably are not known abroad because of such nonscientific reasons as World War II and the period in which Soviet science was cut off from the West. It was noted previously that L. A. Orbeli built in Koltushi the laboratory of insect biology headed by S. I. Malyshev. There is no available evidence about the relationship between S. I. Malyshev and V. A. Vagner. It seems strange, however, that Malyshev's papers of that time make no references to Vagner (the same is true for his collaborators' papers). It is strange because this group of investigators used the same experimental animal as Vagner and used the same approach to the study of behavior as Vagner. Perhaps in the future the reason for this will be made clear. Before the war, Malyshev elaborated in detail the choice of experimental animals that can be used for the study of instinctive reactions (the paper devoted to this topic was sent to a journal in April 1941; it was published only after the war, in 1946). In his opinion, the best subjects for the investigation of instinct are insects, in which instinctive behavior is not complicated with behavior acquired through individual experience. In Malyshev's opinion, instinctive actions manifest themselves in the form of very complicated activities of the animal such as nest building, foraging, and the like. At the same time, they do not need individual experience for their expression. Thus, the main problem for him was studying the evolutionary changes in some instinctive activity in a group of related animals. His research program was based on several groups of Hymenoptera. He was, for example, able to trace the development of elementary instinctive reactions of the Hymentoptera ancestors of the wasp. The results of this work, which took many years to complete, were published in the Magazine of General Biology in 1949. It should be noted that this was a long and thoroughly edited paper containing a large list of references. This paper awaited publication for more than a year. In the same issue was a paper by Maksimova and Genkel (1949) on "The theory of stadial development and its importance for the plant physiology." The reason for including the latter paper is clear to Russian biologists, but it needs some explanation for the Western reader. The main purpose of this article was to promote the propaganda of Lysenko's views and to suppress the views of his opponents. It is obvious that the paper had been written especially for, and approved by, the leaders of the Communist party. I would like to note also a paper of Poljakov in the same magazine of that time entitled "The ideological meaning of the views about periodicity in mass birth of the field mice" (Poljakov 1949). This article was directed against the prominent Russian zoologists Formozov and Kalabukhov. The work of these zoologists was considered by some as "the propaganda of the mechanistic ideas of the foreign ecology"; the author appeared to struggle with these foreign ideas and with the native scientists who support such ideas. I apologize for this short distraction, the purpose of which was to remind you of the time when the Malyshev paper appeared. It is evident that almost


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nobody paid attention to it. As for the main content of Malyshev's paper, he described seven main stages of instinctive behavior necessary to supply larva with the food that is so important for its metamorphosis. The author could explain only a part of this long process. He wrote at the end of his paper: "At this point we are very close to the second part of our investigation: the evolution of instinctive activity of wasp-like insects" (Malyshev 1949:41). However, it was his last publication. There is some indication that Malyshev was forced to stop this work as it neared completion. In the same laboratory there were studies of instinctive behavior of other insects. First of all there is the beetle Deporaus betulae which lives on birch. It cuts a leaf, rolls it around the egg, glues the borders and gnaws through the leafstalk so that the leaf sinks to the ground, where the larva matures (Oksenov 1946). This paper was presented to the magazine in the spring of 1941; it was published only in 1946, with a mourning frame around the author's name. The main conclusion in this article was the presence of a high degree of plasticity of this very complicated behavior. Unfortunately, this work was never continued. Other work was performed on the larval behavior of different species of ant lion (Euroleon europaeus Mak. L., Myrmeleon formicarius L. and Formicaleon lineatus Fabr.). The investigation of their feeding behavior were carried out under natural conditions, with semiartificial arrangements in nature, and in the laboratory. It was noted that the larvae can easily be kept in the laboratory for 9-12 months. The main conclusion of this investigation indicated that there was considerable plasticity of behavior (Puzanova-Malysheva 1947). The work of Puzanova-Malysheva, like so much else in the laboratory of insect biology at Koltushi, was never extended. One may only guess what happened. It is probably not a coincidence that the laboratory disappeared soon after the Pavlovian Session of 1950. Moreover, it is interesting to note that the closing of the laboratory is not mentioned in a history of the Physiological Institute, part of which became the Institute in Koltushi after 1950 (see Zakharzhevsky and Andreeva 1984). Instead, there appeared the laboratory of behavioral genetics, which used insects as experimental animals. Its experimental approach, however, differed significantly from Malishev's. The head of this laboratory, M. E. Lobashov, who was a geneticist, did not approve of Malishev's and Vagner's interest in the evolution of instinctive activity. In his opinion, most insect behavior consisted of conditioned reflexes. He also believed that the heredity of conditioned reflexes can be discovered in invertebrates much more readily than in vertebrates (Lobashov 1951). MAJOR VIEWS CONCERNING BEHAVIORAL SPECIFICITY OF INVERTEBRATES It has been noted above that V. A. Vagner was the prominent specialist in invertebrate behavior. He also had, however, a deep interest in vertebrate behavior (see, for instance, his book about the evolution of nest-building activity in

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swallows). It is obvious that the problem of evolution of psychic (behavioral) activity (from amoeba to human beings) dominated his scientific interests throughout his life. His concepts and ideas concerning the evolution of behavior have been presented several times but most completely in his Essays in comparative psychology (1924-1929). Briefly, the essence of his views can be expressed as: "The excitability as the basement; the reflex as the consequence; the instinct as the hereditary knowledge; the emotions as the adaptation; the mind as the leader of the behavior; the progress as the inevitable result" (this quintessence of Vagner's concept was reproduced in each issue of the Essays). According to his scheme, which resembles a branch, the main stem represents the evolution of life. A branching "twig" begins with protozoa and ends with the infusorians. In multicellular organisms there are two main lines of evolution: the evolution of the excitability of the cells in body tissues responsible for the development of character and temper, and the evolution of the nervous system producing reflexive activity that develops into instinctive and cognitive behavior. Vagner believed that animals with a diffuse nerve net have only reflex reactions. That is, their behavior depends completely on extrinsic factors. He insisted on differentiating between reflexes and instincts (though in evolution the instinctive activity developed from reflexes). Based on some developmental data Vagner concluded that evolution of instinct had five main stages: in the first stage it may be confused with the reflex; in the second there are distinctive instinctive actions which appear due to mutations; in the third there are the instincts which developed from the above-mentioned stages by means of mutations or other events; in the fourth there are the instincts of the higher order, and in the fifth there is the modification of instincts due to environmental contingencies. Vagner insisted that there were important distinctions between the invertebrates and vertebrates and suggested that these distinctions were caused by the organization of their central nervous systems. Vertebrates having brains, especially those having cerebral cortexes, possessed higher psychic abilities, including complex instinctive and cognitive activity, good memory, true learning, a set of emotional reactions, and so on. Invertebrates that possess ganglionic nervous systems exhibit complex instinctive activity but lack emotional reactions, cannot learn "by means of individual experience or by imitation," and have a memory that is qualitatively different from vertebrate memory. It is worth noting that many years ago he observed the heart activity of spiders in different situations and concluded that frequency of heartbeats can vary easily. However, he concluded that "the change in the activity of the invertebrate vasomotor system has nothing in common with the changes which seem to be connected with emotions in higher vertebrates. Today Vagner's concepts and ideas seem rather rigid and mechanistic. They belong to the past. However, we must not forget that the published work of Vagner and of many other scientists of that time, was never reissued for a new generation of students or analyzed seriously. Critical appraisals of Vagner's views concerning invertebrate behavior are also absent. As I noted above, references to Vagner's


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spider work were missing even in Malishev's papers. It is difficult to believe such an omission was an oversight. One may suggest that Malyshev and his colleagues did not want to criticize Vagner and therefore preferred not to cite him at all. This seems possible because of the negative estimations of Vagner's contributions. Presumably any scientist publicly supporting Vagner's position would be subjected to the same criticisms and might be shunned (or worse) in academic circles. The lack of public interest in Vagner during the 1940s-1960s might not mean, however, the absence of private interest in his work. In researching this chapter, I came upon a copy of his major book Biopsychology (published only in 1910 and never reissued) which belonged, during the 1950s, to a prominent specialist in vision research, S. Kravkov. This particular copy contains many notes suggesting that Kravkov had a keen interest in Vagner. An objective evaluation of Vagner's work should be made in the future. Like Vagner, Krushinsky and Beritov also had an interest in the development of central nervous system function across the phylogenetic scale. Differences in the behavior of animals were correlated with the degree of organization in the central nervous system. The more complex the organization, the more complex the behavior. Both of these researchers, however, had only limited experience with invertebrates. Although Beritov did not have any personal experience in invertebrate research, he did develop a scheme describing behavior at all levels of the phylogenetic scale. "The lowest behavioral form is the inborn reflexes of the lower invertebrates. They have only one kind of behavior; that is, reflexive reactions in response to an extrinsic stimulus" (Beritov 1947:22). "With the development of the CNS arises the higher kind of inborn behavior—instinctive behavior which depends strongly on the intrinsic processes in the organism" (Beritov 1947:23). It should be noted that Beritov had a special interest in the spontaneous activity of nervous tissue and conducted electrophysiological experiments with a wide range of animals. As we move up Beritov's scale, we find that the next type of behavior is based on individual experience. He believed such behavior is typical of higher vertebrates and human beings. He also believed some higher invertebrates, for instance, crustaceans, insects, and cephalopods, also profit from individual experience. Beritov's views on the phylogenetic development of behavior are rather similar to those of Vagner. Beritov, however, did not see such a large gap between vertebrates and invertebrates as Vagner did. It is interesting to note that the idea that some invertebrates can profit from experience caused him to be demoted. In 1948 Beritov continued to maintain that some invertebrates can modify their behavior as the result of experience (Beritashvili 1984). After the Pavlovian Session of 1950, he modified his position and now, naturally, agreed with his critics that he was wrong: "I am convinced that my idea about the individual behavior ruled by the representation in the case of invertebrates such as crustaceans and cephalopods is doubtful. I myself did not perform any experiments with them, therefore I had no right to use them (Beritashvili 1984:603). In addition to the work of Krushinsky and Beritov, two other influential Russian

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scientists concerned with the evolutionary analysis of behavior were Fabri and Voronin. We will first briefly discuss the approach taken by Fabri and then conclude with a discussion of Voronin. Fabri's views have been based on the ideas of the prominent Russian psychologist A. N. Leontyev, who proposed two main stages in psychic development: (1) the "elementary sensory" stage and (2) the "perceptive" stage (Leontyev 1959). Leontyev defines elementary sensory psychic activity as that stage of reality representation in which organisms have only a single elementary "feeling." The perceptive psychic activity stage corresponds to the representation of the external world in the form of complex images. Fabri added to this general scheme by subdividing each stage into lower and higher levels. Basing his work on a review of the literature, he was able to superimpose his scheme of psychic development upon a picture of the phylogenetic tree (Fabri 1976). The attempt was not altogether successful. He believed that the primary difficulty was the fact that morphological indices used in constructing phylogenetic trees can not be used to accurately predict psychical development (Fabri 1976). Using a somewhat different approach to the evolutionary study of psychic activity was Voronin. It has been noted throughout this chapter that the dominant method of studying behavior in this country is not from the psychological perspective but rather by the physiological or, more strictly, by the conditioned reflex approach, founded by Pavlov. In later years the Pavlovian approach was broadened somewhat to include comparative analysis. Comparative research of learning phenomena from the Pavlovian perspective had a place for many years at the Department of the Physiology of Higher Nervous Activity, which was headed by Voronin at Moscow University. Using the technique of elaboration of conditioned reflexes similar to that used in Pavlovian experiments with vertebrates, comparative experiments were conducted with various species of worms, decapod crustaceans (Karas 1962, 1963; Burmistrov and Shuranova, this book), and social insects (see Udalova and Karas, this book). An important contribution has also been made by N. A. Tushmalova, who studied the capability to store individual experiences in protozoans and primitive worms (Tushmalova 1987). Based on the experiments of his coworkers, Voronin proposed that there are "levels in the evolution of higher nervous activity" (Voronin 1970, 1984). Combining this with the scheme of Tushmalova, the evolutionary process may be described in the following way. At the first level there are only "summation" responses. These responses are based on an increase of the excitability of the nervous system or (in Protozoa) of the intracellular structures caused by the summation of the traces of previous excitations with the existing state of excitation. Such reactions take place in Protozoa, multicellular animals with a diffuse nerve net, and in the spinal cord of vertebrates. At the second level we see the appearance of habituation. This relates, as does the previous stage, to the "nonsignal reactions of individual adaptation" common to many organisms, from the infusoria to the human being. At the third stage, there is the "non-retrieved (nonstable) conditioned reflex" described in planarians. At the fourth level "true" conditioned reflexes begin to appear. They probably arise first in annelids having


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mushroom bodylike structures in the cerebral ganglia. The insect, whose brain is extremely well developed, demonstrates positive and negative conditioned reflexes, chain reflexes, and instrumental conditioning. It has been shown that the rate of the elaboration of the conditioned reflexes does not depend on the position of the animal on the phylogenetic scale; thus, there is no difference between invertebrates and vertebrates in this sense. These "true" conditioned reflexes are rather stable; they may be stored many hours or days, and even years. The next levels (fifth and sixth) are characterized by much more complicated processes such as reasoning and extrapolation, and are typical only of higher vertebrates and man. CONCLUSION A dramatic and sometimes tragic situation characterized Russian science in the twentieth century. The effect of this situation is readily evidenced in all areas of Russian science, including behavioral research with invertebrates. Of course, there are universal problems confronting scientists and nonscientists alike in all countries. However, additional problems were faced by Russian scientists, specific to the Soviet period. These include ideological and administrative control of science and gradually isolation from the world scientific community, particularly from the 1930s into the 1950s. The time of "the repressed science" ended in essence in the 1950s. However, its influence is still felt even today. Looking back, as I have tried to do in this chapter, one sees clearly how many scientific movements, the reputations of excellent researchers, and the intellectual life of the average individual scientist were damaged or exterminated. Moreover, as time goes on it is doubtful that the scientific work, as reflected in books and research articles, of those who did not follow the "party line" will be found or republished for the next generation of students. It is difficult to predict what results in understanding invertebrate behavior would have been obtained in Russia if the various approaches to the study of behavior had been free to develop. I have tried to show that even under conditions of social and political pressure, some scientific investigation of animal behavior was in progress. In the chapters that follow this historical introduction, the experimental data come predominantly from the Pavlovian perspective (i.e., the position represented by Voronin). The reasons for this are the various social and political factors we have discussed. This "monastic" approach to the study of invertebrate behavior allows the reader to estimate personally the achievements and the mistakes brought about by factors not related to doing science. ACKNOWLEDGMENTS I would like to thank N. Yu. Alekseenko, Yu. M. Burmistrov, M. E. Ioffe, L. G. Leibson, Ya. M. Pressman, and M. E. Varga for reading this manuscript and encouragement. I am also grateful to N. L. Krushinskaya for supplying the photograph of her father.

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nauk SSSR. . 1954. Bibliografiya Trudov I. P. Pavlova i Literatury o Nyem (Bibliography of Pavlov's works and of the materials about him). Moskva, Leningrad: Izdatelstvo Akademii nauk SSSR. Plavilstchikov, N. N. 1928. Observations sur l'excitabilite des infusoires. Russky Arkhiv Protistologii, 7:1-24. Poljakov, I. Ya. 1949. The ideological meaning of the views about periodicity in mass birth of field mice (in Russian). Zhurnal Obshchey Biologii 70:246-260. Pomper, P. 1970. The Russian Revolutionary Intelligentsia. New York: T. Y. Crowell Company. Promptov, A. N. 1947. On the evolutional biological peculiarities of the orienting response in some ecologically specialized bird species (in Russian). In: Trudy Instituta Evolutsionnoy Fiziologii i Patologii Vysshey Nervnoy Deyatelnosti imeni I. P. Pavlova, Tom 1 (Papers of the Institute of the Evolutionary Physiology and Pathology of the Higher Nervous Activity named by I. P. Pavlov, vol.1) 247-258. Leningrad: Izdatelstvo Akademii nauk SSSR. . 1956. Ocherki po Probleme Biologicheskoy Adaptatsii Povedeniya Vorobyinykh Ptits (Essays on the biology of the adaptive behavior in Passerine birds). Moskva, Leningrad: Izdatelstvo Akademii nauk SSSR. Puzanova-Malysheva, E. V. 1947. The Ant-lions and Their Funnel Traps (in Russian). In: Trudy Instituta evolutsionnoy fiziologii i patologii vysshey nervnoy deyatelnosti imeni I. P. Pavlova, Tom 1 (Papers of the Institute of the Evolutionary Physiology and Pathology of the Higher Nervous Activity named by I. P. Pavlov, vol. 1) 259284. Leningrad: Izdatelstvo Akademii nauk SSSR. Roginsky, G. 1940a. V. A. Vagner: K pyatiletiyu so dnya smerti, 1849-1934. (V. A. Vagner: Five years after his death). Sovetskaya Pedagogika, no. 7:96-100. . 1940b. Razvitiye Mozga i Psikhiki. Albom-Vystavka (Development of the brain and of the psychic life. The description of an exhibition). Leningrad: . 1947. Comparative psychology in the USSR (in Russian). Vestnik Leningradskogo Universiteta, 7:60-69. Roitbak, A. I. ed. 1991. Vospominaniya ob Ivane Solomonoviche Beritashvili (Memoirs about I. S. Beritashvili). Moskva: Nauka. Roitbak, A. I., and Bakuradze, A. N. 1988. I. S. Beritashvili: His Contribution into Physiology (in Russian). In: N. P. Bekhtereva, ed., Fisiologicheskiye Nauchniye Shkoly v USSR. Ocherki (Essays on the physiological "schools" in USSR) 157-170. Leningrad: Nauka. Sakharov, D. A., and Turpayev, T. M. 1988. Kh. S. Koshtoyants' School (in Russian). In: N. P. Bekhtereva, ed., Fisiologicheskiye Nauchniye Shkoly v SSSR Ocherki (Essays on the physiological "schools" in USSR), 241-246. Leningrad: Nauka. Semiokhina, A. F. 1991. L. V. Krushinsky. His Life and Work (in Russian). Zhurn. Vyssh. Nervn.Deyat. 47:618-621. Severtsev, A. N. 1922. Evolutsiya i Psikhika (Evolution and Psychology). Moskva: Izdadelstvo M. i S. Sabashnikovykh. Shimkevich, B. M. 1897. Consciouness, instinct and reflex (in Russian). Obrazovaniye 9:1-16. Shlupikova, A. V. 1968. AkademikA. A. Ukhtomsky (1875-1942) (The Academician A. A. Ukhtomsky). Yaroslavl: Verkhne-Volzhskoye Izdatelstvo. Simonov, P. V. 1991. Interaction of the scientific schools at the formation of the investigatory program (in Russian). Vestnik Akademii Nauk SSSR, 7:43-51.

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Sobol, S. L. 1925. Scientific and popular books in biology and zoology (in Russian). In: R. Goldsmit, ed. Ascarida: Obshchedostupnoye wedeniye v nauku o zhizni (Ascaris: Popular introduction in life science), Translated by S. L. Sobol 343-376. Moskva: Gosizdat. Strelchenko, V. I. 1975. V. A. Vagner as a zoopsychologist-darvinist (in Russian). Istoriya i teoriya evolutsionnogo ucheniya (History and theory of evolutionary teaching), 5:101-110. Strelkov, A. A. 1967. General account of the development of the invertebrate zoology (in Russian). In: Razyitiye biologii v SSSR (Development of Soviet biology), 175-189. Moskva: Nauka. Suvorov, N. F., and Andreeva, V. N. 1990. A problem of hereditability of conditioned reflexes in I. P. Pavlov scientific school (in Russian). Zhurnal Vysshey Nervnoy Deyatelnosty 40:3-14. Svidersky, V. L., Veselkin, N. P., and Natochin, Yu. V. 1988. Orbeli's school (in Russian). In: N. P.Bekhtereva, ed., Fisiologicheskiye nauchniye shkoly v SSSR Ocherki (Essays on the physiological "schools" in USSR), 94-106. Leningrad: Nauka. Tushmalova, N. A. 1987. The main regularities in the evolution of invertebrate behavior (in Russian). In: Fiziologiya Povedeniya: Neyrobiologicheskiye Zakonomernosti (The physiology of behavior: Neurobiological problems), 236-264. Leningrad: Nauka. Umrikhin, V. V. 1991. The "beginning of the end" of behavioral psychology in the USSR (in Russian). In: Repressirovannaya Nauka (The oppressed science), 136-145. Leningrad: Nauka. Vagner, V. A. 1890. Nabludeniya nad Araneina (Observations on Araneina). Trudy Sankt-Petersburgskogo obshchestva yestestvoispytateley, Vom 21. . 1896. Voprosy Zoopsikhologii (Problems in zoopsychology). St. Petersburg: Izdaniye L. F. Panteleeva. . 1900a. Vodyanoy pauk (Argyroneta Aquatica Cl), Ye go Industriya i Zhizn kak Material Sravnitelnoy psikhologii (Water spider Argyroneta aquatica CL: Its industry and life as a subject of comparative psychology). Moskva: Kushnerev. . 1900b. Gorodskaya lastochka Chelidon urbica, yeye industriya i zhizn kak material sravnitelnoy psikhologii (Urban swallow Chelidon urbica, its industry and life as a subject for comparative psychology). Zapiski Imperatorskoy Akademii nauk po Fiziko-Matematicheskomu Otdelu, Seriya VIII, 70:1-125. . 1907. Psycho-biologische Untersuchungen an Hummeln mit Bezugnahme auf die Frage der Geselligkeit im Tierreich. Zoologica 46:1-239. . 1910a. Biologicheskiye Osnovaniya Sravnitelnoy Psikhologii (Bio-Psikhologiya). Tom I. (Biological bases of comparative psychology: Bio-psychology, Vol.1). St. Petersburg, Moskva: Izdaniye tovarishchestva M. O. Volf. . 1910b. Biologicheskiye Teorii i Voprosy Zhizni (Biological ideas and life problems). St. Petersburg: Obrazovaniye. . 1913. Biologicheskiye Osnovaniya Sravnitelnoy Psikhologii (Bio-Psikhologiya). Tom II: Instinkt i Razum (Biological bases of comparative psychology: Bio-psychology. Vol. 2: Instinct and mind). St. Petersburg, Moskva: Izdaniye tovarishchestva M. O. Volf. . 1914a. Physiological and biological solution of psychological problems (in Russian). In: Novye Ideyi v Bilogii (New ideas in biology) 6:1-37. . 1914b. Segmentary psychology (in Russian). In: Novye Ideyi v Bilogii (New ideas in biology) 6:38-138.

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. 1923. Biopsikhologiya i Smezhniye Nauki (Biopsychology and related sciences). Petrograd: Obrazovaniye. . 1924-1929. Vozniknoveniye i Razvitiye Psikhicheskikh Sposobnostey (Origin and development of mental abilities). Issues 1-9. Leningrad: Nachatki Znaniy. . 1926. Metodika Nabludeniy nad Zhivotnymi (Posobiye dlya Prepodavateley) (Some ways to observe animals: A manual for the school-teacher). Leningrad: Gosudarstvennoye Izdatelstvo. Voronin, L. G. 1957. Sravnitelnaya Fiziologiya Vysshey nervnoy Deyatelnosti (Comparative physiology of the higher nervous activity). Moskva: Izdatelstvo Moskovskogo Universiteta. . 1970. Phylogenetic evolution of conditioned activity (in Russian). In: Rukovodstvo po Fiziologii. Chast 1 (Textbook of physiology. Part 1), 473-506. Moskva: Nauka. . 1977. Evolutsiya Vysschey Nervnoy Deyatelnosti Ocherki (Essays on the evolution of highernervous activity). Moskva: Nauka. . 1984. Kurs Lektsy po Vysshey Nervnoy Deyatelnosti (Lectures on higher nervous activity). Moskva: Izdatelstvo Moskovskogo Universiteta. Yaroshevsky, M. G. 1985. Istoriya Psikhologii (History of psychology). Moskva: Mysl'. ed. 1991a. Repressirovannaya Nauka (The oppressed science). Leningrad: Nauka. . 1991b. Stalinism and Soviet science (in Russian). In: Repressirovannaya Nauka (The Oppressed Science), 9-33. Leningrad: Nauka. Zakharzhevsky, V. V., and Andreeva, V. N. 1984. Ordena Trudovogo Krasnogo Znameni Institut fiziologii imeni I. P. Pavlova (proshloye i nastoyashchee instituta i yego laboratory) (I. P. Pavlov Institute of Physiology of the Academy of Sciences of the USSR: The past and the present of the institute and its laboratories). Leningrad: Nauka. Zeleny, G. P. 1913. Psychological reactions of the animals as the subject of natural science (in Russian). Priroda 70:1191-1207. Zubkov, A. A., and Polikarpov, G. G. 1951. Conditioned reflex in a coelenterate (in Russian). Uspekhi Sovremennoy Biologii 32:301-302.


Chapter Three Memory and Morphogenesis in Planaria and Beetle Inna M. Sheiman and Kharlampi P. Tiras

This chapter is dedicated to the memory of James V. McConnell.

We will begin with a brief history of the study of memory and morphogenesis in invertebrates. During the 1950s and 1960s James V. McConnell excited the scientific world by his conditioning experiments with planarians. Infected by McConnell's results and enthusiasm, we began to study planarian regeneration, although our experiments have taken a different direction from his. The experiments performed by him and his many colleagues suggested the possibility that memories produced by conditioning survive regeneration following transection. With the interest in nucleic acids during the 1950s and 1960s, McConnell proposed that the memory trace was formed and stored in RNA molecules. These molecules are present in all planarian cells, including those that are critical for the process of planarian regeneration. The conclusions regarding the role of RNA in memory storage in regenerating planaria were not quite verified in our experiments. After our first results on planarian conditioning, the idea arose to study simultaneously planarian memory and morphogenesis. The results of these investigations are presented in this chapter. In our studies memory processes were analyzed by using the common mechanisms of morphogenesis, that is, the behavior of developing cells; their origin, growth, and differentiation; and, finally, how these cells are regulated. Two different models of invertebrate morphogenesis were used: planarian regeneration (regeneration as repeated development) and postembryonic development of insects, specifically the grain beetle. There is a very rich literature describing both planarian regeneration and insect metamorphosis and learning, but the relationship between them has not been studied. Establishing this connection has been the purpose of our investigations for many years, the results of which are reported in the following pages.

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In Russia, planarian investigations were first carried out at Kazan University. These investigations continue to the present (Porfiryeva and Diganova 1987). Planarian regeneration has been the object of investigation in Leningrad University (Krichinskaja 1972), and the study of conditioning and behavior in planaria was studied in Moscow University (Voronin and Tushmalova 1965). PLANARIAN MEMORY AND REGENERATION Planaria are typical flatworms, encased in a longitudinal body covered with ciliated epithelium. About 500 planarian species have been described. Most of them are freshwater animals, but there are also marine and terrestrial species. The length of planarians can vary from 1 mm to several centimeters. The majority are about 10-30 mm long. In Russia, the largest planarians live in Lake Baikal, where there are about eighty endemic planarian species. Planaria are beasts of prey and stalk their potential victims with well-developed chemical senses and photoreceptors. Planaria are hermaphrodites and can breed by sexual interactions. There are also asexual species that breed by fissioning. In this case, both parts of the animal develop into two new planaria. The ability to regenerate, morphogenetic plasticity, is one of the more remarkable features of planarians. In our work the following species of planaria were mainly used: Dugesia lugubris, Dugesia tigrina, Ijimia tennis, and Bdellocephala punctata. D. lugubris and /. tenuis were collected in lakes along the river Oka near the town of Pushchino. Sadly, over the course of thirty years we have witnessed the gradual disappearance of planarians in this lake because of pollution. This is why for the last few years the majority of our work has been performed on laboratory colonies of D. tigrina. In the laboratory our planarians live in large cups at room temperature and semidarkness. They are fed once a week with mosquito larvae. The water in their home containers is changed once every 3-6 months. The Nervous System and Planarian Conditioning Planaria are the first animal in evolution to possess a centralized nervous system. The central nervous system consists of two abdominal nerve cords and anterior paired ganglia. The nerve cords consist of nerve cell bodies and fibers. There are motor and associative neurons. The planarian ganglion represents the beginning of cephalization and consists of several thousand nerve cells surrounding the neuropil. The ganglion in different planarian species has the appearance of a butterfly or a horseshoe. Like other planarian cells, the neurons are characterized by their small size (about 10 pm) and large nucleus. There are multi-, bi-, and unipolar nerve cells. There are also various kinds of ganglion cells. Some of these are neurosecretory, and others resemble glial cells. Ganglion cells send their branches to neuropil and connect with the main sensory

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zones of the planarian body (Lentz 1968a). Planaria have various sensory receptors. The photoreceptors, for instance, enable the animal to detect weak light stimuli (about 1 lux) (Jones 1971); and the chemical receptors are sensitive to some proteins and amino acids: hemoglobin, lecithin, lysin, glutamine, and proline (Karpenko and Seravin, 1973). The various Pavlovian conditioning protocols for planarian research performed in Western laboratories are well known and will be discussed briefly here. Planaria can be conditioned to light or vibration conditioned stimuli when associated with electrical or a chemical-shock unconditioned stimuli. Planarians can also be conditioned in various instrumental situations employing mazes. As pointed out by Western researchers, the claim that planarians can be conditioned has engendered much controversy even today (Corning and Riccio 1970). We feel that the inconsistencies in the data and results can be traced to poor conditioning procedures and the difficulties inherent when one attempts an analysis by lumping together different planarian species. As to Russian studies of conditioning in planarians Krilov and Nasarian (1973) successfully trained planarians to make a correct choice in a cruciform maze. Tushmalova and Gromyko (1968) attempted to condition planaria to reverse their light/dark preferences. However, Voronin and Tushmalova (1965) concluded that planarian conditioned reflexes, when they could be formed, are primitive and probably based on sensitization. Our investigations in planarian behavior, which have spanned almost thirty years, allow us to make some conclusions about the origin of these contradictions and to analyze the properties of planarian conditioned reflexes (Sheiman and Tiras 1984). We have tried various combinations of training parameters, procedures, and species and are confident that we have found the optimal training variables and procedures for the elaboration of true conditioned reflexes in planarians. The majority of our experiments are performed with the species Ijimia tenuis (sexual race from the lakes near Pushchino) and Dugesia tigrina (asexual laboratory race). The planarians Dugesia lugubris and Rhymacephalus pulvinar (the latter is an endemic species from Lake Baikal) were also conditioned. In our first experiments we used the approach taken by McConnell in which subjects received massed training: 120 combinations of light and electrical shock presented in the course of a single training session (Cherkashin, Sheiman, and Sergeeva 1966a). We found essentially the same results as McConnell and observed, as others before us have, that all kinds of stimuli seem to evoked the same motor reaction: turning of the head part or longitudinal contraction of the planarian body. We also observed that the primary effect of the light and shock combinations was to inhibit the reaction to the light conditioned stimulus. This would, of course, underestimate the amount of conditioning and produce an unstable pattern of conditioned behavior (Cherkashin, Sheiman, and Sergeeva 1966b). From the results of our previous massed conditioning experiments, we thought conditioning would be better if a spaced trials procedure were used. We now conditioned animals with 25 trials per day for 10 days. In addition, the intensity of

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light used as a conditioned stimulus was decreased to 170 lux. This level of illumination was practically under threshold and did not evoke any motor responses from the planarian. As the experiment continued, the number of planarian reactions to the light gradually rose and became stable (17-20 reactions per 25 trials) by the fifth or sixth day. Similar results were obtained when vibration and electric shock were used as conditioned and unconditioned stimuli respectively. Figure 3.1 provides a summary of the results. The appropriate pseudoconditioning controls were employed in these experiments (Cherkashin and Sheiman 1967). The next modification of the experimental procedure permitted us to condition planaria in one day by using a mass conditioning situation. There were 40 combinations of light (70 lux) and electrical shock. Under these conditions the planarian Dugesia tigrina were well conditioned (Tiras and Aslanidy 1981). All of our data confirm the notion that the problem of planarian conditioning can be reduced to finding the right combination of species, training variables, and procedures and not to an inability of planarians to form true conditioned reflexes. Figure 3.1 Conditioning of planarians with light as the conditioned stimulus. For Day 9, only the conditioned stimulus was changed to vibration.


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McConnell, in his pioneering works, used a high-intensity light (about 2-3 thousand lux). He observed an increase in the number of planarian reactions to light (sensitization) but did not analyze this phenomenon (McConnell 1970; McConnell, Jacobson, and Kimble 1959; Thompson and McConnell 1955). As mentioned above, we could not form stable conditioned reflexes in planaria using McConnell's procedures (Cherkashin, Sheiman, and Sergeeva 1966a 1966b). Apparently, Tushmalova came across the same phenomena (i.e., unstable conditioned reflexes) when she used a 2000 lux light as a conditioned stimulus (Tushmalova 1973; Tushmalova and Gromyko 1968; Voronin and Tushmalova 1965). This no doubt contributed to her opinion that planarians cannot form true conditioned reflexes. However, as we mentioned earlier, if light intensity is decreased as in our experiments, true conditioned reflexes can be revealed in planarians (Cherkashin and Sheiman 1967). The importance of providing a "weak" CS intensity and using spaced trials in the study of invertebrate learning was mentioned earlier by E. Kreps in his work on ascidians (1925) and by Sokoloff in his work on sea stars (1961). Planarian conditioned reflexes are, however, obviously different from those found in vertebrates. Vertebrates can reveal the formation of conditioned reflexes with a wide range of behavior. Planarians, on the other hand, reply to any stimuli using the same set of behavioral reactions. Thus, conditioning in planaria must be expressed as an increase in the excitability to the conditioned stimulus. The common reaction to different kinds of stimuli promoted disagreements and contradictions in the planarian conditioning literature (Brown, Dustman, and Beck 1966; Cummings and Moreland 1959). Our data suggest several conclusions about the origin and particularities of planarian conditioned reflexes. Planaria have receptor systems that provide a distinct perception of various classes of stimuli such as light, vibration, and chemical. This is why there is an afferent differentiation of the conditioned stimuli, which leads to a specific increase of excitability in planaria. At the same time, however, their effector systems are not sufficiently developed. Thus, although their sensory systems can perceive specific classes of stimuli, their motor systems limit the type of locomotor behavior. Though planarian reflexes have much in common with summation or sensitization reflexes, they still preserve the basic character of classical Pavlovian reflexes. Planarian Regeneration Planarian regeneration is an excellent experimental model for studying morphogenesis (Brondsted 1969). As a classical object for regeneration research planaria have been used for 200 years. T. H. Morgan, for instance, before conducting his investigations in classical genetics published important work on planarian regeneration that is still cited (Morgan 1901). About 30 years ago McConnell was the first to use planarian regeneration for the study of memory. In our laboratory planarian regeneration was studied simultaneously with the investigation of planarian learning and memory.

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Planarian Regeneration: Morphological study The most investigated experimental model in our laboratory was the regeneration of the planarian head. Operations were performed after the animals were food deprived for one week. Morphological and functional investigations were carried out over the 10 day period following surgery. Morphometric Observation of Planarian Regeneration Much of the data was obtained by a combination of intravital computer morphometry and histological methods. The method of computer morphometry of planarian regeneration is based on the fact that intact planaria are covered by pigment cells. After decapitation, however, the regenerating blastema do not have such cells until several days have passed (Tiras and Khachko 1990). Therefore, the photocontrast between old (i.e., pigmented) and newborn (blastema) portions of regenerates can provide an accurate record of the time course of regeneration. The resulting photographs can be digitized for computer analysis. A computer program has been developed that constructs an image "skeleton" of the planarian and on the basis of this image computes the following primary traits: the area, length and width of the planaria and the area and length of the growing blastema. On the basis of these traits, a regeneration criteria can be computed. We used two regeneration criteria that reflect the restoration of the body proportions. The first reflects regeneration of the original proportions of the length of the head and the whole body. The second considers the area, including both linear indices (length and width). These criteria reflect the regeneration of the proportions of the areas of the head and the whole body (Tiras and Khachko 1990). The criterion of length, Cr(l), and criterion of area, Cr(a), were calculated according to the formulas:

where A(0) is the length of the intact planaria; C(0) is its area; A(i) and C(i) are the length and area of the regenerates at i- day after sectioning; B(0) and D(0) are the length and the area of the head part of the intact planarian body; B(i) and D(i) are the length and area of the blastema; m is the number of animals in the experimental groupand S is a constant. After the planarian was sectioned, an intensive reorganization of all the dimensions of the body was initiated. The length of the blastema increased during the experiment and reached a stable level after 20 days. Meanwhile, the total length of the body, which contracted after sectioning, gradually decreased over the whole period during which measurements were made. The width of the regenerated body


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increased immediately after sectioning because of the contraction of the body; it gradually decreased during regeneration down to 70% of the initial level. The length of the planarian body changes to a significantly lesser degree than its width, and the decrease in the total length of the body occurs during the increase in length of the regenerating head part of the body. The area of the anterior part of the body grew gradually during regeneration, but the total area of the regenerating body decreased. The area of the planarian profile decreased in exactly the same way. As the planaria were not fed during regeneration, obviously the observed changes are directly related to the morphogenetic reorganization of the regenerates. On the basis of the computed regeneration criteria collected during the 30 days of the experiment, Cr(l) reaches a level corresponding to 100% of the body proportions of intact animals by the 10th day of regeneration, but Cr(a) does so only by the 30th day. Thus, periodicity during regeneration is reflected in different criteria. The results permit an identification of the sequence of stages in the regeneration of the planarian body at a macromorphological level of organization. Stage I consists of the regeneration of the proportions of the length of the head and the whole body of the planarian. Stage II consists of the reestablishment of proportions related to area. The first stage takes approximately 10 days, and the second takes 30 days. Histological Investigations of Planarian Regeneration Histological studies of planarian regeneration were made in our laboratory using a modification designed to diminish preparation time (Bogorovskaya 1969). It was mentioned earlier that in a transected planaria the nervous system is the first to regenerate (Pedersen 1958). It takes about 5 days to form the frame of the new planarian ganglion. Twenty-four hours after the operation, the cut nerve cords lie free under the epithelial surface. The next day, the growth of thin nerve fibers were observed. On the 3rd day of regeneration, the number of these nerve fibers increased and their growth moved toward the median. On the 4th day, this tendency was clearer and the pioneer fibers established contacts. Finally, on the 5th day, these contacts developed into a strong junction, which then became thicker. The cellular composition of the new ganglion forms in parallel with the regeneration of the nerve cords. First of all, the small round or slightly extended cells concentrated around the remaining nerve cords. Usually, in planaria, such cells are called neoblasts. Two days after the operation small groups of such cells appeared under the wound surface. Over time, their numbers increased and they concentrated around the growing nerve fibers and formed the blastema. On the 5th day, the shape of the planarian ganglion, with the cell and fiber composition, was established. Thus, the planarian ganglion restores its structure in 4 or 5 days. The source of its formation is the growth of nerve fibers from the remaining nerve cords and neoblasts that are concentrated in the blastema.

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Cellular Destruction During Regeneration It has been previously mentioned that some planarian cells are destroyed during regeneration (Sheiman and Efimov 1973). The cell destruction is more pronounced in the remaining head area but not in the remaining tail area. The percentage of damaged cells (cell nucleus deformations) was calculated as a function of time since the operation. In the first hours following the operation, cell destruction in the remaining ganglion is pronounced. The maximum destruction (more than 50%) occurred 4 hours after the operation. As time continued, the number of intact cells increased and the number of destroyed cells decreased. After 12 hours postregeneration, the cell destruction increased again. Thus, this process has an oscillatory character. In the days following the operation, cell destruction in the ganglion slowly decreased. It must be mentioned that cell density in the ganglion during regeneration was not diminished but actually increased slightly. Although the whole structure of the anterior part of the regenerating planaria lost cells, cells from other parts of the body migrated to the ganglion region. Functional Changes in Regenerating Planaria It may be concluded on the basis of the morphological investigations that the primary ganglion structure formed on the 5th day after the operation. The functional differentiation of the planarian cells during regeneration will be illustrated by analyzing some cellular properties. Neurosecretion Neurosecretory cells have been described in planarian ganglion and nerve cords (Lender and Klein 1961, 1962; Sauzin-Monnot, Lender and Gabriel 1970). It is known that such cells play an important regulatory role in planarian regeneration (Grasso 1966; Lender 1964; Lindh 1959). N. Sakharova and R. Gordon (1974) from our laboratory described the neurosecretion in Dugesia tigrina using the Gabbe method. Apart from neurosecretory cells, some vacuoles filled with neurosecretion were also found. They were located at the periphery of the ganglion and also near the nerve cords. Two days after the anterior portion of a planarian was amputated, neurosecretion was found directly under the blastema and along the remaining nervous trucks. During the following 2-day period the supply of neurosecretion was exhausted and the vacuoles gradually disappeared. On the 5th day of regeneration, new neurosecretory cells appeared in the blastema. Simultaneously they were also found in the nerve trucks. Neurosecretory cells and vacuoles containing neurosecretion also appeared in both the parenchyma and along the nerve trucks. Apparently neurosecretion does play a role in planarian regeneration, as it is exhausted in the first days after the operation. The main conclusion of this work,


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however, was that new neurosecretory cells appeared in the new ganglion on the 5th day. This time period nicely corresponds to the morphological formation of the ganglion as described above. Acetylcholinesterase Activity Another histochemical characteristic of the new nervous structure in regenerating planarians is neuromediatory activity. Among other kinds of mediators, planaria of different species have acetylcholine and acetylcholinesterase (AChE) (Lentz 1968b; Tiras 1978; Tiras, Sheiman, and Sackharova 1975; Welsch 1946; Wolff 1962). The presence of AChE was revealed by the method of Koelle and Fridenvald, using the Gerebtzoff modification. The number of cells containing AChE particles in different regions of the regenerating planaria Dugesia lugubris and Planaria torva was calculated. In the ganglion region located in the head part of the operated on planaria the AChE cells increased in the first 2 days from 64% to 86%. In the remaining nerve trucks of the regenerating tail portion, few AChE cells were found. In the blastema such cells appeared only on the 4th day, and on the 5th day their quantity became considerable. At this time the normal picture of the ganglion AChE activity was established. The AChE and neurosecretion dynamics during the 1st day of regeneration indicate their important role in this process. Perhaps they are the elements regulating regeneration. Immediately after cutting, both of these systems are exhausted and are reestablished in the differentiating nerve elements. They indicate the functional maturation of the ganglion. Factors of Regeneration We would suggest that besides ACh and neurosecretion some specific factors of regeneration also take part in regulation. Such factors, as a special kind of embryonal inductor were described in many models of regeneration. For example, in hydra the specific neuropeptide activating the head regeneration was found (Schaller and Gierer 1973). Lender established that planarian ganglion cells excrete a factor that compels neoblasts to differentiate in "eyes" (Lender 1952, 1955), and Sengel has shown that the mechanisms of ganglion and pharynx regeneration are similar (Sengel 1959). Simultaneously, the pharynx region excretes the inductors for the pharynx itself (Sengel 1951, 1953). Lender also demonstrated the existence of both inhibitors and simulators of ganglion regeneration (Lender 1956a, 1956bb, 1960). The active factors stimulating and inhibiting morphogenesis are known as morphogens (or morphogenetically active substances) (Sheiman 1984). Their existence was known earlier from experiments in which planaria regenerated in homogenates (Lender 1960). There are also some data about the inhibitor of ganglion regeneration. It is glucoproteide with m.m. 2.4 * lOd (Steele and Lange 1977).

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The pharynx stimulator and inhibitor correspondingly have m.m. lower and higher than 15 kda (Wolff 1974). For revealing planarian morphogenetic factors, the homogenates of regenerating planaria were cut on different days and fed to intact recipients; 24 hours later, the recipients were sectioned, too. The regeneration of these recipient planaria were investigated using the computer morphometry method described earlier. The regenerates of the 1st to the 5th day were used as food and sources of regeneration factors (groups R1-R5) (Tiras and Sheiman 1984). The control animals were fed with intact planaria homogenates (group CI), with neutral food—mosquito larvae—(group CM), or not fed (group C). The results of the experiment indicate that in the control groups the restoration of the body proportions of the regenerates occurred as follows. Throughout almost the entire experiment, unfed animals (group C) regenerated the most slowly of any of the groups, while regenerates feed neutral food (group CM) developed more intensively. Planarians feed with intact planaria homogenates (group CI) outstripped both the preceding control groups and all groups of experimental animals. Planaria of the CI group significantly outstripped the animals of the C group from the 2nd to the 8th day, and the CM group on the 3rd, 4th, 7th, 8th and 11th days of the experiment. Regeneration in the CM group outstripped growth in the C group from the 3rd to 5th days following decapitation. Despite the fact that on individual days the differences between the control groups were insignificant, it may be concluded that feeding itself before decapitation stimulated regeneration. Feeding with homogenates from the intact planarians also stimulated regeneration. Moreover, feeding with intact planaria homogenates stimulated regeneration most effectively. It can be concluded that intact planaria Dugesia tigrina contains factors that stimulate regeneration. An essential factor in the course of regeneration of the head end of the planarian body is the formation and functioning of a new ganglion. This begins to occur on the 4th and 5th days following decapitation. Therefore, the experiments to be discussed will be analyzed in two stages: before and after the 5th day of regeneration. The animals of the Rl group regenerated more intensively than the animals of the CM group throughout the entire experiment. This difference was observed before and after the formation of a new ganglion in the recipients on the 5th day of regeneration. Significant differences in the value of the index Cr were observed on the 4th, 5th, 7th, and 11th days following decapitation. The most substantial difference in growth in comparison with the control planaria was on the 7th and 11th days (i.e., on the second stage of regeneration). The regeneration of planaria from group R2 in the first 5 days of the experiment was at the same level or below the level of group CM. In the second stage of regeneration, group R2 rapidly outstripped the CM group in development and significantly outstripped the CM group from the 7th to the 11th day. Planaria from group R3 regenerated similarly during the first 5 days of the experiment. However, a tendency was manifested here for a decrease in regen-


53

eration rates, and on the 4 and 5 days after cutting, the Cr in this group was significantly lower than in the CM group. After the 6th day, planaria of group R3 overtook regenerates from the CM group. The development of animals in group R4 resembles the dynamics of restoration in group R3. The tendency for slow growth in this experimental group was enhanced. In the second phase of regeneration, from the 7th to the 11th days, the regeneration of planaria in group R4 was worse than that of the animals from all other experimental groups. The value of Cr in group R4 remained at the level of the control group CM. Planaria in group R5 outstripped the animals of the CM group in terms of development from the 2nd to the 5th day. This was especially noticeable at the 4th day. After the 5th day the rate of development of the animals of this group slowed down, but it was again intensified on the 8th to the 11th day of the experiment. On the basis of these data we can made the following conclusions. The regenerates of the 1 st day contained a factor that stimulates regeneration of planaria. The regenerates of the 2nd and 5th days also stimulate this regeneration. In regenerates of the 3rd and 4th days, a factor inhibiting regeneration was detected; this inhibition was more stable after feeding of 4th-day homogenates. On the 5th day after decapitation and in intact planaria, stimulators of regeneration were active, especially in the intact planaria. The stimulation of regeneration in the CI group was the least pronounced in comparison with all other experimental groups of animals. The main conclusions to be made regarding the existence of regeneration factors (RF) and of their dynamics during planarian restoration are the following. On the 1st and 2nd days following decapitation, an activator of regeneration turns on, thereby triggering the recovery process. On the 2nd day the effect of activation gradually decreases, and on the 3rd and 4th days the action of inhibitory factors predominates in the regenerates. Then, on the 5th day, after the formation of a new ganglion in the recipients, activity of the regeneration stimulators is again manifested. The intact planaria also contain a regeneration stimulator (Tiras and Sheiman 1984). The regeneration of planaria can be represented in the form of an active interaction of two types of regulators: stimulators and inhibitors, so that the real course of the process is determined by the preponderance of activity of the factor of one sign or the other. MEMORY TRACE IN REGENERATING PLANARIA Storage of Conditioned Reflexes After Planarian Regeneration McConnell was the first to find that conditioned reflexes in intact planaria were preserved after the animal was sectioned and regenerated (McConnell, Jacobson, and Kimble 1959; McConnell, Jacobson, and Maynard 1959; Murphy 1963). The conditioned reflexes to light were elaborated over several sessions. Then planaria

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were sectioned transversely into two parts and tested again 2 weeks later. Both regenerated parts reproduced the conditioned reflexes. The same results were obtained if the light was replaced with other conditioned stimuli. Similar results were obtained using conditioned stimuli other than light and in situations employing T mazes (Ernhart and Sherrick 1967; Nazarian 1973). McConnell believed that memory was stored in the neoblasts and was influenced by RNA (McConnell 1965). As mentioned previously, such an interpretation was popular in the 1950s and 1960s, when the idea that memory may be coded on the RNA as genetic information was suggested. Not enough control experiments were performed, however, to test this suggestion. Thus the phenomenon of memory storage during regeneration remained unexplained. An additional question concerns how the memory trace is dependent upon the planarian central nervous system. It was also of interest to know the relationship between the neoblasts, which play such an important role in regeneration, and memory formation in planarians. In an attempt to provide answers to these questions, experiments were conducted to analyze the storage of the conditioned reflexes in regenerated planaria (Cherkashin, Sheiman, and Bogorovskaya 1966). Fifty planaria Ijimia tenuis were conditioned to vibration combined with electrical shock. Then all the animals were sectioned across the midline. In the following days groups consisting of 10 animals (corresponding to an intital group of 5 intact planarians) were tested for the presence of conditioned reflexes. The results indicate that the level of conditioning in head regenerates did not fluctuate during regeneration. In the tail region (which regenerated a new ganglion), during the first 4 days of training the level of conditioned reflexes was significantly lower. This indicates that conditioned reflexes were not manifested. On the 5th day of regeneration, however, the number of reactions sharply increased and was stable for the remaining sessions of the experiment. Our results indicate that tail regenerates restore their conditioned reflexes on the 5th day after cutting. In the next series of experiments, different types of operations on the central nervous system were made to study the role of nervous system function in the storage of the memory trace. Planaria were conditioned to light, operated on, and checked on the 2nd and 14th days after the operation. The reults indicated that as in the previous experimental series, the conditioned reflexes were not manifested immediately after ganglion removing but were restored following a 2-week regeneration period. Following removal of the nerve trunks, the conditioned reflexes remained intact. After removal of both the ganglion and nerve cords (the lateral cords remained intact), conditioned reflexes were not restored. The final experiment from this series consisted of double sections. Intact planarians were conditioned to light and operated on in the usual manner. After 14 days the regenerated parts were removed again, and only a month after the first operation each of the 4 regenerated planaria (obtained from one intact animal) were tested. In this experiment 2 animals from every 4 consisted of the original planarian tissue, and the remaining 2 animals came from tissue regenerated at two


55

stages. The results indicated that the number of conditioned reactions in the regenerated planarians was lower than for the intact planarians but reliably higher than in the corresponding control animals. These experiments confirmed the results of McConnell (McConnell, Jacobson, and Kimble 1959), who found that the memory of conditioned reflexes is present in regenerated animals. Moreover, our experiments indicate the dependence of such storage on central nervous system function. The memory of conditioned reflexes was not destroyed in the head parts that kept the ganglion. In the tail regenerates, memory of conditioned reflexes did not appear until the 5th day following the operation, that is, before the ganglion regenerated. Memory was not observed in regenerates who had only intact lateral nerve trunks. The memory trace was also found in planaria that had completely regenerated following a double section. This last result is evidence of the transfer of information during CNS regeneration and its fixation in new neural elements. Conditioned Reflex Formation During Ganglion Regeneration The previous series of experiments shows the possibility of memory storage for conditioned reflexes in planaria after cutting and regeneration. We will now present some data that indicate the possibility of forming new conditioned reflexes during regeneration (Cherkashin, Sheiman, and Bogorovskaya 1966). In these experiments planaria were cut across and conditioned for 5 days with a vibratory conditioned stimulus. These animals were tested for the memory of conditioned responses 2 weeks after the operation. The control animals were sectioned and then treated the same as experimentals, with the exception that they received only the conditioned stimulus. The results indicated that the number of responses to the conditioned stimulus did not change over 5 days of regeneration in both experimental and control groups. It was low. There was more activity, however, in the head regions than in the tail. However, in another experiment in which the presence of conditioned reflexes were tested following 14 days of regeneration, conditioned reflexes in trained animals, but not in controls, were revealed. The number of conditioned responses was similar to that found before amputation. The number of responses in the control group was constantly low throughout the entire experiment. The present experiments show that it is possible to condition planaria in the absence of the ganglion and during its regeneration. The results of such conditioning will appear only after ganglion regeneration. These results raise the question of whether conditioning depends on the maturity of the new nervous elements, the state of the remaining nervous structures, or perhaps some other factor. The following experiments were carried out to answer these questions. It was necessary to create a new experimental device and a new method of conditioning. To conduct this study, it was necessary to keep training times brief. Mass conditioning was therefore carried out intensively throughout a day. Forty

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combinations of light and electrical shock were given to a planaria restricted to a "cell." Learning was conducted in two daily sessions separated by a 2-hour interval. The basic illumination of the cell was 40 lux. The conditioned stimulus was increasing the light to 70 lux, and the unconditioned stimulus was electric impulses generated by electronic stimulator (current strength 10 uA; duration 10 ms). Progress in the formation of conditioned reflexes was monitored by an optical-electrical registration designed to record planarian movements automatically (Tiras and Aslanidi 1981). A light flux fell perpendicular to a sensor containing two photoresistors connected to different arms of a balanced bridge. Movements of the animal resulted in alterations of the illumination difference of photoresistors and, correspondingly, changed the signal of the bridge. Motor responses of planaria were registered in the course of the experiment as "bends" on a paper recorder. The ratio between the number of bends obtained under the action of a conditioned stimulus and the total number of bends for a combination of stimuli were calculated. The average value of 10 subsequent stimuli combinations was taken as a coefficient of conditioning (CR) and expressed as a percentage:

where A is the number of bends in the course of the action of a conditioned stimulus, B is the total number of bends for a stimuli combinations, and S is a constant. In the first set of experiments, planaria were trained after various numbers of days of regeneration (Sheiman and Tiras 1983; Tiras and Sheiman 1981). After sectioning of the head, experiments were carried out daily (up to the 5th day) in different group of animals. In 2 weeks, when the regeneration was practically completed, the memory trace preservation was tested using 10 conditioned stimuliunconditioned stimuli pairings. As controls, intact animals that had acquired the conditioned response 2 weeks previously were used, as was a second control group whose members were cut and subjected to the conditioning without receiving any preliminary conditioning. Each group contained 20-30 planaria Dugesia tigrina. As the activity of regenerated planaria was variable, it was impossible to compare the results of learning in different experimental groups. The results of conditioning were judged according to the a test session. The coefficients of conditioning in learned planaria on the 1st, 3rd and 5th days of regeneration are close to the level of responses of intact learned control planaria. Conditioning was very poor in planaria who received training on the 2nd and 4th days of regeneration: they were at the level of control planaria who also regenerated but were not trained. This set of experiments led to the following conclusions. The first group of control animals is the standard of learning, while the second one reflects those alterations in planaria motor activity and excitability that because of regeneration.


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Comparison between experimental and control groups showed that planaria subjected to the experiment on the 1st, 3rd and 5th days of regeneration appeared to be trained at the end of this process, while those trained on the 2nd and 4th days manifested motor activity characteristic of untrained regenerated planaria. As the deviations described are not connected with the linear character of ganglion development, it is natural to suppose that they are caused by some other factors operating in the course of regeneration. We believed that such a factor might be, in particular, the regenerating factors (RF). To test this supposition, the regenerative material was to be transferred to intact planarians prior to training. An adequate method of introducing this material was developed, and procedures were designed that made it possible to separate the source of RF and the object of action from the location where the formation, storage, and reproduction of the memory trace occurs. Planaria of different groups obtained the homogenates from planaria after different numbers of days (from 1st to 5th) of regeneration (groups H1-H5). The control groups were fed either with Tubifex worms or with homogenates from intact planaria (CT and CI correspondingly). On the next day after feeding, planaria-recipients were conditioned, and a week later they were tested for preservation of the memory trace. In all the experiments, the results of learning and reproduction of a memory trace were taken into account. The results indicate that the responses of planaria in different groups vary in the course of training. In all groups, deviation of the level of responses occurred in the same direction: In the second part of the experiments the response level was higher than in the first part. This indicated that motor activity increased in the course of training. The level of responses differed within experimental groups as well as between experimental and control groups. Motor activity of planaria fed with homogenates of the 1st and 3rd days of regeneration were higher than those in control group CT throughout the course of the experiment, although the differences were not statistically significant. The level of activity in animals obtaining homogenates of the 2nd and 5th days of regeneration was higher at the end of the experiment than in the control group CT. Finally, the group H4 obtained homogenates from the 4th day differed from all others (including the control groups) by a significantly lower level of responses throughout training. Hence, all experimental groups (except the H4 group) more or less expressed stimulation of learning compared to the control group. Only in one experimental group was an inhibitory effect revealed. To determine the preservation of the memory trace in the learned planaria of the previous experiments, these animals were tested one week after training. The response level in all groups of experimental animals were higher than those in the first experiment (after 40 training trials) and in control animals. Statistical analysis of the data allowed us to divide experimental and control groups as follows. The lowest level of motor activity was revealed in the control group CT; a higher level characterized the group of planaria fed with homogenates of intact planaria and regenerates of the 1st and 4th days. The highest level of activity was observed in

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planaria from the groups H2, H3, and H5. All experimental groups were reliably different from control group CT. There were no significant differences between the experimental groups H2, H3, H5 and HI, H4. The results show that in the interval between learning and testing the memory trace is enhanced. This is specific to experimental animals rather than to controls and is more pronounced in those obtaining homogenates of the 2nd, 3rd and 5th days and less in animals fed with those of the 1st and especially the 4th day. The results supported the hypothesis that in the course of regeneration there arise regenerating factors that stimulate or inhibit the formation of memory traces. An attempt was made to characterize these factors and reveal their effects on reproduction of memory traces. Intact planaria were trained by the standard method. Preservation of memory traces was tested a week later. Prior to testing, the trained planaria were divided into experimental groups; each group was fed with homogenates from the regenerating planaria (1-5 days) and Tubifex worms. The results indicate that in most experimental and control animals the response levels during the test differed only slightly. However, the group of planaria who obtained the regenerates of the 1st and 4th days manifested lower responses as compared with controls. In all groups except that of the recipients fed with homogenates from the 4th day, the level of responses in the test experiments were higher than in the course of training (Sheiman and Tiras 1983). Training of planaria with the amputated head containing ganglion showed the possibility of memory trace formation, but the reproduction of the memory trace requires the ganglion (Cherkashin, Sheiman, and Bogorovskaya 1966). The experiments on training regenerates at different days demonstrated the possibility of memory trace formation in decapitated planaria. The supposition was made that during days 1-3 of regeneration learning proceeds because residual nervous elements of the longitudinal nerve trunks and that on days 4-5 of regeneration, when the of base of the ganglion is practically formed, the function of memory trace formation should be carried out by a new ganglion. In such a case one could expect that, in the first set of experiments, the memory traces were preserved throughout the whole course of regeneration. The results of our experiments on memory trace reproduction after completing the process of regeneration indicated that the capacity of learning in the course of regeneration fluctuates and is not directly dependent on maturation of nervous structures. The memory trace can be formed in the absence of the ganglia (1st and 3rd days of regeneration) and may not arise even when the ganglion is developed (4th day of regeneration). These data support the idea that in the course of ganglion regeneration learning is affected by some additional factors (for example, RF) rather than by the substrate providing the fixation of a memory trace. We believe these factors to be directly related to the process of regeneration and its stages. The possibility of their transmission from one animal to another by means of corresponding homogenates indicates their chemical nature. Similar morphogenetically active factors that regulated regeneration of the ganglia were


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described, for example, in our own experiments (Tiras and Sheiman 1984). In the study described above, the activity of RF was observed under various conditions. This allows us to distinguish between their endogenous and exogenous effects and its influence on memory trace formation, its reproduction, and its time course. Experimental groups of animals were compared with control animals under all experimental conditions. The results revealed positive (stimulatory) and negative (inhibitory) and neutral effects of RF. Stimulatory effect of RF was most pronounced in the course of reproduction of the memory trace in intact planaria obtaining the homogenates of various groups prior to training (1-5 days of regeneration). This delayed effect, apparent after an interval, resulted from exogenous introduction of RF. Endogenous RF effects that were inhibitory were observed in experiments with regenerating planaria on the 2nd and 4th days of regeneration. Exogenous RF effects on the 4th day of regeneration was also observed to be inhibitory in intact planaria. Direct inhibitory RF effects on the 1st and 4th day of regeneration homogenates were observed under exogenous introduction and in the course of memory trace reproduction. The inhibitory RF effect was manifested mainly immediately. However, delayed effects were also observed: the total level of stimulation diminished in the intact planaria trained and tested a week after being fed with homogenates of the 1st and 4th days. The most distinct effects were those produced by RF in the last days: regenerates of the 3rd and 5th days had a stimulating effect, and those of the 4th day had an inhibitory one. As for RF of the 1 st and 2nd days of regeneration, the effects were not similar in different experiments. These results suggest that, at different days of regeneration, planaria contain active substances inducing different effects and that, moreover, different substances may exist together at the same stage of regeneration. Attention was drawn to the difference between the effects of RF of the early (1-2 days of regeneration) and the late (3-5 days of regeneration) stages. The latter group contains both stimulating and inhibiting agents and possesses some direct features relating to nervous system functioning. As these effects were revealed during the the process of differentiation and maturation of a ganglion, it was reasonable to suggest the existence of neurogenous factors specific to these stages of regeneration. At the same time, the indistinct character of the RF effect during the first 2 days of regeneration indicated their polyfunctionality and nonspecificity, at least as it relates to the function of the nervous system. At this stage of regeneration, such processes prevail as the healing of damaged surfaces and proliferation and migration of neoblasts, and it seems natural to expect that RF effects are directed toward the regulation of these processes (Sheiman and Tiras 1983). Earlier some indications were made that the factors arising in the process of regeneration of planarian ganglion have a morphogenetic effect (i.e., they act as embryonal inductors). They originate from the longitudinal nerve cords and the ganglion (Lender 1955; Wolff 1962), as well as from other parts of planarian body (Lender 1956a, 1956b). With respect to regeneration of homological structures they are inhibitors (Lender 1956a, 1956b).

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The results obtained here and the small amount of data relating to this question allow us to relate the regenerative factors we have described to the substances currently classified as neuropeptides and, on the other hand, as morphogens. This assumption received support from the recently revealed similarity between hydra head activator and the hypothalamus neuropeptide obtained from the rembryonal brain of a rat (Shaller 1975). To summarize our results thus far, the conditioned reflexes formed in planaria before cutting are stored during regeneration but are reproduced only after the formation of new ganglia. It is possible to condition planaria during regeneration, but the memory trace is not revealed until new ganglia are regenerated. Regenerator factors (RF) regulate the creation of the memory trace, its storage, and its reproduction. The most active RF occurs on the 4th day of regeneration. This RF was able to influence all stages of memory trace functioning. There are many different RFs, and their characterization requires additional biochemical investigations. INSECT MEMORY AND METAMORPHOSIS Part two of this chapter is concerned with a parallel analysis of development and memory on another preparation, insect metamorphosis. The complex multistage process of insect postembryonal development includes several larval stages separated by periodical molts, a pupa stage, and finally the appearance of the adult organism, the imago. All these forms differ sharply from each other in exterior structure and, more important, in inner structure. It is proposed that the insect genome consists of two programs. The first one is realized in the system of larval structure and behavior, and the other in the imagos inner organs and behavior. During metamorphosis one genetic system is regularly replaced by the other. The cyclic organization of insect development and metamorphosis is regulated by a system of hormones: activation hormones, molting hormone (ecdysone), and juvenile hormone. The data on insect development and metamorphosis are extensive, and we used these processes as a biological model for the study of memory. Accordingly, they initiated the following questions. First, how is a memory stored in the larval stage recalled after metamorphosis? This problem was solved in our laboratory for the first time. But it raised the following questions: (1) How does the periodicity in larval development influence the memory trace? (2) How do hormones produced during metamorphosis affect the insect memory trace? Unfortunately, it is not known exactly what happens to larval organisms during metamorphosis. It is also unclear if all larval cells are replaced or if some remain. The grain beetle Tenebrio molitor was used in our experiments (Cherkashin, Sheiman, and Stafekhina 1968). The animals were tested on a T-maze instrumental task. The stem and arms of the plexiglas maze were 50 mm in length. The animals crawled through the maze in a semicircular section of 10 mm diameter. The first trial of training was a preference test to determine if the right or left


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arm was preferred. In succeeding trials the animal was run against its preference. An incorrect choice was punished by a "light mechanical blow" delivered to the anterior portion of the animal. The correct response was rewarded with access to a cup of bran. A session consisted of 10 runs with an intertrial interval of 2-4 minutes. Such sessions were repeated 5 times for each insect, with a 1-hour interval. In some experiments animals received 10 sessions of training, in other experiments only 1 day of training. Sometimes insects showed prudence and preferred either to go back into the starting area or, simply, to stop. In such cases they were prevented from retreating or encouraged to continue (after 1 minute of inactivity) by a slight mechanical prodding. Metamorphosis Influence on the Storage of the Memory Trace More than 100 beetle larvae (about 25 mm long) were conditioned in a T-maze for 10 days. Some time after training they pupated and test experiments were conducted on the adult beetles 1 month after training. Another group of larvae about 10 mm long were trained similarly but tested as adult beetles 3 months after training. The results of this experiment indicate that the number of correct choices gradually increased both in larvae and in adult beetles and stabilized after 8 days of training. After the 1 month interval there were a large number of correct choices in all animals, and those animals in the experimental group performed better than controls. Small larvae learned as well as larger animals, although they moved more slowly. Moreover, the memory trace in small larvae was more stable than in adult beetles. After metamorphosis, trained animals retained the correct response (Cherkashin et al. 1968). The adult insect brain differs sharply from the larval brain. The adult brain is formed after the destruction of larval brain cells, and there is a proliferation, migration, and differentiation of new nerve cells. These processes are under genome control. Behavior of nerve cells in larvae and pupae depend on the stage of development (Pipa 1973). The mechanisms of postembryonal brain development are like those found in the embryonic stage. The number of cells in the insect nervous system significantly increased during the transformation into an imago. Neuroblasts and ganglion cells constantly divide (Panov 1963; Sbrenna 1971) the number of glial cells increase (Gymer and Edwards 1967), and the neuropil is enlarged (Afify 1960). The most straightfoward explanation of why the memory trace persists into the adult stage is that some nerve cells are preserved during the change from larva to adult. It is unclear, however, if the mechanisms of the memory trace is solidified during metamorphosis. Memory Trace Formation in Larvae and Cyclic Development To study this phenomenon one must investigate the dynamics of memory trace

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consolidation during metamorphosis in connection with larvae development. Larvae were used because the changes in their internal structures are not as pronounced as in pupae. This makes it possible to separate the metamorphosis processes into definite phases. The periodical larvae molts were used as a marker separating neighboring cycles. Memory traces were formed in different phases of one larvae instar (Sheiman, Khutzian, and Ignatovitch 1980). The white larvae of Tenebrio molitor (mealworms) obtained just after molting were selected for training, and this initial molt served as the zero-point for the developmental cycle. This insect is characterized by a great but variable number of molts, and tests were performed on the 3 last instars. The larvae were selected from the bulk of animals developing under constant conditions in bran. Mealworms, 20-22 mm long, were kept separately in cups containing bran at approximately 22° C. Under such conditions the intermolting period lasts about 20 days. Tests were conducted on 5 subgroups on day zero (i.e., the day of the molt), and on days 4, 8, 12, and 16 thereafter. Each developmental stage was represented by 10 larvae with the exception of the day 16 subgroup, which included 20 larvae. In order to reduce to a minimum the developmental changes of larvae within the time of an experiment, we used intensive training during carried out in single day. Because the behavior of untrained larvae at different stages of the intermolt period did not differ at all, we used as a control group 10 larvae selected randomly in regard to stage. They were studied in the same maze as the test animals but, in contrast, were not subjected to any reinforcement and were left to their own devices. The tested larvae that were not given training showed a preference for a definite direction, as was observed with the control group: 5 larvae of this group chose the right and 5 chose the left direction on their first run and during the rest of the test preferred their original direction with a probability greater than 0.95. The mean number of correct choices was 4.0 for the first session and 3.7 for the last session in this group. In all experimental subgroups the mean number of correct choices for the first session was somewhat more than that in the control group (group C), fluctuating from 4.5 to 5.3 in the different subgroups. In the second session the distinction became apparent between the mean number of correct choices for different subgroups: much greater for the day zero and day 16 subgroups than for the other subgroups, with the differences increasing in the subsequent sessions. In the last session the mean number of correct choices was approximately 7 for the day zero and day 16 subgroups but remained only slightly different from the original value for the other subgroups. Based on the obvious differences in the behavior of the experimental larvae at the various stages, two large experimental groups were formed. The day 0 and day 16 animals comprised the first such group (A); the second group (B) included all the others. Both Groups A and B differed from Group C by the larger numbers of correct responses beginning with the 1st session. A comparison of Group A with Group B revealed that the number of correct responses increased for Group A over the 5 sessions of the test but for Group B remained close to its first session value.


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The subsequent analysis by the bifactorial proportional complex scheme with the use of the modified Fisher's criterion confirmed the appropriateness of the division of the experimental larvae into 2 groups. In this scheme we considered the course of the test, namely, the number of a session as the 1st factor and the larvae's inclusion in either Group A or Group B as the 2nd factor. Thus, we derived a 5 X 2 design with 60 animals in all. We calculated the variances for each square, along each factor and for the whole design, and then compared them, taking into account the number of the degrees of freedom, of course. We used the numbers of correct choices during a session as the basic parameter because we found no reliable increase in number of correct responses within each session, either for each run taken separately or for the first 5 runs compared with the last 5. It was found that the stage of the instar period on the worm's aptitude for learning was significant. Thus, the training of larvae at different stages of their development revealed a clear distinction: learning was most efficient when approaching the molt and immediately after it. During most of the instar, learning was hampered. In order to determine more accurately the bound of the outlined phenomenon tests were conducted on 2 additional groups (day 2 and day 14) of larvae. The day 2 group showed hampered learning resembling that of the day 4 subgroup, and the day 14 group performed similarly to the day 16 larvae. Subsequent observations of the experimental larvae showed them to be mixed population in regard to the number of molts from pupation. Some belonged to the last instar and others to the second to last, but some were earlier instars. A comparison of larval behavior at different instars has shown that the periodicity in their aptitude for learning exist for all the investigated instars; no accurate data on instar influence on aptitude were obtained. The mealworms showed a different aptitude for learning depending on the stage of the instar period, a peculiarity inherent in different instars of larvae. Therefore, the dynamics of memory trace formation must exhibit a cyclic character. A large literature exists about the learning of adult insects, but little information is available regarding learning at the larval stage of insect development. Larvae possess such pronounced peculiarities, distinguishing this stage from both embryonic and adult stages, that one must consider it independently. The larval stage is a postembryonic stage of insect development and larvae can live, feed, and adapt themselves as well as adults do. The larvae are characterized by the active development of most of their organs and systems, in particular the nervous system, and by the cyclic nature of this development. A relationship may exist between the cyclic facilitation of learning in larvae and the sensitive periods in the development of vertebrate behavior. However, a comparison of the larval stage of insect development with the early period in vertebrate ontogenesis is extremely difficult to draw because the latter does not possess the genetic rigidity notable in insects. Only incidental information exists about the cyclic changes in the structure and function of the insect nervous system. An investigation by Tyshchenko (1969) of spontaneous electrical activity in nerve ganglia of caterpillars has shown that this

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activity abruptly decreases at the time of a molt and then increases gradually during the intermolting period. Motor activity also decreases at the time of the molt. Beetsma, de Ruiter, and Wilde (1962) described a change in the reaction to light between molts of the larvae of Smerinthus ocellata L. These animals show photopositive behavior at the beginning of the instar that is gradually supplanted by a photonegative response by the end. The knowledge about periodical changes in the structure of the nervous system of larvae is no more abundant. The process of reproduction of neuron bodies and their branches presumably goes on monotonously during the larval development, and only mitosis of the glial and neurolemmal cells possesses a cyclic character and occur at the time of a molt. At the same time the neuron population is replenished with continuous fission of neuroblasts and ganglion-mother cells (Nordlander and Edwards 1968, 1969; Panov 1960). Unfortunately, data on this matter are not numerous enough to explain the cyclic features in learning, and they need some explanation. The broadly manifested periodicity in the development of insect larvae is governed by a group of hormones related to metamorphosis. Interaction of the hormones affects the course of each cycle of larval development. By the end of each instar the molting hormones of metamorphosis, namely, ecdysones, may have an effect upon these processes. The experiments verifying the effect of hormonal extracts upon the cockroach's nervous system (Ozbas and Hodgson 1958) indicated that the hormones may have a direct influence upon the nervous system of insects. Ecdysone is responsible not only for molting but for a whole series of changes in the larval organism connected with a molt. However, there are no data about such a causal connection. There also are no data regarding the dependence of learning on ecdysone or other hormones. It follows from the results of our previous series of experiments that changes in the efficacy of memory trace formation may depend on the ecdysone content and its activity in the larvae organism. That is why the following special series of experiments were performed to investigate the direct effect of ecdysone on larval learning. Memory Trace and the Hormones of Metamorphosis Larvae 20 mm long were used immediately after molting. They were placed in cups containing bran at 25° C. The next molt at this temperature occurred 12-13 days later. The T-maze training was intensive. In accordance with the previous results of successive larvae training, this series of experiments was made 3, 6 or 9 days after molting. Two hours before the experiment larvae were injected with 0.6 ul mixture containing 2 ug of ecdysterone, dissolved in insect physiological solution. Control animals obtained only the physiological solution. Each experimental group contained 10 larvae (Sheiman and Ignatovich, 1977). The results of the experiment indicate that the control animals from the 9th day differ from the other animals both in the total number of correct choices and in the


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dynamics of the increase in correct choices as the experiment progressed. A quite other situation was seen in the experimental groups. In the 6th day, group training was the most effective. At the same time, the 3rd and 9th day groups did not significantly differ from the control groups. In a special series of experiments the effect of injecting large doses of hormone were tested. Larvae from the different groups obtained 4 and 8 ug of ecdysterone. The results showed that increasing the dose of hormone improved the training in the 3rd- and 9th-day groups (Sheiman and Ignatovitch 1977). This experiment with exogenous ecdysone confirmed the supposition about the dependence of the cyclic recurrence of larvae learning on the endogenous ecdysone content. This investigation was made on the larvae from different stages of instar (Sheiman 1976). It was found that degeneration of the protharacal gland (which secretes ecdysone in insects) was revealed just after molting. On the 3rd day, together with the destroyed cells, appeared many small cells that did not separate from the tracheal wall. On the 6th day mature gland cells were observed concentrating around the gland cavity. On the 9th day the gland was ready for secretion. Thus, in accordance with the changes in the gland secreting ecdysone, it may be concluded that on the 9th day of instar ecdysone is active in the larvae body. It is precisely at this time that the larvae reveal the best abilities to learn. Clearly, ecdysone facilitates conditioning in beetle larvae. In parallel with the gradual increasing of endogenous hormone titre, larvae conditioning sharply improves at the end of the instar. Injection of high doses of exogenous hormone improves learning in different phases of the instar. The larval reaction to hormone depends on its inner state, which changed during the intermolting period. Small doses of hormone effect only the middle phase of the instar. Perhaps at this time larvae tissue are ready to react to ecdysone coincidentally with prothoracal gland maturation. Probably the corresponding receptors become active in larvae tissue at that time. To summarize our results thus far, during complex morphogenesis, characteristic of postembryonal development in insects, the specific picture of memory trace functioning is shown. Grain beetle larvae may be conditioned to choose the correct direction in a T-maze, and this memory trace is preserved during metamorphosis and manifest in adult beetles. It was found that different abilities to learn were related to the different stages of one intermolting cycle. This ability does not manifested itself in the beginning and middle of the instar. However, larvae may easily learn in the end of the instar and immediately after it. The larvae ability to learn correlates well with the content in their body of the metamorphosis hormone ecdysone. REGULATORS OF MEMORY AND MORPHOGENESIS In two previous sections, the substances closely connected with regulation of development and also with memory trace functioning were revealed and outlined. These are planarian RF and the insect molting hormone ecdysone. As the main

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function of these substances is the regulation of morphogenesis, they belong to the morphogens. Naturally, the question is raised of whether such common effects on development and memory are characteristic of other morphogenetic regulators. To answer this question it is necessary to find morphogenetic properties among the active regulators. The most attractive were the neuropeptides. There are true regulators of morphogenesis, such as different growth factors and hormones, components of tissue-specific factors, invertebrate growth regulators (as hydra head activator, hydra morphogen, or insect activation hormones) (Sheiman, Balobanova, Martinovich, Polikarpova, and Slobodchikova 1984, 1990). All these substances may be directly classified as morphogens on the basis of their general function. The first task was to reveal morphogens among peptides, in particular, among neuropeptides. The second task was to reveal the effects of morphogens on memory processes. Neuropeptide Effects on Invertebrate Morphogenesis In our experiments, neuropeptides were obtained from the laboratories of the Russian Cardiological Center and in the Institute of Bioorganic Chemistry, Byelorussian Academy of Sciences. We used neuropeptides in concentrations ranging from 10(-7) to 10(-11) M. The peptides were added to the water containing the planaria. The peptides (1 ul) were injected into insects with a Hamilton microsyringe or were mixed in with food that was given to both larvae and adult beetles (Sheiman, Tiras, and Balobanova 1989). We use different neuropeptides: luliberin (LHRH), vasopressin, somatostatin (SRIF), dalargin (analog of leu-enkephalin), D-sleep peptide (DSIP), and hydra head activator (HA), specific for lower invertebrates. Among them, HA and SRIF have direct morphogenetic functions, LHRH only partially, and the remainder do not. The results indicate that neuropeptides may stimulate or inhibit development. There is a stimulating effect on planarian regeneration with hydra morphogen (HA), vasopressin, dalargin, and SRIF. There is an inhibitory efect with LHRH, no effect with Delta-sleep peptide (DSIP). Insect metamorphosis was stimulated with HA and LHRH. In the majority of cases, neuropeptides had stimulative effects. However, the dynamics of these effects differ considerably. Vasopressin and dalargin were active throughout the experiment, and the effect of dalargin even intensified by the end of the experiment. HA was reliably effective in the beginning and in the end of regeneration. SRIF also acted positively, but only in the second part of experiment. The data allowed us to conclude that the various morphogenetic mechanisms of neuropeptides have an effect on planarian regeneration (Sheiman, Tiras, and Balobanova 1989). Stimulation of Insect Development Only two peptides, HA and LHRH, were used in the experiments on insect


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development. Both of them proved to be stimulators of larvae development. If the neuropeptide is presented in the beginning of the instar, the molting in treated animals occurs earlier than in controls. However, this stimulating effect of neuropeptides is not always revealed. There were no differences in molting between experimental and control larvae after LHRH injection in the second instar (8th day after molting). Inhibiting Effect of Neuropeptides The effects of LHRH and DSIP on planarian regeneration differ from the effects mentioned above. LHRH had an inhibitory effect on planarian morphogenesis during the entire experiment. The next peptide, DSIP, had no effect on planarian regeneration. Thus, apart from stimulation, neuropeptides may also inhibit planarian morphogenesis. There is also no influence of neuropeptides on planarian regeneration. Effect of Neuropeptides on Development and Memory in Insects Two neuropeptides (HA and LHRH) were tested in experiments on development and memory of insects. Hydra Head Activator Effects on Development in Grain Beetles Experiments on development were performed using 300 larvae injected with hydra head activator (HA) following various days post molt (Sheiman and Bespalova 1988). There was an equal number of control animals. The larvae and pupae that molted were collected every day. Some of the larvae received HA in concentrations of 10 (-7) M and the others received 10(-9) M. The results indicate that molting and pupation were unsynchronized both in experimental and control groups; this is characteristic for grain beetles development. In the beginning of molting the number of larvae in experimental groups was larger than in controls. HA had the same effect on pupation: Treated larvae that molted to pupae developed more quickly than controls. Molting in larvae with injection on the 7th-9th day of instar was delayed in comparison with larvae with earlier injection. Both experimental groups differed from controls. The results of this experiment indicate that application of hydra head activator accelerates the molting and metamorphosis of mealworms to pupae. Hydra Head Activator Effects on Memory in Grain Beetles T-maze instrumental conditioning was carried out on adult beetles over the course of five training sessions (10 trials per session). Experimental (treated with HA) and control groups (untreated) contained 35 animals each. We predicted that

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animals treated with HA would perform better than controls over a series of training sessions. The results of the experiment supported this prediction (Sheiman and Bespalova 1988). The number of correct choices over the course of the experiment was significantly higher in treated animals than in untreated controls, although both groups improved. The data indicates that in treated beetles the number of correct choices within a session is also higher than in untreated controls (with the exception of the first and third training sessions). Improved performance of treated versus untreated animals was observed when 1 pi HA was injected in a concentration of 10(-9) M, but no effect was observed at concentrations of 10(-8) and 10(-10) M. The HA morphogenetic action on larvae observed in previous experiments is similar to the effect of ecdysone or prothoracotropical. It is possible that HA has a direct influence on molting or works indirectly on prothoracal glands, producing ecdysone. A third possibility is that HA exerts influence on ganglion secretory cells, excreting ecdysone (Sheiman and Bespalova 1988). HA was discovered and intensively studied in Professor H. C. Shaller's laboratory. HA is excreted by hydra neurosecretory cells and regulates regeneration and budding in hydra (Schaller 1976). HA morphogenetic function manifests itself in some cases in the activation of budding in hydra and in other cases in the acceleration of insect metamorphosis. The common result of administering HA is to increase the rate of development. HA effect on development can be seen, as we have done here, by making crossspecies comparisons. Another function of HA was revealed in our experiments on adult beetles, the stimulation of learning. We did not find a direct connection between rate of development and learning because development was studied in larvae but learning was investigated in adult animals. However, it was shown that the administration of ecdysone stimulated learning in larvae. This suggests the possibility that the action of HA on the memory of adult beetles and its effect on larval development in beetles is regulated by the same mechanism. Effects of Luliberin and its Fragments on Development in Grain Beetles and Larvae LHRH has a predominantly gonadotrophic effect in mammals. The purpose of this experiment is to test the effect of this peptide on grain beetles in the course of their normal development. Eggs were placed on fresh bran and, after they turned into larvae, were weighed every day, and the number of molts were counted. The peptide was administered to the larvae by mixing it in their food. The effects of LHRH and its fragments were studied. Fragments L(l-2) and L(9-10) were used. Every experimental group contained 100 larvae, and experiments were repeated 2-3 times. Most experiments were performed with L(9-10), the C-end fragment of LHRH. In 3 series of experiments, 5 mg of peptide was added to the animals' food. Larvae


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weight and weekly growth were calculated. The results indicated that the control larvae grew faster than treated larvae. The average number of molts before pupation in the experimental group were found to be lower than in untreated controls. This difference was considerable in the first period of development and then disappeared. After pupation, molting in both groups continued. By the end of the experiment, the average number of molts in the experimental group was 6.7 and in the control group was 7.4. This difference was due to the lack of stable instars in the mealworm population. The effect of neuropeptides in such unstable systems produced molts in experimental animals that were, in average, 1 molt less than in controls. The duration of the instar period during metamorphosis gradually increased in experimentals. We calculated only the average duration of one instar. This was found to be 9.1 for the control group and 10.7 for the experimental group. The first pupae appeared in both groups simultaneously. The pupation of the whole population took about a month. The dynamics of pupae appearance was calculated every 10 days. The onset of pupation in the experimental group was later than in control animals. The conclusion of the experiments is that L(9-10) delays growth and development in mealworms. This is reflected by a delayed growth in weight, an increase in both instar duration and time of pupation, and a decrease in the number of molts. These data were replicated in the next experiments. Just before the new experiments were performed, large larvae were selected and injected with peptide. Injection of peptide, rather than by administeration in food, appears to traumatize the animal. Despite this limitation, injection has the advantage that it allows us to know precisely the stage of development when the peptide was administered and its dosage. In these experiments the dose was 1 pi of 10 (-5) M. The injections were made at different stages of development, 2 or 8 days after molting (i.e., in the beginning or close to the end of instar). Accordingly, there were 2 experimental and 2 control groups of larvae in each set of experiments. The type of molt (to larvae or pupae) was a variable. Each group contained about 100 animals. The results indicated that the percentage of pupation in the first molt in both groups was similar. However, when the peptide was injected 8 days after molting, the number of larval molts were more than that observed in pupae molting. A molting criterion (Mc) was established to analyze these results, in which the ratio between the total number of animals molting into larvae and the total number of animals in the experimental population was calculated. In two control groups these values were 0.78 and 0.79. In the two experimental groups, however, the values were 0.9 (in the group injection on the 2nd day) and 1.34 (in the group injection on the 8th day). The results indicate that the peptide stimulated larvae to molt. The effect of L(9-10) was slight following the day 2 injection but was considerably stronger after the day 8 injection. Additional results indicated that pupation "intensity" following day 8 injection was a little lower than in the control group. However, in the group that received the injection on the 2nd day there were more pupae than in control group.

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The results indicate that L(9-10), whether injected or administered in the larvae's food during the last instar, inhibited development. Also of importance was the time of peptide action (i.e., the stage in the intermolting period). Peptide introduced in the end of instar was more effective than when introduced just after molting. Experiments with L(l-2) were carried out using the same methods just described for fragments L(9-10). The peptide introduced in the food (0.5mg/ml) increased larvae growth in experimentals relative to control animals. Increasing peptide concentration to 1 mg/ml led to a delay of larvae growth that was lower than in a control group. The rate of pupation did not differ in experimental and control animals. L(l-2) (1 pi, 10(-5) M) was also injected on day 2 and day 8 after the initial molt. In control groups half the animals molted into larvae, and the other half into pupae. In the experimental groups, however, an injection in the beginning of the instar period produced 75% larvae molts and 25% pupa molts. In the day 8 group, 36% produced larvae molts and 64% pupa molts. The molting criterion was 0.79 in control groups and 1.43 and 0.62 in experimental groups (2nd and 8th day injection). These results indicated that L(l-2) stimulates growth and development of larvae. LHRH was also given by injection in another series of experiments conducted at the same time as the previous one. There was a visible increase in the development of experimental larvae after injection on day 2. Intensive molting and pupation occurred in experimental groups. There was no effect after day 8 injection of LHRH. LHRH did not effect the molt and pupae ratio, which was equal in experimental and control groups. Effects of Luliberin and its Fragments on Memory in Beetles LHRH and its fragments were tested on groups containing 30 subjects each. LHRH injection improved learning compared to untreated controls early in training, but as training continued the controls reached the levels of experimentals. N-end peptide L(l-2) had a stimulating effect in the beginning of training that was not sustained as training continued. In fact, the number of correct choices decreased. The C-end fragment L(9-10) had the most expressive effect on conditioning. Experimental animals made significantly more correct choices than controls throughout the experiment. It should be noted that a high number of correct choices were made in the beginning of training. LHRH and L(9-10) had stimulative effect on the number of correct choices, and L(l-2) had an inhibitory, effect which was revealed as training continued. It is characteristic that by the end of the experiment (after 4-6 hours of training) the results of different peptides were similar. It seems that the effect of peptides on memory was transitory. Our results suggest that LHRH was able to influence insect behavior. This is one of the more intriguing examples of the effects of neuropeptides. To understand the morphogenetic effect of LHRH, it is important to analyze its


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influence and interactions with specific insect regulators of morphogenesis, (i.e., the hormones influencing metamorphosis such as ecdysone, juvenile, and activating hormones). The results of LHRH fragment injection at different stages of instar indicate that injection of LHRH did influence development when it was injected in the beginning of instar. It is possible that when LHRH did increase molting it limited the effects of juvenile hormone. Thus, the effect of L(l-2) injection on day 2 corresponds to some extent to the effects of L(9-10) injected on day 8 and vice versa. The first phenomenon—increasing the total number of molts and molts to larvae coupled with a decrease of pupations—probably is connected with the reinforcement of juvenile hormone action or weakening of ecdysone action. The second phenomenon expressed after L(l-2) injection on day 8 but weakly revealed after L(9-10) injection on day 2 demonstrates the limitation of juvenile hormone action and intensification of the action of ecdysone. LHRH and its fragments generally had a stimulative effect on grain beetle conditioning; only the N-end peptide had an inhibitory effect. The inhibitory action of this peptide, however, did have an initial stimulating effect on conditioning. The same biphasic influence of peptides was found for ACTH fragments (Sheiman, Ponomareva-Stepnaya, and Maksimova 1980). The positive effect of LHRH fragments on beetle training was revealed as an acceleration in learning but not in terms of the total number of correct choices. The end fragments of LHRH had different effects on adult beetle memory and larval development. However, based on these effects it can be concluded that the influence of LHRH is realized through the interrelations with its fragments. This may explain why the effect of LHRH administered alone is weak in comparison with the effects of its fragments. Finally, it should be mentioned that the possible mechanism of LHRH action on learning is either direct or perhaps indirect via the hormones that regulate metamorphosis. It is possible that this conclusion is applicable not only to larval development but also to adult beetles conditioning. For after all, adult beetles contain the hormones necessary for metamorphosis (Sheiman, Balobanova, and Martinovich 1984, 1990). CONCLUSIONS The comparative study of memory and morphogenesis in the simple biological models reported here and an analysis of their interrelations suggests the following conclusions. 1. The memory trace formed in developing animals is more stable than in intact (planaria) or adult (insects) animals. Retention of the memory of conditioning is preserved during the morphogenetic processes. 2. Memory of conditioning is retained despite considerable destruction of nerve cells during morphogenesis. It must be taken into account, however, that the destruction of these cells is gradual and occurs simultaneously with the develop-

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ment of new nerve cells. These new cells via neoblasts make contact with old nerve cells prior to the destruction of these old cells. The new cells obtain the specific properties of their precursors. The most remarkable data convey the contribution that these new cells make to the memory trace. The transmission of memory information from "old" cell to "new" cell resembles the process of embryonal induction as defined by Weiss (1961). In planarian regeneration, for example, the transmission of certain information takes place from cell to cell. 3. Regulatory factors are active in the planarian body during the course of regeneration. These factors as well as insect hormones regulating metamorphosis can stimulate or inhibit the unfolding of development sequences. These factors also influence the formation of a memory trace. 4. Planarian regeneration factors, like insect hormones, are not homogeneous. There are many regulators of regeneration. These can be classified as inhibitors or simulators. They can also be classified acording to their effects on such cellular behavior as proliferation, migration, destruction, and differentiation. During the various phases of morphogenesis, it appears that different regeneration factors can dominate. These factors are also active in the process of memory. 5. Some substances (neuropeptides) were found to regulate morphogenetic processes in much the same way as regeneration factors and those hormones related to metamorphosis. These substances may form a common group of regulators the morphogens that may be related to memory. ACKNOWLEDGMENTS We wish to thank our colleagues A. M. Cherkashin, N. Ju. Sakharova, G. I. Bogorovskaya, E. P. Sergeeva, I. A. Efimov, G. S. Ignatovich, R. Ya. Gordon, S. S. Khutzian, and E. Ph. Balobanova who took part in many of these experiments. REFERENCES Afify, A. M. 1960. Uber die postembryonale Entwicklung des Zentralnervensystems (ZNS) bei der Wanderheuschrecke Locusta migratoria migratorioides (R.u.F.) (Orthoptera, Acrididae). Zoologische Jahrbucher, Abt. fur Anatomie und Ontogenie der Tiere 75:1-38. Beetsma, J., de Ruiter, L., and Wilde, J. 1962. Possible influence of neonetine and ecdysone on the sign of phototaxis in the eyed hawk caterpillar (Smerinthus ocellata L.). J. Insect. Physiol. 5:251-256. Bogorovskaya, G. I. 1969. Regeneration of the planarian nervous system. Tsitologia 77:964-972. Brondsted, H. V. 1969. Planarian regeneration. London: Pergamon Press. Brown, H. M , Dustman, R. E., and Beck, E. C. 1966. Sensitization in planaria. Physiol. and Behav. 7:305-310. Cherkashin, A. N., and Sheiman, I. M. 1967. Peculiarities of conditioned reflexes in flatworms (in Russian). In: E. M. Kreps, ed., Evolyutsionnaya Nejrofiziologiya i Nejrokhimiya (Evolutionary neurophysiology and neurochemistry), 31-35.

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Leningrad: Nauka Cherkashin, A. N., Sheiman, I. M., and Bogorovskaya, G. I. 1966. Conditioned reflexes in planaria and regeneration experiments (in Russian). Zhurnal Visshey Nervnoy Deyatelnosti 76:1110-1112. Cherkashin, A. N., Sheiman, I. M., and Sergeeva, E. P. 1966a. Effect of combination of light and electrical shock on planaria (in Russian). Zhurnal Vysshey Nervnoy Deyatelnosti 76:266-273 . 1966b. Experimental reproduction of integration in planaria (in Russian). Zhurnal Vysshey Nervnoy Deyatelnosti 76:858-863. Cherkashin, A. N., Sheiman, I. M., and Stafekhina, V. S. 1968. Storage of conditioned reflexes during metamorphosis in insects (in Russian). In: E. M. Kreps, ed., (Fiziologiya i Biokhimiya Bezpozvonochnykh (Physiology and biochemistry of invertebrates), 117-120. Leningrad: Nauka. Coming, W. C, and Riccio, D. 1970. The planarian controversy. In: W. L. Byrne, ed., Molecular Approaches to Learning and Memory, 107-149. New York: Academic Press. Cummings, S. B., and Moreland, C C 1959. Sensitisation vs. conditioning in Planaria: Some methodological consideration. Amer. Psych. 14:591-600. Ernhart, E. N., and Sherrick, C, Jr. 1967. Retention of habit following regeneration in planaria (D. maculata) In: J. V. McConnell, ed. A Manual of Psychological Experimentation on Planarians, 85. Worm Runner's Digest: Ann Arbor: ML Grasso, M. 1966. Sui fenomeni di neurosecrezione durante la rigenerazione di dischetti isolati di Dugesia lugubris. Arch. Zool Ital. 51:321. Gymer, A., and Edwards, J. S. 1967. The development of the insect nervous system. 1. An analysis of postembryonic growth in the terminal ganglion of Acheta domesticus. J. of Morphology 725:191-197. Jones, F. R. 1971. The response of the planarian Dendrocellum lacteum to an increase in light intensity. Animal Behaviour 79:269-276. Karpenko, A. A., and Seravin, L. N. 1973. Role of chemical factors in feeding behavior of Dugesia tigrina (in Russian). Zoologichesky Zhurnal 52:1142-1148. Kreps, E. M. 1925. About the reaction of ascidians to external stimuli (in Russian). Arkhiv Biologicheskikh Nauk 25:191-221. Krichinskaya, E. B. 1972. The structure of nervous system in Turbellaria (in Russian). Archiv Anatomii, Gistologii i Embriologii 63:99-110. Krilov, O. A., and Nazarian, O. A. 1973. Effects of homogenates of regenerating planarians on their retention (in Russian). Zhurnal Vysshey Nervnoy Deyatelnosti 25:1303-1305. Lender, T. 1952. Le role inducteur du cerveau dans la regeneration des yeux d'une Planaire d'eau douce. Bull. Biol. Fr. etBelg. 56:140-215. . 1955. Sur l'inhibition de la regeneration du cerveau de la planaire Polycelis nigra. CR. Acad. Set, 247:1863-1865. . 1956a. Analyse des phenomenes d'induction et d'inhibition dans la regeneration des planaires. Ann. Biol. 52:473-485. . 1956b. L'inhibition de la regeneration du cerveau des Planaire Polycelis nigra et Dugesia lugubris en presence de broyats de tetes ou de queues. Bull. Soc. Zool. France 57:192-199. . 1960. L'inhibition specifique de la differenciation du cerveau des Planaires d'eau douce en regeneration. J. Embryol. Exp. Morphol. 5:291-301

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. 1964. Mise en evidence et role de la neurosecretion chez les Planaires d'eau douce en regeneration. Ann. Endocrinol. 25:61-70. Lender, T., and Klein, N. 1961. Mise en evidence de cellules dans le cerveau de la Planaire Polycelis nigra. Variation de leur nombre au cours de la regeneration posterieur. C.R.Acad. Sci. 253:331-333. . 1962. Les cellules neurosecretices de D. lacteum. Bull. Soc. Zool. France 57:380-384. Lentz, T. L. 1968a. Primitive Nervous Systems. New Haven: Yale University Press. . 1968b. Histochemical localization of acetylcholinesterase activity in planarian. Comp. Biochem. Physiol. 27:715-718. Lindh, N. O. 1959. Heteroplastic transplantation of transversal body sections in flatworms. Arkiv. Zool. 72:183-195. McConnell, J. V. 1965. A tape recorder theory of memory. Worm Runner's Digest 7:3-10. . ed. 1970. A Manual of Psychological Experimentation on Planarians. Worm Runner's Digest: Ann Arbor, MI. McConnell, J. V., Jacobson, A. L., and Kimble, D. P. 1959. The effects of regeneration upon retention of conditioned response in the planarian. J. Comp. Physiol. Psychol. 52:1-5. McConnell, J. V., Jacobson, A. L., and Maynard, D. M. 1959. Apparent retentioned response following total regeneration in the planarian. Amer. Psych. 74:410-417. Morgan, T. H. 1901. Regeneration. Chicago: University of Chicago Press. Murphy, J. 1963. Learning in the planarian. Worm Runner's Digest 5:37-41. Nazarian, O. A. 1973. Storage of traces about experience after regeneration in planaria (in Russian). Zhurnal Vysshey Nervnoy Deyatelnosti 25:120-125. Nordlander, R. H., and Edwards, J. S. 1968. Morphological cell death in the postembryonic development of the insect optic lobes. Nature 275:780-781. . 1969. Postembryonic brain development in the monarch butterfly, Danaus plexippus. 2. The optic lobes. Wilhelm Roux Archiv fur Entwiklungsmechanic der Organismen 762:197-217. Ozbas, S., and Hodgson, E. S. 1958. Action of insects neurosecretion upon central nervous system in vitro and upon behavior. Proc. Natl. Acad. Sci. (USA) 44:825-830. Panov, A. A. 1960. The character of reproduction of the neuroblasts, neurolemma and neuroglial cells in the brain of the Chinese oak silkworm larvae (in Russian). Doklady Akademii Nauk SSSR 752:689-692. . 1963. The origin and fate of neuroblasts, neurons and neuroglial cells in the central nervous system of the Chinese oak silkworm Antheraea pernyi Guer. (Lepidoptera, Attacidae) (in Russian). Entomologicheskoe Obozrenie 42:186-191. Pedersen, K. J. 1958. Morphogenetic activities during planarian regeneration as influenced by triethylene melamide. J. Embryol. Exp. Morphol. 6:308-334. Pipa, R. L. 1973. Proliferation, movement and regression of neurons during the postembryonic development of insects. In: D. Young, ed. Developmental Neurobiology of Arthropods, 105-130. London: Cambridge University Press. Porfiryeva, N. A., and Diganova, R. Ja. 1987. Planaria of the European part of the USSR (in Russian). Kazan: Izdatelstvo Kazanskogo Universiteta. Sakharova, N. Ju., and Gordon, R. Ja., 1974. Neurosecretory activity during planarian regeneration (in Russian). In: Pervaya Vsesoyuznaya konferentsiya po neyroendokrinologii (First All-Union Neuro-Endocrinological Conference), 152-154. Leningrad.

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Sauzin-Monnot, M. J., Lender, T., and Gabriel, A. 1970. Etude autoradiographique ultrastructurale des neoblastes dans le blasteme de regeneration de 24 h. et dans les tissus en arriere du blasteme, chez la Planaire Dugesia gonocephale (Turbellarie, Triclade). CR. Acad. Sci. 277:1892-1895. Sbrenna, G. 1971. Postembryonic growth of the ventral nerve cords in Schistocerca gregaria Forsk (Orthoptera, Acrididae). Boll. Zool. 38:49-74. Schaller, H. C 1975. A neurohormone from hydra is also present in the rat brain. J. Neurochem. 25:187-188. . 1976. Head regeneration in hydra is initiated release of head activator and inhibitor. Wilhelm Roux's Archives 750:287-295. Schaller, H. C, and Gierer, A. 1973. Distribution of the head-activating substance in hydra and its localization in membranous particles in nerve cells. J. Embryol. Exp. Morphol. 29:39-52. Sengel, P. 1951. Sur les conditions de la regeneration normale du pharynx chez la Planaire Dugesia (Euplanaria). lugubris O. Schm. CR. Soc. Biol. 745:1381-1384. . 1953. Sur Finduction d'une zone pharingienne chez la planaire d'eau douce Dugesia lugubris. Arch. Anat. Microsc. et Morphol. Exp. 42:57-66. . 1959. La region caudale d'une planaire est elle capable d'induire la regeneration d'un pharynx? J. Embryol. Exp. Morphol. 7:73-85. Sheiman, I. M. 1976. Memory in insects and neurometamorphosis (in Russian). In: Structural and Functional Basis of the Memory Mechanisms, 188-204. Moskva: Nauka. . 1984. Regulatory Morfogeneza i ikh Adaptivnaya Funktsiya (The regulators of morphogenesis and their adaptive function). Moskva: Nauka. Sheiman, I. M., Balobanova, E. F., Martinovich, V. P., Polikarpova, V. P., and Slobodchikova, L. K. 1984. The effect of LHRH and its fragments on memory of beetle Tenebrio molitor (in Russian). Zhurnal Evolutsionoy Biokhimii i Fiziologii 20:314-319. . 1990. Morphogenetical effect of LHRH and its fragments on the metamorphosis of grain beetle (in Russian). In: I. M. Sheiman, ed., Morfogeneticheski Aktivniye Veshchestva (Morphogenetical active substances), 145-154. Pushchino-na-Oke: Biological Research Center. Sheiman, I. M., and Bespalova, Zh. D. 1988. Effect of hydra morphogen on the development and learning of the grain beetle Tenebrio molitor (in Russian). Zhurnal Evolutsionnoy Biokhimii i Fiziologii 24: 420-425. Sheiman, I. M., and Efimov, I. A. 1973. The importance of cell destruction in planarian ganglion during regeneration (in Russian). Tsitologiya 75:1215-1221. Sheiman, I. M., and Ignatovitch, G. S. 1977. Moulting hormone effect on training of Tenebrio molitor larvae (in Russian). Zhurnal Obshchey Biologii 55:627-633. Sheiman, I. M., Khutzian, S. S., and Ignatovitch, G. S. 1980. Periodicity in the behaviour of grain beetle larvae. Developmental Psychobiology, 75:585-590. Sheiman, I. M., Ponomareva-Stepnaya, M. A., Maksimova, L. A, Nezovibatko, V. N., and Ashmarin, I. P. 1980. Effect of adrenocorticotropin fragments on memory in the grain beetle Tenebrio molitor (in Russian). Zhurnal Evolutsionnoy Biokhimii i Fiziologii 76:359-364. Sheiman, I. M., and Tiras, Kh. P. 1983. Influence of endogenous factors of regeneration on the memory of Planarians (in Russian). Zhurnal Obshchey Biologii 44:94-104. . 1984. Formation and properties of planarian conditioned reflexes (in Russian). In: V. F. Konovalov, ed. Nejrofiziologicheskiye Mechanismy Pamyati i Obucheniya

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(Neurophysiological mechanisms of memory and learning), 46-57. Sheiman, I. M., Tiras, Kh. P., and Balobanova, E. Ph. 1989. Morphogenetic effects of neuropeptides (in Russian). Fisiologichesky Zhurnal SSSR 75:619-626. Sokoloff, V. A. 1960. The conditioned reflex to light in Asterias rubens (in russian). Trudy Murmanskogo Morskogo Biologicheskogo Instituta 2:236-248. Steele, V. E., and Lange, C 1977. Characterization of an organ-specific differentiator substance in planarian Dugesia etrusca. J. Embryol. Exper. Morphol. 57:159-172. Thompson, R., and McConnell, J. V. 1955. Classical conditioning in the planarian Dugesia dorotocephala. J. Compar. Physiol. Psychol. 45:65-68. Tiras, Kh. P. 1978. Acetylcholinesterase activity in the planarian nervous system during regeneration (in Russian). Ontogenez 9:262-268. Tiras, Kh. P., and Aslanidy, K. B. 1981. Device for graphic recording of planaria behavior (in Russian). Zhurnal Vysshey Nervnoy Deyatelnosti 57:874-877. Tiras, Kh. P., and Khachko, V. I. 1990. Criteria and the stages of regeneration in planaria (in Russian). Ontogenez 27:620-624. Tiras, Kh. P., and Sheiman, I. M. 1981. Effect of regeneration on planarian conditioning (in Russian). Ontogenez 72:635-638. . 1984. Chemical factors of morphogenesis in planaria (in Russian). Ontogenez 75:374-379. Tiras, Kh. P., Sheiman, I. M., and Sakharova, N. Ju. 1975. Acetylcholinestarase activity in the nervous system of several triclads (class Turbellaria) (in Russian). Zhurnal Evolutsionnoy Biokhimii i Fiziologii 77:427-429. Tushmalova, N. A. 1973. The types of memory of some lower invertebrate (in Russian). In: M. N. Livanov, B. N. Veprintsev eds. Kletochnye Mekhanizmy Pamyati, 40-48. Pushchino-na-Oke: Biological Research Center. Tushmalova, N. A., and Gromyko, N. M. 1968. The conditioned reflexes on chemical stimuli in planarian Policelis nigra (in Russian). In: Fiziologiya i Biokhimiya Bespozyonochnykh (Physiology and biochemistry of invertebrates), 34-39. Leningrad: Nauka. Tyshchenko, V. P. 1969. The spontaneous electrical activity in insect nervous system (in Russian). Trudy Vsesoyuznogo Entomologicheskogo Obshchestva 55:148-181. Voronin, L. G., and Tushmalova, N. A. 1965. About the so-called conditioned reflexes in planarians (in Russian). Zhurnal Evolutsionnoy Biokhimii i Fiziologii 7:98-103. Weiss, P. 1961. Interactions between cells (in Russian). In: G. M. Frank, A. G. Pasynsky eds. Sovremennye Problemy Biofiziki, Tom. 2. (Modern problems in biophysics, vol 2), 176-184. Moskwa: Izdatelstvo Inostrannoy Literatury. Welsch, G. H. 1946. Evidence of a tropic action of acetylcholine in a planarian. Anat. Rec. 94:421-432. Wolff, E. 1962. Recent researches on regeneration of planaria. In: Regeneration, 53-84. New York:: Ronald Press. . 1974. Analysis of substances inhibiting regeneration of freshwater planarians. In: Biology of the Turbellaria, 446-459. New York: McGraw Hill.

Chapter Four Innate and Acquired Behavior of Mollusks Pavel M. Balaban and Igor I. Stepanov

NEUROBIOLOGY OF MOLLUSCANS: AN HISTORICAL OVERVIEW The study of the behavioral neurobiology of molluscans started in Russia with a classic paper of I. P. Pavlov (1885), in which he investigated the role of different ganglia in the reflex closure of valves in Anodonta. The investigation was carried out during his visit to the laboratory of Professor R. Heidenhain in the Physiological Institute in Breslau. Using two kinds of preparation with ablated cerebral and pedal or visceral ganglia, Pavlov demonstrated quite different effects of these ganglia on the muscles that close the valves. Results described in this work suggested the existence of motor fibers (originating from visceral ganglia) that elicit muscle contraction, and of inhibitory fibers originating from cerebral ganglia that cause muscle relaxation. Pavlov investigated reflex responses to adequate stimulation of mantle and gill and demonstrated the necessity of certain ganglia for behavioral responses. Investigating the role of environment, he changed the quality and temperature of the water in which the molluscans were maintained, used pharmacological blockers to alter the behavior (morphine), and described the dependence of behavior on environment. A "summation" phenomenon showing the effect of previous stimulation on subsequent responses was described. In fact, this work in invertebrates was a prelude to the investigation of the principles of learning and brain functioning that Pavlov carried out later, after a large interval of time devoted to the investigation of stomach functioning and brain regulation of visceral functions, for which he won a Nobel prize in 1904. It is interesting to note that this pioneering work has almost all the components of a contemporary neurobiological study: (a) investigation of behavior (b) ablation experiments (c) usage of pharmacological tools (d) modeling of brain functioning (e) application of external stimuli, and (f) change of the conditions of experiment

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(conditioning may have appeared as a concept in these experiments!). Surprisingly, another publication that had a great impact on invertebrate neurobiology in Russia appeared the same year: N. P. Vagner (1885), Invertebrates of the White Sea. This large book with perfect illustrations was simultaneously published in Russian in St. Petersburg and in German in Leipzig. Vagner was one of the founders of the first biological marine station on the Solovetzky Islands in the White Sea where, between studies of other animals, he thoroughly investigated the pteropod mollusc, Clione limacina. The level of investigation of ecology, behavior, and morphology was so high that without new technologies there is nothing to add to this description. He described individual giant neurons and traced some of their branches and spoke about how it is possible to provide a complete investigation of the nervous system in this animal using the simplicity and clarity of its structure. Investigation of molluscan neurobiology on a large scale was started in Russia by Kh. S. Koshtojants, who himself was a student of a famous Dutch comparative physiologist, Hermann J. Jordan. In the laboratory of Koshtojants in Moscow University, the first studies concerning the influence of a "nerve factor" on snail muscles and behavior were carried out. Now we know the nerve factor under the name of neurotransmitter. Findings in invertebrate neurobiology were summarized in the extensive volume Comparative Physiology of the Nervous System (Koshtojants 1957), which was and still remains a brilliant insight into the comparative physiological approach. Investigation of transmitter functioning traditionally has remained one of the main themes in Russian neurobiology of invertebrates. Two students of Koshtojants (K. Rosza and J. Salanki), established a well-known laboratory in Hungary, while D. Sakharov continued to study the role of transmitters in invertebrate brain organization and behavior using electrophysiological, cytochemical, morphological, and phylogenetical approaches. Comparative investigation of structure and function of nervous system in pteropod Clione (Sakharov 1960) and later in different species of nudibranch mollusks (Sakharov 1962; Veprintsev, Krasts, and Sakharov 1964) provided evidence of the existence in two quite different species of identifiable cells with completely similar properties including specificity in biochemistry. This notion shifted the scientific interest of D. Sakharov to considering the problem of the genesis of multiplicity of transmitters in the brain. The questions he posed were: Why do we have a strikingly similar set of transmitters in phylogenetically very different animals? How does a similar set of transmitters appear in different animals, by analogy or homology? His findings and a hypothesis considering homology as a principle underlying the appearance of numerous transmitters in different animals are summarized in the book Genealogy of neurons (Sakharov 1974), which became a rarity the next day after its appearance, not only because of the presence in it of a transmitter poligenesis theory but implicitly because of the author's logical style and intelligible and expressive language. With the onset of an era of intracellular physiology, the quantity of laboratories

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working in molluscans flourished. Mostly to obtain an understanding of biophysical properties, giant nerve cells were chosen as the focus of many experiments. Among the first investigators was B. N. Veprintzev, who worked on giant neurons of a pond snail and succeeded in the investigation of ionic channels in isolated neurons; in his laboratory, isolated snail neurons were kept in culture (Kostenko, Geletyuk, and Veprintsev 1974). In the Ukraine, the membranes of snail neurons were used for investigation of ionic channels; the method of dialyzed neurons was introduced by P. G. Kostyuk and his collaborators (Kostyuk, Krishtal, and Doroshenko 1975). Behavioral and electrophysiological investigation of simple forms of plasticity in pond and terrestrial snails was started at the end of the 1960s in a newly formed Department of Psychophysiology (Moscow University). This group of researchers was directed by E. N. Sokolov, who switched from studies of orienting reflex and plasticity in humans and vertebrate animals to cellular mechanisms (Sokolov 1969). The main goal of this group of researchers was to provide a connection between simple networks investigated on an intracellular level and behavior. Habituation of neuronal membrane to repeated electric stimuli was one of the models used to investigate cellular and subcellular mechanisms of plasticity (Sokolov and Jarmizina 1970). A group of devoted snail researchers (to which P. Balaban belonged) in the 1970s moved from Moscow University to the Institute of Higher Nervous Activity headed by a collaborator of Pavlov, E. A. Asratjan, to investigate neural mechanisms of learning in invertebrates. It is important to note that, at the end of the 1970s in Leningrad, a group in the Institute of Experimental Medicine (I. Stepanov among them) started investigating factors influencing learning in terrestrial snails. Their laboratory was located in the famous "Tower of Silence" constructed specifically for the behavioral experiments of Pavlov at the beginning of the century. Thus, we can watch how a loop of a giant helix of scientific knowledge started by Pavlov turned a 100-year wheel. Principles of analysis of behavior and investigation of underlying brain structures have not changed too much but are completed by new technological tools like immunochemistry and electrophysiology. In the present chapter we will try to describe the approaches used to investigate behavior and its modifications in terrestrial snails Helix. BEHAVIORAL REPERTOIRE OF HELIX In the behavior of snails one can find all the major forms of behavior characteristic of higher vertebrates, including humans. They are feeding, escape (avoidance), exploratory, and sexual behavior. We will describe briefly all these behavioral forms before beginning an analysis of behavioral plasticity. Defensive Behavior Terrestrial snails demonstrate withdrawal reactions accompanied by mucus re-

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lease to all kinds of noxious stimuli. Defensive and feeding behavior of terrestrial snails (Helix pomatia, Achatina fulica) is easy to investigate if an animal's shell is fixed with plasticine to a holder as shown in Figure 4.1. A snail can freely crawl upon the surface of a polyethylene ball floating in water, and the behavioral responses can be recorded. A tactile stimulus, irritating chemical stimulus, or electric shock initiates defensive withdrawal responses. It includes a contraction of optic tentacles (ommatophores), small tentacles (rhinophores), a closure of pneumostome opening, and a withdrawal of a foot into the shell. Some mucus is secreted if noxious stimulus intensity overcomes the threshold. A normal snail exerts well-coordinated defensive behavior. Chemical substances injected into the body cavity can evoke separate components of withdrawal reaction. However, this behavior may not be coordinated. For example, glutamic acid, injected into the cephalopedal sinus in the dose of 0.1 mg/g of an animal's weight, induces the withdrawal of tentacles and foot and mucus secretion, but the snail can not fully withdraw into its shell because of the absence of coordination (Stepanov, Smirnova, and Sapronov 1994). The ethological analysis of snail defensive behavior may serve as one of the tests for screening of different environmental pollutants. Two elements of a defensive behavior are suitable for a detailed physiological analysis. First is the defensive reflex of pneumostome closure. This reflex in snails is controlled by the pneumostome circular muscle. The muscle contraction consists of three partly independent components. The first component is the tonic contraction. It depends on the background motor neurons impulse activity. The second type is spontaneous contractions. These contractions are mainly caused by spontaneous synchronous discharge of motor neurons. The third type is a reflex contraction elicited by sensory stimulation. The most suitable method for recording the pneumostome movements proved to be a differential photoelectric device (Stepanov and Poszinsky 1982). A pneumostome opening is transilluminated with light through a glass fiber bundle. When the pneumostome opening is opened, a red spot of an irregular shape appears on the shell opposite to the pneumostome. A photodiode in the lightproof cylinder is fixed to the shell. It receives the light passing through the pneumostome opening plus an environmental light. The second photodiode receives only the environmental light. A differential amplifier picks out only the signals from the pneumostome opening movement. The device is valuable if environmental light is changing. That may be the case if, for example, an action associated with turning off a light is investigated. It was found that the defensive behavior can be initiated when the intensity of the light is stepwise decreased. The hungry state of the animal suppresses the tonic type of circular muscle contraction. A pneumostome is opened to its maximal dimension. This was used to evaluate the level of maximum opening. After the level of the closed pneumostome is also recorded on a pen recorder, the difference between the two levels gives the maximal value of the amplitude of a behavioral response in a given snail. A ratio of amplitude of reflex closure to the maximal amplitude gives the relative amplitude as a percentage of maximal response. This approach provides an

Figure 4.1 Experimental setup for behavioral experiments. The animal is fixed by its shell to a holder (H), but in such a manner that it can move freely on a plastic ball (B). I: indifferent carbon electrode, T: electrically driven tapper, S: electrode for manual application of electric shock to the skin.

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opportunity to compare the responses in different behavioral states (Stepanov, Smirnova, and Sapronov 1994). The withdrawal of optic tentacles constitutes an independent component of withdrawal behavior. A method for quantitative recording of this behavioral response has been suggested (Stepanov and Poszinsky 1982). A polyethylene ball, floating in an aluminum basin, is covered with aluminum foil. The aluminum basin is connected with the negative pole of a DC constant current source. The active electrode (stainless steel wire) is connected with the positive pole and is positioned manually on the skin of the animal. The current range is 1-20 microamperes. The current intensity must be chosen individually for every snail. It has to exert the full contraction of stimulated tentacle and the hardly visible contraction of a contralateral one. After a withdrawal the tentacle begins to protrude. At a certain moment an eye appears on the tip of the tentacle. This appearance is clearly visible. An experimenter can record the time interval between the touching of the tentacle with the electrode and the appearance of the eye. This time interval was called the tentacle reflex time. It is interesting to mention that a histogram of tentacle reflex time is asymmetrical and that the distribution is not normal. The left side is short, and the right side resembles a tail (this shape of the histogram was found both in Helix and in Achatina). Any influences move the histogram along the time axis or changes its shape. The tentacle withdrawal reflex may be used as an objective and quantitative criterion of any influences on the central nervous system. A component of the phototactic behavior may be regarded as defensive behavior. Snails prefer darkness, and this preference can be quantitatively estimated in a special chamber, which is designed in the following way. Half of the chamber is covered with a lightproof cover, and the second part of the chamber receives light from an electric bulb containing a heat filter. An animal or a group of animals at the beginning of the experiment is situated in the middle of the bright part of the chamber. Every 30 minutes the number of animals that crawl into the dark compartment is counted, over 4-hour period. It was shown that if both parts of the chamber are equally illuminated, the animals are distributed equally in the whole chamber, meaning that there is no preference for any part of the chamber. The redistribution of more than 85% of animals into the dark compartment, as was shown, points to a strong dark preference. As the percentage of animals moving to the dark compartment was found to be exponentially fitted, so regression analysis may be used. It is interesting to note that even after removal of the eye such preferences remained (Stepanov and Sidelnikov, unpublished data). This finding suggests that extraretinal receptors found in the mantle and skin of snails mediates this behavior. Feeding Behavior Terrestrial snails are usually generalized herbivores faced with the problem of locating food and assessing its nutritional value or possible harm. Foraging snails


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detect food odors from a distance of 20-50 cm via olfactory receptors located at their tentacles. Helix inverts its lips and protracts small tentacles (rhinofores) to taste the food. In the feeding behavior of snails, appetitive and consummatory phases are easily observed. The appetitive phase contains active locomotion in the direction of food and light touches by either pair of tentacles. We consider the behavior after the contact of rhinophores with food as the consummatory phase, which contains lifting of the head, contact with food by the lips, and rhythmic buccal mass movements, resulting in scraping the food. The final decision to begin feeding is based on the chemosensory stimulation received at the lips. It will be described in the following sections that Helix is capable of associating the odor of food with a subsequent electric shock. It is possible to record the duration of the appetitive phase and the latency of the consummatory phase in the setup described above (Figure 4.1). A piece of food (carrot, cabbage, apple, etc.) weighing 2-4 g was attached to the stainless steel wire, which may be used as an electrode. The food was presented to the animal's mouth at a distance of 2-5 mm. The hungry snail captures the piece of the food with its mouth and pulls the food from the needle. The time interval from the presentation of food to obtaining it from the needle can be measured by an experimenter. It was found during a session consisting of 5-10 presentations of food that the above-mentioned time interval decreased exponentially. Experimental data are well fitted with the mathematical model, which describes the reaction of a firstorder linear system to a steplike external stimulus (Stepanov 1983):

where X is a number of a trial, Y is a time interval, B3 is the coefficient reflecting an initial level of the feeding status, B4 is the coefficient reflecting the asymptotic level of the feeding behavior, and B2 is the coefficient reflecting the velocity of the system transition from initial level to asymptotic one. The coefficient B3 reflects the food preference. The less B3, the more preferable this kind of food is to the animal. In Helix pomatia, B3 for carrot was less than for cabbage. Figure 4.2 shows the performance of snails given 10 trials with an intertrial interval of 6-10 minutes. Curve 1 represents the time needed to capture a piece of cabbage. The coefficients of the mathematical model are as follows: B2 = 0.62, B3 = 87, B4 = 18. Curve 2 represents the time needed to capture a piece of carrot. The coefficients of the mathematical model are as follows: B2= 0.60, B3= 46, B4= 16. The coefficients B3 differ significantly (p < 0.001), indicating that B3 can be used to reflect food preference. This result confirmed the experimental observations on snail feeding behavior in a terrarium, which indicated that animals preferred to eat carrot. Later it was shown that the feeding behavior of Achatina fulica also fits with the abovementioned mathematical model (Stepanov, Nikolaev, and Borodkin 1991). During the consummatory phase of feeding behavior, defensive behavior changes; 15 minutes after the first chewing movement, a generalized defensive

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Figure 4.2 Performance of snails in a food capture task. Curve 1 represents the time needed to capture a piece of cabbage. Curve 2 represents the time needed to capture a piece of carrot.

reaction can be elicited by a weaker stimulus (Shevelkin 1989). It is interesting to mention that the neurochemical basis of feeding behavior of snails and mammals can be very close. For example, a peptide pentagastrin stimulated snail's feeding behavior (Sudakov and Kozyrev 1986). Exploratory behavior It is difficult to distinguish between the appetitive phase of feeding, during which the snail locomotes actively, and exploratory locomotion. During all types of locomotory activity, even satiated snails contact the substrate with lips and tentacles, occasionally trying to eat it. It should be noted that satiated snails do not usually locomote, but rather stay immobile in shells. An interesting case of exploratory behavior can be easily seen in response to a weak repeated tactile stimulation. Instead of withdrawal, the snail turns its head to the stimulated place and tries to actively contact the stimulating object. High humidity and water elicit a high level of locomotory activity in snails, which can be regarded as exploratory


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at least in part. Sometimes they travel quite a long distance in such conditions. Sexual Behavior The sexual behavior of Helix is far from dull. Courtship in these hermaphroditic snails lasts for about 8 hours and consists of 12 distinct stages (Lind 1976). The highlight of courtship is the reciprocal shooting of calcareous "love darts" from and into both members of the mating pair. A short time later the penises are everted and reciprocal copulation ensues. Prior to dart shooting, the degree of eversion can be reliably classified into six successive stages from its size and shape (Adamo and Chase 1988). A group of neurons in the metacerebral lobe of the cerebral ganglion was identified as responsible for releasing the love dart and everting the penis (Chase 1986). Normally, in the behavioral hierarchy of Helix, avoidance behavior suppresses feeding and exploratory locomotion. Recently it has been shown that courtship can suppress feeding (Adamo and Chase 1991). Locomotion is suppressed during copulation, but avoidance responses are not suppressed significantly. Information about the behavioral repertoire of Helix is necessary for investigation of behavioral plasticity, which will be described in the following sections. NONASSOCIATIVE MODIFICATIONS OF BEHAVIOR Habituation and sensitization of a behavioral response are two forms of modification of behavior that, by definition, are not associative. Habituation refers to a decrement of responsiveness due to repetition of a stimulus. Habituation should be distinguished from fatigue, when the response is not restored by the presentation of a novel stimulus. Sensitization is a form of nonassociative learning in which an animal learns to strengthen its defensive reflexes and to respond with escape to a variety of previously weak or neutral stimuli after it has been noncontingently exposed to potentially dangerous or noxious stimuli. In behavioral experiments carried out in a specially designed snail setup (Figure 4.1), we investigated plastic properties of the avoidance reaction in Helix. Repeated tactile skin stimulation of moderate intensity elicits certain dynamics of avoidance response amplitude. Normally, the response to the second and third stimuli was greater than the first response in a series. After this short period of amplitude increase, which can be considered to be sensitization, a decrease of response amplitude to successive repeated stimuli was observed. We tried to establish whether these changes in amplitude are plastic, by using the parametric features described by Thompson and Spencer (1966). Sixty-five adult animals were used for these experiments, and the results are similar for 85%-90% of snails. In 10%-15% of cases the results differed from the average, but we presume that this difference, which sometimes disappeared during the experimental session, is due to some uncontrolled changes by the experimenter

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in the functional state of the animal. In the majority of the experiments the decrement of the response amplitude was more rapid if: (1) intensity of the stimuli is increased; (2) interstimulus interval is decreased. Extrastimulus (3) or a rest period (4) restores the amplitude. Effectiveness of the extrastimulus declines with several applications (5). Response amplitude decreases more rapidly during the successive series in one experimental session (6). Change of the stimulation site of more than 2 mm led to partial restoration of response to repeated stimulation (7). Decrease of the interstimulus interval led to a slower decrement of the response amplitude (8). A necessity for amplitude restoration (to a given value), a period of rest is significantly increased if stimulation is continued after full extinction of the response. Therefore, all parametric features of habituation are fulfilled in Helix, and we can call the decrement in question habituation. Habituation persisted throughout the experimental session, but never was seen 2-3 days after the elaboration. A noxious electrical shock applied to the foot of Helix, evokes not only restoration of habituated response of tentacles withdrawal and pneumostome closure but also an increase above the initial value in some cases (Balaban 1983). This suggests that sensitization can be independent from habituation. ENVIRONMENTAL CONDITIONING It was shown in a marine snail Aplysia that sensitization persists from minutes to weeks, depending on the number and intensity of the sensitizing stimuli (Pinsker, Hening, Carew, and Kandel 1973). In the Pavlovian school the test for existence of environmental conditioning is obligatory, especially in cases when nonassociative processes are studied (Kupalov, Voevodina, and Volkova 1964; Pavlov 1927). Environmental (context) conditioning is a form of associative learning in which the reinforcing stimulus is made contingent on environmental properties. Several series of experiments aimed to investigation of existence of long-term sensitization and environmental conditioning in terrestrial snails are described. In the experimental setup, the snail was tethered by its shell in a manner allowing it to crawl on a ball that rotated freely in a 0.01% solution of NaCl (Figure 4.1). The ball was laced with bare stainless steel wire to complete an electrical circuit between the animal's foot and a carbon electrode placed in the water. Electric shock was delivered using a 1-10 mA, 0.5 s current through a macroelectrode applied manually to the dorsal surface of the snail's foot. Punctate mechanical stimuli were applied with calibrated von Frey hairs, permitting delivery of pressures ranging from 6 to 68 g/mm2. The snails were injected with 5,7-DHT (dihydroxytryptamine creatinine sulphate from Sigma) in a volume of 0.1 ml, in doses of 15 mg/kg dissolved in physiological solutions containing 1 mg/ml ascorbic acid as antioxidant. Control animals received an injection of the same volume of vehicle. After several preliminary series, the behavioral response, the intensity (25


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gm/mm), and the location of tactile stimulation were chosen. Tactile stimulation of the middle part of the foot appeared to be sensitive to previously applied noxious stimuli, and it was chosen for investigation of long-term effects. Each snail was exposed for 20 minutes daily to the experimental setup. Only snails from two experimental groups received 2 electrical shocks per day for 5 days. The shocks were explicitly unpaired with testing tactile stimulation. Three days after completion of sensitizing treatment (animals were fed during 3-day periods of rest), the responsiveness to the same tactile stimuli was compared in control and experimental snails. An experimenter blind to the experimental histories of animals applied the tactile stimulus to the skin of the foot and measured the withdrawal amplitude in percentages of the maximal withdrawal, taken as 100%. Testing was performed in the experimental setup and also in the nonreinforced environment, on the glass lid of a terrarium in which the animals were kept between sessions. To reduce possible effects of recent handling, the test was administered no sooner than 5 minutes after the subjects had been placed in the environment. Only actively locomoting animals were tested. Five tests per day for 2 to 3 days were scored for each animal. No shocks were delivered during the test sessions. Six days after the completion of sensitizing treatment and testing sessions, 4 control and 4 experimental snails were injected with 5,7-DHT, while others were injected with vehicle. Three days after the injection, a second testing session was performed using the same procedure as described before. Before noxious reinforcement, no significant difference in amplitudes of tentacle withdrawal to the testing tactile stimuli existed. Three days after a 5-day session, during which experimental snails received 2 shocks per day, testing of responsiveness performed in the setup used for sensitizing revealed a significant increase of the median response amplitude in sensitized animals. The difference between control groups and the difference between sensitized groups of animals were not significant. The amplitude of withdrawal was greater in the context previously paired with the shock than the response of the snails that did not receive the shock and were tested in the same setup. Our next step was to compare responses of control and shocked snails in another environment. Similar results were obtained with the same animals on the glass lid of the terrarium in which animals were maintained continuously between training sessions. Testing was carried out for three days, alternating with testing in the experimental setup. Besides a change in the surface texture, intensity of light was lower, and the snail was not fixed by its shell during testing on the glass. No significant difference in responsiveness was found between control groups or experimental groups. In fact, snails displayed a heightened defensive reaction only in the environment that had a history of pairings with shock. This outcome is consistent with the assumption that the snails can differentiate the environment in which shocks were scheduled to occur. The specificity of that enhancement is extremely important because it allows us to rule out a sensitizing effect of the shock as the sole result of sensitization training.

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As to the effect of 5,7-DHT injection, it was shown previously in Helix that after 5,7-DHT treatment (ablating the serotonergic neurons) both the sensitization of the withdrawal reaction and associative aversive conditioning are impaired (Balaban, Vehovszky, Maksimova, and Zakharov 1987). The feeding behavior of 5,7-DHTinjected intact animals was visually normal, as were the electrophysiological responses of the investigated neurons to single feeding and noxious stimuli. This is indicative of the fact that the absence of associative learning of this type is not due to changes of responsiveness of the neurons taking part in the feeding and aversive behavior, respectively. Further, 5,7-DHT treatment after elaboration of aversive conditioning in Helix does not impair the conditioned responses (Balaban et al. 1987). This result suggests that 5-HT-containing cells participate in associative learning during the consolidation phase of the conditioned reflex but are not necessary during its reproduction. In the present work we tested the possibility of the involvement of 5-HTcontaining neurons, which modulate the network underlying avoidance responses (Zakharov and Balaban 1991), in environmental conditioning. Injection of 5,7DHT led to disappearance of the effects of training. Only responses of vehicleinjected sensitized snails differed significantly from responses of snails from other groups in both environments. The difference between vehicle-injected sensitized snails and 5,7-DHT-injected snails also was significant. This result suggests that 5HT-ergic neurons are necessary for the reproduction and/or maintenance of environmental conditioning. ASSOCIATIVE MODIFICATIONS IN BEHAVIOR The history of attempts to elaborate conditioned responses in snails started with the experiments of E. Thompson (1917) using the pond snail Physa gyrina. He associated tactile stimulation of foot with food presentation, and after 250 paired trials in 2 days tactile CS elicited feeding responses in 39.6% of cases compared to 3.3% before conditioning. The changes were maintained for 4 days, and no controls with nonpaired presentation or differential conditioning were made. In Russia, experiments in Physa acuta were carried out by V. Sokolov (1959), who paired light with water application of noxious 0.2% KC1 solution. Conditioned responses (snails moved to a dark compartment) were noted after 8 paired presentations and became frequent after 30 trials. However, this reflex was not stable, and fast extinction was seen. The first report on conditioning in terrestrial snails was published in 1976. The pneumostome closure evoked by strong noxious stimuli was used as an unconditioned response in these experiments (Litvinov, Maximova, Balaban, and Masinovsky 1976). As the conditioned stimuli, weak noxious stimuli were used that normally do not evoke pneumostome closure: tactile stimulation, local heating of the foot epithelium, or tapping on the shell. Elaboration was slow, and more than 100 trials were necessary for conditioning. However, the conditioned stimulus


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(CS) used was of the same modality as the reinforcement; therefore, the question about nonassociative sensitization as the mechanism of observed changes in behavior was not elucidated. Pavlov wrote that in principle any stimulus initiating any response may serve as the conditioned stimulus (Pavlov 1951). The only requirement is that this stimulus should not initiate the same reflex that is initiated by the unconditioned stimulus (UCS). In the experiments of O. A. Maksimova (Maksimova 1979), a tactile stimulus was used as the CS, and food as the UCS. An animal had to turn to the right when CS was delivered in order to receive a piece of food. Before learning, the conditioned stimulus elicited only withdrawal reactions. After 100-150 paired trials, snails began to react to tactile stimulus with feeding behavior (details are published in a book: Maksimova and Balaban 1983), turning to the right side, where they usually found food. Conditioning with food reinforcement is easy to elaborate, but such reinforcement cannot be given on a preparation suitable for intracellular investigation. Therefore, another behavioral approach was chosen. Food-Aversion Conditioning The primary purpose of behavioral experiments is to generate rapid and obvious modification in motor responses, reproducible in neurophysiological preparation, and to demonstrate specificity of elaborated behavioral modifications for the pairing of conditioned and unconditioned stimuli. The withdrawal reaction as an unconditioned response meets these criteria, and the underlying neuronal circuitry has already been investigated in Helix (Balaban 1983; Zakharov and Balaban 1991). Aversive learning appeared to be one of the most suitable paradigms for investigation of learning in snails, because it usually concerns two competitive behavioral acts, one of which changes dramatically because of the pairing of two stimuli. To be suitable for neurophysiological investigation, learning should occur rapidly, and as in a study on the carnivorous mollusc Pleurobranchaea (Davis and Jillette 1978; Mpitsos and Collins 1978), we reinforced food presentation by strong electric shock, which evoked generalized withdrawal reaction of the snail (Maksimova and Balaban 1983). A similar procedure was used in the experiments of Stepanov and Lokhov (1986). Snails chosen for experiments were deprived of food for a week prior to conditioning. A day before conditioning, the snails were tested for their feeding behavior. Subjects who refused food were excluded from the experiments. During 5 days, 1 session per day was performed with 10 trials per session. When a food piece (carrot, cabbage, etc.) is offered to an animal at a distance of 4-5 mm from the mouth, the snail begins to search for the food (the appetitive phase of feeding behavior). It lowers its optic tentacles and tastes the food. Then the animal opens its mouth, captures the food piece, and pulls it away from the needle (consummatory phase). The time interval between the presentation of the food and a hungry snails removing it from the needle is 20-50 sec. This time never exceeds 1 minute

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in active animals. The reinforcement consists in turning on the current when the animal captures the food piece. The intensity of the current is selected in such a way as to evoke the withdrawal of tentacles and a half of the anterior portion of the foot. The pneumostome often does not react to this stimulation. It is necessary to use a moderate current intensity to avoid a generalized escape reaction. In these conditions every animal begins to avoid the food at the end of the first session. The criterion of the conditioned reaction in a trial was food avoidance for 3 minutes after food presentation. The food avoidance behavior manifests itself in different ways. At first, a snail ceases to lower its optic tentacles. Then the animal raises the most anterior part of its foot up to prevent contact of its lips with the food. Further, the snail withdraws both pairs of tentacles and the anterior part of the body. Finally, the animal crawls away from the needle with the food after the first contact with it. In other words, a fully conditioned animal behaves as a satiated one. It is important to emphasize that an experimenter can observe and estimate the individual conditioning of every subject. It is enough for this purpose to compute a percentage of conditioned reactions (CR) as a number of food refusals during a session divided by 10 (because 10 trials per a session are used) and multiplied by 100. This percentage increases from the first session to the fifth session. Properties of Conditioning and Two-Way Conditioning Intensity of Reward In full accordance with Pavlov's (1927) data, intensity of reward in our experiments had great influence on speed of elaboration of conditioned reflex. In a series of experiments in which we used a weak reinforcement, the dynamics of elaboration were quite different. We consider the reinforcement as weak if it elicits only tentacles and head withdrawal, while strong reinforcement, used in some experiments, elicited complete withdrawal of the body in the shell and release of mucus. It was possible to deliver up to 20 trials per day to one snail if a weak reinforcement was used. In all other respects the experiments were similar to experiments with a strong reinforcement. In the cases when the snail did not take food for 150 sec, no reinforcement was given, thus encouraging the snail not to respond. Unpaired control received the same number of food presentations and reinforcements. It appeared that usage of a weak reinforcement requires about 60-100 paired trials for exceeding the 60% level of conditioned responses, and only 5-15 trials when a strong reinforcement was used. But in both cases all stimuli should not be given in one session (day); no less than 3 days should pass from the beginning of the experiment. Optimal timing is 5-8 days with 1-2 paired trials for experiments with strong reinforcement, and 10-20 trials for experiments with weak reinforcement. It means that, independently of intensity of reward some optimal timing exists. Retention of Conditioned Reaction In a major part of learning experiments in snails, we tested responsiveness


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during the first week after the end of a learning session that lasted 5-10 days. Average percentage of aversive reactions (withdrawal and refusal) during the first week was 85%-90%. Measuring the level of responses in trained snails 2, 4 and 6 weeks after the training showed that even after 6 weeks the percentage of conditioned responses was about 60% (Maksimova and Balaban 1983). This result was consistent throughout several independent series of experiments in 28 snails. Therefore, the duration of behavioral changes is long enough to consider it as a long-term modification of behavior. Differential Conditioning Besides employing unpaired controls, which was conventional for all experiments, in some series we used a different odor (or type of food), which was presented without the reinforcement. At first, when the nonreinforced odor was presented, a conditioned response can be seen in about 50% of the presentations. Very quickly, however, this percentage decreased to 10%-15%. An example is given in Figure 4.3 of such an experiment in which pneumostome closure was monitored using a light-beam and a photodiode. In this experiment the presentation of watermelon odor (cotton was soaked in watermelon juice and presented at a distance of 5 mm from the tentacles) before pairing did not evoke any withdrawal response (upper trace, Figure 4.3). After 10-20 pairings with a moderately intense noxious stimulus, only a transient response is seen (second trace, Figure 4.3). After several hundred pairings the conditioned withdrawal response to food in hungry snails is long lasting and stable (third trace, Figure 4.3), but presentation of an unpaired odor (carrot) evokes no response (lower trace, Figure 4.3). Differential conditioned response confirms specificity of the elaborated behavioral changes. Extinction and Spontaneous Recovery Active extinction of conditioned aversion response to food is possible in one day. In a series of experiments, 5 conditioned snails were presented with food offered at small intervals (3-5 min). After 30 presentations the number of aversive responses decreased to 50%, and continuation of presentation of food led to zero aversive reactions. It should be remembered that the snails are hungry and feeding motivation is high. Presentation of the same food to the same animals 24 hours after the extinction trials revealed spontaneous recovery of the conditioned response, but the rate of extinction was higher. At 72 hours after the second extinction series, the level of conditioned responses to the first 10 stimuli was even higher than 24 hours after learning (about 70%), and extinction was rapid. These results are in full conformity with results obtained in vertebrates (Pavlov 1927) and allows us to consider the observed modifications of behavior as classical conditioning. Two-way Conditioning One of the most interesting paradigms introduced in science by Pavlov's student

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Figure 4.3 Pneumostome closure responses in the snail during paired presentation of watermelon odor (tilled dot) and moderately intense noxious stimulus (triangle, upper trace) across training trials. The upward shift in the trace represents pneumostome closure. The unfilled dot represents the presentation of carrot. Carrot odor was not paired with the noxious stimulus.


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E. Asratjan (1970) is "two-way conditioning." The operational definition of twoway conditioning is clear and simple: after elaboration of a conditioned feeding response in dogs to light by repeated contingent presentation of light and feeding reinforcement, presentation of food only elicits before normal feeding an anticipation of light stimulus—eye-blinking and turning of the head to the light source. In our opinion, this notion of formation of associative connection not only between the conditioned stimulus (CS) preceding the unconditioned stimulus (UCS) but in the reverse direction as well undermines a part of Pavlovian theory that presumes that a connection is formed only when the CS precedes the UCS. Discussion of this point was taboo in Russian literature until recently, but the phenomenon of two-way conditioning was abundantly described in the literature and is considered a necessary criterion for an animal capable of associative learning. Therefore, we carried out several series of experiments on the behavioral and cellular level aimed at demonstrating the possibility of the formation oftwo-way connections in snails. In fact, all experimentation turned to testing what will happen in the animal with elaborated aversive conditioning, if a noxious stimulus (similar to one used as a reinforcement, but weaker) is applied. It appeared that weak noxious (tactile) stimuli, which before learning evoked no response or withdrawal, after elaboration of aversive conditioning elicited activation of exploratory activity and feeding behavior. Such behavioral response never was observed before learning. In responses of identified cells involved in feeding the absence of response to noxious stimuli changed to activation of the pattern accompanying feeding (Balaban, Maksimova, and Galanina 1985). These results suggest that two-way conditioning can be formed in snails. Results presented in this section confirm the suggestion that Helix can associate sensory inputs and adaptively modify its behavior for a period of time substantially greater than the time needed for protein turnover, thus suggesting involvement of genomes. Mathematical Model of Conditioning During a search for an adequate mathematical model of conditioning dynamics, the above-mentioned reaction of the first-order linear system to the stepwise external stimulus has been found to be good enough to fit all experimental data (Stepanov 1983):

This model is one of the best initial approximations of the real physiological process of learning. All regression coefficients may be interpreted in physiological terms. B3 is the initial level before the beginning of conditioning, and B4 is an asymptotic level of a conditioned state. B2 is the velocity of an achievement of the asymptotic level. It is necessary to pay attention to B3. Nearly all previous mathematical models of learning were based on the probability theory approach. It is

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well known that probability values must lie in the interval between 0 and 1. However, many experimental data could not be significantly fitted if B3 had to be equal to zero. This model allows B3 to receive any value—positive, zero, or negative. The negative values of B3 point to an inhibitory influence on the elaboration of the conditioned reflex, and the positive values provide information about facilitation of learning taking place prior to conditioning itself. This method not only gives an opportunity to calculate coefficient values but also computes a confidence interval for every coefficient (Stepanov 1983). Moreover, if an experimenter uses a group of animals he can calculate the variance of measurement error and, with the help of Fisher's F-criterion, be convinced of statistically significant fitting of experimental data. Food avoidance conditioned reflex was found to be fitted significantly by this model. The analysis of the three coefficients gave additional information about the learning process. For example, if a preferred food was chosen as a conditioned stimulus, then B3 values were negative. If non-preferred food was used as a conditioned stimulus, then B3 values were close to zero. B3 corresponded well to real behavior of the animal. If another kind of food was used as the differential stimulus (for example, cabbage as CS carrot as DS), then the asymptotic level (B4) was greater in comparison with these coefficients than in animals conditioned without the differential stimulus exposure. Figure 4.4 shows an application of the model to this situation. The coefficients of the mathematical model in the first curve are: B2 = 0.078, B3= 0, B4= 98; for the second, B2 = 0.078, B3= 0, B4 = 91; and for the third, B2 = 0.049, B = -32, B4 = 93. The coefficients B3 for curves 1 and 2 differ significantly (p < 0.05). The coefficients B2 and B3 of curve 3 differ significantly from the same coefficients of the curves 1 and 2 (p < 0.05). It is interesting to note that during the first session there were food refusals during differential stimulus exposure. However, the animal did not refuse DS at all to the end of conditioning (5th session) and some weeks later. This result indicates the continuation of memory formation after the end of conditioning itself. This may be a sign of a biochemical process involvement in the memory consolidation stage. This process strengthens only the temporal connections between CS and US. At the same time the temporal connection between DS and US is inhibited and decreased. This model allows us to estimate the level of memory retention some time after original learning. As a rule, food avoidance of CS is maintained up to one month. When a second conditioning experiment was undertaken with the same CS, the coefficient B3 received positive values. This value can be used as a quantitative parameter of conditioned reflex maintenance and stability. Conditioned Tentacle Withdrawal It is necessary to mention that food avoidance conditioning strongly depends on the level of satiation. An experimenter must carefully control all details of food consumption. As mentioned above, the initial level of feeding motivation must be


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Figure 4.4 The dynamics of food avoidance learning in Helix fitted to the mathematical model. 1: conditioned stimulus—carrot, differentiated stimulus—cabbage; 2: conditioned stimulus—cabbage, no differentiated stimulus; 3: conditioned stimulus—carrot, no differentiated stimulus.

created by 7-10 days of food deprivation. During conditioning the animal should not receive any food at all; the differential stimulus becomes more preferred because it allows animals to eat the food used as DS. During any testing for memory consolidation and retention, animals must receive restricted amounts of food. Another reflex, which is not dependent on food motivation, is conditioned tentacle withdrawal. An interesting version of this kind of learning, used first in the lab of G. Kerkut (Emson, Walker, and Kerkut 1971), was designed by G. Christoffersen (Christoffersen 1981). He described the main properties of this reflex. In our experiments, a snail's optic tentacles are extended when an animal is crawling on the surface of a ball. The first touch of the optic tentacle tip with the wire electrode initiates a rapid and complete tentacle withdrawal into the body cavity. Several seconds later the tentacle protrudes and at some moment the eye, clearly visible as a black dot, appears on the tentacle's upper surface. Each time when the eye appears on the tentacle surface an experimenter touches the tentacle

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with the electrode. As the animal tends to hold its tentacle contracted longer and longer, the time of the response becomes larger and larger, and the number of electric shocks becomes less and less. A session lasts 80, minutes and the number of electric shocks during every 4 minutes is counted. The exponential decrease of the number of stimulations during every session was found to fit significantly with the regression model (Stepanov, Kuntzevich, and Lokhov 1989). For this reflex the coefficient B3 means the initial value of stimulation prior to the beginning of conditioning. In other words, it means the reflex time before conditioning. B4 is the asymptotic level of the reflex time, and B2 is the velocity of a transition from the initial to the asymptotic level; the elaboration of the conditioned reflex takes place during every session. The memory fixation displays itself in a lowering down of curves from session to session. This was due to a decrease of B3 and sometimes B4. Figure 4.5 presents an application of the model to this situation. The intersession intervals were 24, 96, and 192 hours between every consecutive session. Curve 1 represents the first session. The regression coefficients are as follows: B2 = 0.28, B3 = 34.9, B4 = 8.3. The regression coefficients of session 2 are: B2= 0.29, B3 = 32.3, B4= 6.5; of session 3: B2= 0.17, B3 = 14.6, B4= 5.0; and of session 4: B2= 0.20, B3 = 16.7, B4= 3.5. The coefficients B2 of the second and the third curve and of the third and the fourth curves differ significantly (p < 0.001). The coefficients B3 of the second and the third curves and of the third and the fourth curves differ significantly (p < 0.001). The coefficients B4 of the first and the second curves and of the second and the third curves and of the third and the fourth curves differ significantly (p < 0.01, p < 0.05, p < 0.02). The advantage of the tentacle conditioned reflex is an opportunity to analyze the short-term memory formation during a session and the long-term changes reflected during an intersession interval. It is important to note that the length of the intersession interval is a crucial parameter for long-term memory formation. Christoffersen used intervals of 24, 96, and 192 hours between consecutive sessions. In our experiments, when sessions were run every day, only the short-term memory formation during every session could be observed (Stepanov et al. 1989). The memory consolidation during the intersession interval was absent, and learning curves were superimposed on each other. This result suggests that the memory consolidation needs rest and the absence of neuronal activity in the reflex arc. It is interesting that Pavlov himself used 2-3 day intervals between sessions of classical salivary feeding reflex elaboration in dogs (Pavlov 1951). That may be one of the very important features of memory "fixation" process. The retention of tentacle conditioned reflex lasts more than 3 weeks. As in the case of food avoidance, the coefficient B3 gives a quantitative parameter for reflex retention. There is one feature inherent to this reflex. If current intensity was chosen in the range that was enough to excite unilateral tentacle withdrawal only, then the second tentacle could be used as a control. All tentacle learning dynamics during a 20-minute session were found to be very similar to one another. This feature enlarges the opportunities to use this reflex.


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Figure 4.5 An application of the model to the instrumental conditioning of left tentacle withdrawal reflex in Helix after 1,2,3, or 4 training sessions.

HUMORAL COMPONENT OF LEARNING In spite of a large number of investigations, the problem of the role of chemical substances in the maintenance of memory and, consequently, memory transfer has not been solved (Mitchell, Beaton, and Bradley 1975). An investigation of the existence of a humoral component of conditioned reflexes in the snail Helix lucorum was therefore undertaken (Stepanov, Lokhov, Satarov, Kuntsevich, and Vartanian 1987a, 1987b). It was presumed that this problem might be easier to solve using mollusks because there is the possibility of linking behavioral experiments with neuronal ones performed on the identified neurons. An experiment of that kind was undertaken with the left and right tentacle withdrawal conditioned reflex. The idea of the experiment was that any condi-

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tioned animal at the same time is both donor and recipient. Five days after the last left tentacle conditioning session, the right tentacle first session was performed. The right tentacle learning curve was found to be lower in comparison with the first session curve of the left tentacle. The facilitation of contralateral tentacle learning could be due to the action of some chemical substance secreted into the blood during the first tentacle conditioning. A facilitation of learning was also observed in experiments in which donors were conditioned as usual and naive recipients were conditioned after being injected with trained donors' blood (Stepanov 1991). Their blood in the volume of 200-300 mcl was collected after every session. The part of the snail's foot, hidden under the shell in the normal position of the animal, was found to be nearly insensible to the insertion of a surgical needle, if the shell is turned along its longitudinal axis to the right side until this foot, which is lighter, comes out from under the shell. The needle can be inserted at a 45° angle into the left side of the foot. A normal snail partially, and only weakly, withdraws and very soon (2-3 minutes later) begins to crawl. A loss of 200-300 mcl of blood does not hurt the animal. This method gives us an opportunity to sample some blood after every session (intersession intervals were 2 and 4 days). All samples were double blind labelled, and 200 mcl was at once injected into naive recipients, using the same technique of injection. Recipients had their first learning session 24 hours later. The donors' blood, taken after the first, second, or third session, facilitated the first learning session of recipients. Their learning curves were displaced down in comparison with the first learning session curve of the donors. However, there was an important difference: the blood samples taken after the first session inhibited memory consolidation. This inhibition manifested itself in the raising of the recipients' second learning curve, so that both curves practically coincided. Figure 4.6 presents these data. The regression coefficients are as follows: Curve 1: B2= 0.18, B3 = 15.8, B4 = 3.5; curve 2: B2= 0.20, B3 = 11.3, B4 = 2.2; curve 3: B2= 0.14, B3 = 12.7, B4 = 3.6. The coefficients B3 and B4 for the first and the second curves differ significantly (p < 0.001 and p < 0.1 accordingly). The coefficients B2 and B4 for the second and the third curves differ significantly (p < 0.001). The blood sampled after the fourth donors' learning session, however, exerted only strong facilitation. Their second-session learning curve remained in the same position and did not rise at all. These data are presented in Figure 4.7. The regression coefficients are as follows: curve 1: B2= 0.18, B3 = 15.8, B4= 3.5; curve 2, B2 = 0.27, B3 = 9.7, B4 = 2.7; curve 3, B2 = 0.20, B3 = 9.2, B4 = 2.6. The coefficients B2 and B3 for the first and second curves differ significantly (p < 0.001). The coefficients B2 for the second and third curves differ significantly (p < 0.001), but others did not differ at all. Curves 2 and 3 nearly coincide. In this series of transfer experiments, the first session of the contralateral tentacle of recipients was also facilitated. In the control group of recipients, naive donors' blood did not change the dynamics of learning. These results were interpreted as proof of the existence of the secretion of some chemical substances into the blood


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Figure 4.6 The influence of blood, sampled from donors after their first left tentacle withdrawal reflex learning session, on the learning of naive recipients in the snail Helix. Curve 1: the learning curve of the donors' first session. Curve 2: the learning curve of the recipients' first session. Curve 3: the learning curve of the recipients' second session.

during learning. The inhibitory activity of samples taken after the first session suggests the inhibitory influence of the conditioning session itself on memory consolidation. Moreover, it may be assumed that a brain, being a stable system, tries to maintain its current state. In other words, at the beginning of conditioning, a brain is "struggling" against the influence of conditioning. However, as the brain is an adaptive system, if conditioning lasts long enough, the brain changes its state to the conditioned one. These experiments clearly show that the humoral component related to learning does really exist. However, there exists a possibility for facilitation of learning, but not a direct memory transfer.

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Figure 4.7 The influence of blood, sampled from donors after their fourth left tentacle withdrawal reflex learning session, on the learning of naive recipients in the snail Helix. Curve 1: the learning curve of the donors' first session. Curve 2: the learning curve of the recipients' first session. Curve 3: the learning curve of the recipients' second session

MOTIVATION, REWARD, AND LEARNING One of the most important questions that arises in the course of the analysis of learning in invertebrates is, Is it possible for the snail to consider a certain situation as emotionally positive or negative? In other words, can the snail feel pleasure? It is evident that the human experimenter can never place himself in the snail's brain, but in neurobiology there exists an experimental technique—the self-stimulation method—that can provide an answer to whether the situation is pleasant for the animal or causes distress.


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In the phenomenon of self-stimulation, an animal receives direct electrical stimulation of the brain as a consequence of its operating a manipulandum. If the electrode is implanted in certain areas of the brain, the animal repeatedly selfstimulates (Olds 1958; Olds and Milner 1954). Since its discovery (Olds & Milner, 1954), numerous experiments have confirmed the phenomenon of self-stimulation in various vertebrate species (Olds 1977). Self-stimulation itself remains to be remains to be explained, however, by mechanisms at the cellular level, as well as its relation with learning and reward (Stellar and Stellar 1985). To develop a new approach to this problem, we investigated whether a snail, with its relatively simple and technically advantageous nervous system, will self stimulate. Freely moving animals with chronically implanted electrodes were used, and the rewarding properties of a contingent extracellular stimulation of certain cellular groups in semi-intact preparation were investigated. Sexually mature specimens of Helix aspersa (Marinus, Long Beach, Calif.) were anaesthetized with 0.012 mg succinylcholine chloride per gram of body weight dissolved in 60 mM MgCl2. An incision was made in the skin above the brain, and a single 20 um Teflon-coated platinum-iridium wire was inserted into the connective sheath overlying either the right mesocerebrum or the right parietal ganglion. Insulation was removed from the tip of the wire. A suture (monofilament nylon, 22 um) held the electrode in place. The other end of the wire was led out from the snail's mantle and soldered to a connector pin that was glued to the shell. The skin wound was sutured. The position of the electrode was verified weekly by X-ray photography. The precise location of the electrode was determined by dissection after the behavioral experimentation. The experiments were contained in 40-minute sessions, one per day. The snail was tethered by its shell in a manner allowing it to crawl on a ball that rotated freely in a 0.01% solution of NaCl (Figure 4.1). The ball was laced with bare stainless steel wire to complete an electrical circuit between the animal's foot and a carbon electrode placed in the water. To receive stimulation, the tethered snail was required to displace the end of a rod, thus closing a switch. Usually, the snail would first sense the rod with its tentacles, then raise its head to explore the rod with its lips and mouth, displacing the rod during exploration. Each session began with a 20-minute period without reinforcement, followed by a 20-minute period with reinforcement. An electrical timer automatically switched reinforcement conditions. The general activity of the snail was monitored using a light beam and a photocell. Experiments in semi-intact preparation were carried out using adult snails of two species: Helix aspersa and H. lucorum (Crimea population). Cellular map and functional significance of the identifiable cells is similar in these species. No significant difference in results was found in H. aspersa, which is active sexually all year. All animals were kept in an active state. Details of preparation are published elsewhere (Balaban and Chase 1990). To determine the rate of touching of the rod in the absence of reinforcement, snails were allowed free access to the rod in a 40-minute session prior to any stimulation. The animals were slightly more active in touching the rod during the

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beginning of a 40-minute session than at the end, but there was no significant departure from a sustained frequency of contact. When reinforcement was delivered to snails having electrodes implanted in the parietal ganglion (avoidance behavior neurons are located in the place of stimulation), there developed a strikingly consistent pattern of behavior. After one or two reinforcements, the snails remained active but appeared to avoid the rod. Although there was some recovery between sessions, the mean response rate fell to nearly zero by the end of each session with reinforcement. Quite different results were obtained when reinforcement was delivered via electrodes implanted in the mesocerebrum (sexual behavior cells are located in the place of stimulation). After just a few reinforcements, these snails began to contact the rod with increasing frequency. The magnitude of the effect was variable in different animals, but in no case was there a decline in response frequency such as was seen in the snails having parietal electrodes. Pooled results are shown in Figure 4.8 in terms of the average frequency of bar contacts. Each histogram shows the number of responses in 5-minute periods, expressed as a percentage of the mean number of responses recorded during all nonreinforced periods. One averaged score was taken for each snail for each 5 minutes. Panel A represents the performance of nonreinforced controls in 40minute sessions, showing the absence of any systematic change in the number of bar contacts during the course of test sessions prior to brain stimulation, in 7 snails. Panel B shows the results of self stimulation of parietal ganglion in 3 animals. Panel C shows self stimulation of mesocerebrum, in 7 snails. In B and C the data are from two consecutive 20-minute periods, the first without reinforcement, the second with reinforcement. The error bars show standard errors of the mean (SEM). The dotted line separates reinforced from nonreinforced periods. A comparison of changes during 40 minutes without reinforcement in two different groups of snails (electrodes in mesocerebrum and in parietal ganglia) shows a significant effect of reinforcement on the ongoing behavior. Neural substrates mediating reward, or reinforcement, have been identified in vertebrate brains using the self-stimulation procedure. The medial forebrain bundle at the level of the hypothalamus is the most effective site (Gallistel, Gomita, Yadin, and Campbell 1985). However, no complete neural circuits have been delineated, nor has it been possible to identify any individual neurons whose participation is essential. These difficulties have so far prevented any mechanistic explanation of self-stimulation at the cellular level. The identification of the mesocerebrum as a site of reward in snails offers the possibility of cellular studies because the neurones in this region are large (diameters up to 80 um) and easily accessible for intracellular investigation (Chase 1986). Interrelations between mesocerebral cells and parietal giants were investigated (Balaban and Chase 1990), and it was shown that stimulation of the mesocerebrum causes a suppression of spiking in the parietal command neurons in response to tactile stimulation of the skin. In addition to the effects observed at the level of command neurons, a second, independent control over withdrawal is brought about by the


Figure 4.8 Average frequency of bar contacts. Pooled results.

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inhibition of neurons that are capable of sensitizing the afferent excitation of the same parietal command neurons (Balaban and Chase 1990). Unfortunately, these data cannot be directly linked to emotionally positive effects exerted by the same mesocerebral cells. This impossibility is caused by the absence of a cellular hypothesis for emotional processes, by only a small overlap of behavioral physiology and cellular physiology. We hope that further investigation of emotionally dependent behaviors in animals with relatively simple behaviors and nervous systems accessible for cellular analysis will increase this overlap. The experiments reported here suggest the possibility that the snails learn the rewarding properties of electrical stimulation during the course of a session or a series of sessions. However, these data are not sufficient to demonstrate learning. Further, and more extensive, experimentation is required at this point. AGE-DEPENDENT CHANGES IN THE ABILITY TO LEARN Avoidance reflexes are one of the earliest forms of behavior to be formed in ontogeny. Developmental analysis of avoidance reflexes and underlying neuronal circuitry can be conveniently undertaken in mollusks, in which the nerve cells, participating in organization of these reflexes, have been identified (Carew 1989; Pawson and Chase 1984). Withdrawal reflexes of pneumostome closure and tentacle withdrawal in Helix provide an excellent system for the analysis of the neural mechanisms of learning and development, because these reflexes participate in associative and nonassociative learning (Balaban 1983; Maksimova and Balaban 1983 1984). To develop a suitable model for studying development at the neuronal level, we have used the approach of examining behavior in snails of different ages (Zakharov and Balaban 1987). Experiments were carried out in snails (Helix lucorum L.) of three age groups: snails of the first age group were taken from the nest and were under 1 month old; those of the second group were 4-6 months old, and in the third age group were adult snails (4-6 years). Snails of the first two groups were laboratory-reared. All animals were kept in an active state. Before learning sessions, the animals were deprived of food for 3-5 days. Age-dependent Changes in Avoidance Behavior Avoidance reactions in the adult terrestrial snail Helix can be evoked by tactile stimulation, local heating, vibrational stimuli, and light-off stimuli. Tactile stimuli were applied in all experiments at the base of the left ommatophore with frequency 1 per 20 seconds. Three consecutive zero responses were taken as the criterion for cessation of stimulation. A video recording was used for quantitative analysis. Snails from all groups responded with tentacle withdrawal to tactile stimulation. Amplitude of withdrawal depended on stimulus intensity. Repeated presentation of tactile stimulation of moderate intensity (1.3, 2.4, and 6 gm/mm2 for the three mentioned groups respectively) elicited changes in reaction amplitude with


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different dynamics in snails of different ages. Sensitization of response to the second and third stimuli in a series was characteristic of the adult animals (Zakharov and Balaban 1987). No sensitization in responses of snails under 1 month old was noted. The rate of response decrease was significantly greater in juvenile snails due to the absence of sensitization. An increase of responses to the second and third stimuli, similar to the increase in adults, was seen in 4-6-monthold snails in spite of the fact that their size was very close to the Group I animals. This result allows us to discard the suggestion that the huge difference in the size of snails in Groups I and III is the cause of the difference in response dynamics. Aversive Conditioning in Juvenile Snails Using the same methods of conditioning as described in the previous sections, the aversive conditioning paradigm was applied both to a group of hatchlings under 1 month old and to a group of 4-5-month-old snails. Before training sessions all animals exhibited feeding responses in 90%-100% of trials. After 9-20 pairings of the food with the noxious stimuli, the hungry snails aged 4-5 months refused to take the reinforced type of food in 90% of test trials, while the snails under 1 month old ate food in 90%-100% of trials, even after 20-24 reinforcements of food with electric shock. Unpaired control snails from either group exhibited feeding responses in 95%-100% of test trials. Thus, in the early stages of postnatal development, not only is sensitization absent from snail's behavior, but the capacity to acquire aversively conditioned responses is absent as well. The coincidence of these deficiencies in the behavioral repertoire of juvenile snails and in 5,7-DHT-treated adults allowed us to suggest that it is the difference in 5-HT levels in juvenile, as compared to adult, snails that explains the absence of sensitization and aversive conditioning. Developmental Changes in Serotonin Levels In order to look for a correlation between serotonin levels and behavioral performance, the central nervous system (CNS) of the snail was examined with the glyoxylic fluorescence histochemical technique modified for cryostat sectioned nervous tissue (Lindwall and Bjorklund 1974). Serially sectioned (40 um) wholeanimal preparations from juvenile snails and CNS preparations from adults were examined with a fluorescence microscope using a 405 nm light wavelength for excitation. In accordance with previously reported results for Helix (Sakharov 1974), 5-HTcontaining neurons were found in cerebral, pedal, visceral and right parietal ganglia of adult snails. However, in the CNS of newborn animals, 5-HTcontaining neurons were not found. It was thought that the cells in the newborn animals might be too small to be revealed by this technique, but in the CNS of the 4-month-old snails the 5-HT cells were readily seen in cerebral and pedal ganglia (but not in visceral and parietal ganglia), while the CNS size at this age is only

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slightly larger than in newborns: Maximal diameter of giant parietal cells ranged from 40 to 60 um in hatchlings, from 50 to 70 um in snails aged 4-6 months, and from 220 to 280 um in adults. The absence of 5-HT-containing neurons in parietal and visceral ganglia of 4-month-old snails points to the possibility of heterochronical development of a transmitter-producing neuronal system. It is assumed that the age dependence of behavioral and neuronal reactions may be due to some delay in the development of the serotonergic systems. Thus, in the early postnatal period in Helix, the 5-HT content is very low in the nervous system of the snail, suggesting dependence of the absence of sensitization and the inability to be aversively conditioned from the 5-HT levels. PERSPECTIVES FOR FUTURE RESEARCH Intracellular recording in the 1960s, molecular neurobiology in the 1970s, and the neurogenetical approach in the 1980s gave the impression that the problem of mechanisms of learning will be solved soon. In the 1990s, behavioral analysis is still crucial, and our knowledge about underlying learning mechanisms is meager. In spite of the fact that behavioral analysis of learning in snails is relatively easier, no significant advancement concerning mechanisms of associative learning has been made. Mostly mechanisms of synaptic plasticity underlying changes in synaptic effectivity were thoroughly investigated. Certainly, these mechanisms constitute a hardware of the nervous system adaptive functioning, but the software concerning the whole network is still unknown. What kind of progress can be anticipated in the "invertebrate learning" field? If we consider learning as an emergent property of the whole network, it is theoretically not possible to achieve any substantial result concerning this behavioral phenomenon by investigating one neuron. (This, of course, is true only if we speak about behavioral learning, not about "cellular plasticity," which includes relatively long-term (minutes) changes in functioning of the neuron more or less correlating with behavioral changes.) To our knowledge, a breakthrough in this field of neurobiology can be made with an optical recording of tens and hundreds of cells during learning. Such "optical imaging" of the brain allows us to monitor changes, not in several arbitrarily selected points, but in whole regions. It seems that today our understanding of network functioning is based more on mechanical models than on biological knowledge. Optical imaging, like other types of brain imaging, may help us to acquire the biological knowledge necessary for understanding the principles of the brain functioning as an integrative unit. ACKNOWLEDGMENTS The authors are indebted to their collaborators Drs. O. A Maksimova, I. S. Zakharov, A. P. Sidelnikov, M. I. Lokhov and N. I. Bravarenko for their participation and helpful criticism.


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REFERENCES Adamo, S. A., and Chase, R. 1988. Courtship and copulation in the terrestrial snail Helix aspersa. Can. J. Zool. 66:1446-1453. . 1991. "Central arousal" and sexual responsiveness in the snail, Helix aspersa. Behav. Neural Biol. 55:194-213. Asratjan, E. A. 1970. Ocherki po Fiziologii Uslovnykh Refleksov (Essays on Physiology of Conditioned reflexes). Moskva: Nauka. Balaban, P. M. 1983. Postsynaptic mechanism of withdrawal reflex sensitization in the snail. J. Neurobiol. 14:365-315. Balaban, P. M. and Chase, R. 1990. Inhibition of cells involved in avoidance behavior by stimulation of mesocerebrum. J. Comp. Physiol. A, 766:421-427. Balaban, P. M., Maksimova, O. A., and Galanina, G. N. 1985. Cellular responses during elaboration of two-way conditioned reaction in the snail (in Russian). Zhurn. Vyssh. Nervn. Dejat. 35:491-503. Balaban, P. M., Vehovszky, A., Maksimova, O. A. and Zakharov, I. S. 1987. Effect of 5,7dihydroxytryptamine on the food aversive conditioning in the snail Helix lucorum L. Brain Res. 404:201-210. Carew, T. J. 1989. Developmental assembly of learning in Aplysia. TINS 72:389-394. Chase, R. J. 1986. Brain cells that command sexual behavior in the snail Helix aspersa. Neurobiol. 77:669-679. Christoffersen, G. R. J. 1981. Short- and long-term retention in the tentacle reflex of Helix pomatia: Kinetics and interspecimen transferability. Compar. Biochem. and Physiol. 68:461-415. Davis, W. J. and Jillette, R. 1978. Neural correlates of behavioral plasticity in command neurons of Pleurobranchaea. Science 799:801-804. Emson, P., Walker, R. J. and Kerkut, G. A. 1971. Chemical changes in a molluscan ganglion associated with learning. Compar. Biochem. Physiol. 40B:223-239. Gallistell, C. R., Gomita, Y., Yadin, E., and Campbell, K. A. 1985. Forebrain origins and terminations of the medial forebrain bundle metabolically activated by rewarding stimulation or by reward-blocking doses of pimozide. Journal of Neuroscience 5:1246-1261. Koshtojants, Kh. S. 1957. Osnovy Sravnitelnoj fiziologii. Tom 2: Sravnitelnaja Fisiologija Nervnoj Sistemy (Principles of comparative physiology. Vol. 2: Comparative physiology of the nervous system). Moskva: Izdatelstvo Akademii Nauk SSSR. Kostenko, M. A., Geletyuk, V. I. and Veprintsev, B. N. 1974. Completely isolated neurons in the mollusc Limnaea stagnalis: A new objective for nerve cell biology investigation. Comp. Biochem. Physiol. 49A:89-100. Kostyuk, P. G, Krishtal, O. A. and Doroshenko, P. A. 1975. Outward currents in isolated snail neurons. I. Inactivation kinetics. Comp. Biochem. Physiol. 57C:259-263. Kupalov, P. S., Voevodina, O. N., and Volkova, V. D. 1964. Situatsionnyje Uslovnyje Refleksy u Sobak v Norme i Patologii (Situational conditioned reflexes in normal and pathological dogs). Leningrad: Meditsina (in Russian). Lind, H. 1976. Causal and functional organization of the mating behavior sequence in Helix pomatia. Behaviour 59:162-202. Lindwall, O. and Bjorklund, A. 1974. The glyoxylic acid fluorescence histochemical method: A detailed account of the methodology for the visualization of central catecholamine neurons. Histochemistry 39:91-121.

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Litvinov, E. G, Maximova, O. A., Balaban, P. M. and Masinovsky, B. P. 1976. Defensive conditioned reaction in the snail Helix lucorum (in Russian). Zhurn. Vyssh. Nervn. Dejat. 26:203-206. Maksimova, O. A. 1979. Elaboration in the snail of an instrumental food-searching conditioned reflex with a two-way connection. Zhurn. Vyssh. Nervn. Dejat. 29:793-800. Maksimova, O. A. and Balaban P. M. 1983. Nejronnye mekhanizmy Povedencheskoj Plastichnosti (Neural mechanisms of behavioral plasticity). Moskva: Nauka. . 1984. Neuronal correlates of aversive learning in command neurons for avoidance behavior of Helix lucorum L. Brain Research 292:139-149. Mitchell, S. R., Beaton, J. M. and Bradley, R. J. 1975. Biochemical transfer of acquired information. Internal Rev. Neurobiol. 77:61-83. Mpitsos, G J., and Collins, S. D. 1978. Learning: A model system for physiological studies. Brain Research 799:497-506. Olds, J. 1958. Self-stimulation of the brain: Its use to study local effects of hunger, sex, and drugs. Science 727:315-324. . 1977. Drives and Rewards: Behavioral Studies of Hypothalamic Functions. New York: Raven Press. Olds, J. and Milner, P. 1954. Positive reinforcement produced by electrical stimulation of septal area and other regions of the rat brain. J. Comp. Physiol. Psychol. 47:419-421. Pavlov, I. P. 1885. Wie der Muschel ihre Schaale offnet. Versuche und Fragen zur allgemeinen Muskel- und Nervenphysiologie. Pflug. Arch. Ges. Physiol. 37:6-31. . 1927. Conditioned Reflexes, and Investigation of the Physiological Activity of the Cerebral Cortex. Oxford: Oxford University Press. . 1951. Lektsii o rabote bolshikh polusharij (Lectures about the work of brain hemispheres). Polnoe Sobranije Sochinenij Moskva-Leningrad: Isdatelstvo Akademiji Nauk SSSR. Pawson, P. A. and Chase, R. 1984. Developmental changes in the passive membrane properties of an identified molluscan neuron. Devi. Brain Res. 77:296-300. Pinsker, H. M., Hening, W. A., Carew, T. J. and Kandel, E. R. 1973. Long-term sensitization of a defensive withdrawal reflex in Aplysia. Science 182:1039-1042. Sakharov, D. A. 1960. Automatism in pedal ganglia of pteropod mollusc Clione limacina L (in Russian). Nauchnye Doklady Vysshey Shkoly (Biologitcheskije nauki) 3:60-62 (in Russian). . 1962. Giant nerve cells in nudibranch molluscs Aeolidia papillosa and Dendronotusfrondosus (in Russian). Zhurn. Obshchej Biologiji 23:308-311. . 1974. Genealogija Nejronov (Genealogy of neurons). Moskva: Nauka. Shevelkin, A. V. 1989. Feeding sensory satiation induces defence behavior activation of garden snails (in Russian). Zhurn. Vyssh. Nervn. Dejat. 39:379-381. Sokolov, E. N. 1969. Mekhanizmy Pamjati (Mechanisms of memory). Moskva: Izdatelstvo Moskovskogo Universiteta. Sokolov, E. N. and Jarmizina, A. L. 1970. Habituation of a giant molluscan neuron to repeated intracellular stimuli. In: E. N. Sokolov and O. S. Vinogradova, eds., Nejronnyje Mekhanismy Orientirovochnogo Refleksa, (Neuronal Mechanisms of the Orientation Reflex), 111-117. Moskva: Isdatelstvo Moskovskogo Universiteta. Sokolov, V. A. 1959. A conditioned reflex in gastropod mollusk Physa acuta (in Russian). Vestnik Leningradskogo Universiteta, Serija Biologicheskaja 2:82-86. Stellar, J. R. and Stellar, E. 1985. The Neurobiology of Motivation and Reward. New York:


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Springer. Stepanov, I. I. 1983. An approximate approach to estimation of learning curve parameters (in Russian). Fiziologija Cheloveka 4:686-689. . 1991. The nootropic effects of hemolymph of the land snail Helix lucorum with elaborated tentacle conditioned reflex (in Russian). In: Simpler Nervous Systems. Regional meeting of the Internal Soc. Invertebr. Neurobiol, 96. Minsk. Stepanov, I. I., Kuntzevich, S. V. and Lokhov, M. I. 1989. Regression analysis of instrumental conditioned tentacle reflex in the snail Helix lucorum (in Russian). Zhurn. Vyssh. Nervn. dejat. 39:890-897. Stepanov, 1.1., and Lokhov, M. I. 1986. Dynamics of elaboration of conditioned reflex and differentiation in the snail (in Russian). Zhurn. Vyssh. Nervn. Dejat. 36:698-706. Stepanov, 1.1., Lokhov, M. I., Satarov, A. S., Kuntsevich, S. V. and Vartanian, G A. 1987a. Humoral link in the mechanism of formation of conditioned food refusal in snail (in Russian). Zhurn. Vyssh. Nervn. Dejat. 37:489-497. . 1987b. Specific and non-specific components of neurohumoral link of conditioned food refusal reflex in the snail (in Russian). Zhurn. Vyssh. Nervn. Dejat. 37:935-945. Stepanov, 1.1., Nikolaev, Yu. V. and Borodkin, Yu. S. 1991. Effects of ethanol upon feeding behavior of the land snail Achatina fulica (in Russian). Zhurn. Vyssh. Nervn. Dejat. 47:573-580. Stepanov, I. I., and Poszinsky, A. M. 1982. A device with a differential photogauge for a registration of Helix pomatia pneumostome contractions (in Russian). Zhurn. Evol. Biokhim. i Fiziol. 75:200-201. Stepanov, I. I., Smirnova, A. G and Sapronov, N. S. 1994. Glutamic acid influence on defensive and feeding behavior of the land snail, Helix lucorum (in Russian). Zhurn. Vyssh. Nervn. Dejat. (in press). Sudakov, S. K., and Kozyrev, S. A. 1986. Restoration by pentagastrine of feeding behavior modulated by learning (in Russian). Zhurn. Vyssh. Nervn. Dejat. 36:978-980. Thompson, E. L. 1917. An analysis of the learning process in the snail Physa gyrina Say. Behav. Monogr. 3:89-110. Thompson, R. F. and Spencer, W. A. 1966. Habituation: a model phenomenon for the study of neuronal substrates of behavior. Psychol. Rev. 173:16-34. Vagner, N. P. 1885. Bespozvonochnyje Belogo Morja (Invertebrates of the White Sea). St. Petersburg. Veprintsev, B. N., Krasts, I. V. and Sakharov, D. A. 1964. Neural cells of a nudibranch mollusc Tritonia diomedia Bergh (in Russian). Biofizika, 9:321-335. Zakharov, I. S. and Balaban, P. M. 1987. Neural mechanisms of age-dependent changes in avoidance behavior of the snail Helix lucorum. Neuroscience 23:721-729. . 1991. Serotonergic modulation of avoidance behavior in Helix. In: D. A. Sakharov and W. Winlow eds., Simpler Nervous Systems, 316-329. Manchester, N.Y.: Manchester University Press.


Chapter Five Individual Features in Invertebrate Behavior: Crustacea Yuri. M. Burmistrov and Zhanna P. Shuranova

"Die Entwickelungsgeschichte des Individuums ist die Geschichte der wachsenden Individualitat in jeglicher Beziehun." (The history of the development of an individual is the history of the rise of its individuality in all the directions.) (K. E. von Baer 1828:263)

RUSSIAN INVESTIGATIONS OF CRUSTACEAN BEHAVIOR There are a number of different species of Crustaceans in the former USSR, but only a small percentage have been studied from the behavioral point of view. Primarily, this is due to the low degree of exploitation of the basins situated at the Arctic or Far East, locales where the climate makes it difficult to conduct research effectively. Although some species of crustaceans in this country's oceans are large (the crab Paralithodes camtschatica has an interleg distance of about 1.5 m and weighs up to 7 kg), the great majority of indigenous crustaceans are small. They are abundant in both salt and freshwater basins. For example, in the Chukotsky Sea there are about 40,000 amphipods Gammarus and Anisogammarus per one sq. meter of the bottom, and in Lake Baikal there are 240 endemic species of freshwater amphipods (Dogel 1975; Tsvetkova 1975). Among these we find Acanthogammarus maximus, whose body length reaches 7 cm (Kozhov 1963). As for the large crustaceans, their species diversity is small even in the seas; in freshwater the only crayfish to be found is one of the genus Astacus. Several centers of scientific interest in the former USSR have focused on conducting multidisciplinary investigations of crustaceans, including their behavior. These centers include the Institute of the Biology of the Southern Seas of the Ukrainian Academy of Sciences (Sebastopol, Odessa); the Institute of the Freshwater Biology of the Russian Academy of Sciences (Borok, Yaroslavl Region); the Far-East Scientific Center of the Russian Academy of Sciences (Vladivostok); and

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the Institute of Zoology and Parazitology of the Lithuanian Academy of Sciences (Vilnius). The Behavior of Lower Crustaceans The behavior of the sea crustaceans Copepoda, which compose 40%-80% of the plankton population, has been investigated in detail at the Institute of the Biology of the Southern Seas. The main goal of this research was to elaborate the quantitative estimation of crustacean behavior and to reveal its ethological bases (Piontkovsky 1985). Specifically of concern was the delineation of Copepoda behavior into its structural elements for ethologically based classification. The consideration of Copepoda behavior led Piontkovsky (1985) to suggest that the smallest unit of behavior is the "elementary movement," while the "behavioral act" would represent sets of "elementary movements." The "behavioral complex:" would represent the whole behavioral repertoire of the animal, comprised of behavioral acts. Also developed in this institute were several techniques for measuring both locomotion of freely swimming animals and feeding movements. This was accomplished via clamping the animals at their dorsal side and using high-speed microfilming of their behavior. This technique has enabled investigation of the structure of locomotor activity, the elements of which consist mainly of jumps, glides, and manipulatory movements. These data are represented in the form of ethograms with concurrent mathematic models of behavioral complexes, falling under the influence of factors such as temperature, illumination level, barometric pressure, the content of feeding materials and the concentration of the food, and so on. It has been demonstrated that there is an ontogenic development of these behavioral acts, with such acts becoming more complex and efficient over time. The number of appendages participating in elementary movements increase, such that many appendages become multifunctional. The behavioral study of small freshwater crustaceans belonging to the Cladocera has been centered at the Institute of Freshwater Biology. Polyphemus pediculus, a typical crustacean for shallow-water fauna, was chosen for research. This species is a very important food source for fish and may also be useful in the artificial cultivating of fish (Butorina 1980, 1986a, 1986b). The behavioral repertoire of P. pediculus, considered from the ethological point of view regarding hierarchical classification of behavior, yields the following observations: The adult animal is an active predator; during hunting its speed may reach up to 16 mm/s (this is about 30 times its body length!). One characteristic feature of Polyphemus pediculus is the development of "schools" (clearly defined groups consisting of many individuals without any single dominant animal). It is suggested that these schools are analogous to schools of the octopods and squids, as well as those of pelagic fish and some terrestrial vertebrates. Within the schools there may exist subgroups of animals whose cohesion is short-lived. It is further believed that Polyphemus pediculus is capable of engaging in imitative behavior. Behavioral relationships

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between individual animals are based on a continuous exchange of different signals (chemical, visual, and vibrational). The structure of Polyphemus populations is highly complex: It is based on the combination of many behavioral acts, the social-like structure of the schools, and the animals' sensory environment. The structure of their groups and the behavior of the individual animals in them are well-suited for the limited and unstable conditions characteristic of shallow water basins. The Behavior of Higher Crustaceans Although there have been no specific behavioral investigations of small higher crustaceans, in some research their behavioral reactions have been used for studying the effects of various water pollutants (Cherkashin and Ternovenko 1983; Udalova, Karas, and Zhukovskaya 1990), as well as the effects of strong artificial electric and magnetic fields (Burba 1983; Shuranova, Sadauskas, and Vekhov 1988). The crab Carcinus maenas, which lives in the Black Sea, has been used in experiments on the comparative investigation of conditioned reflexes (Karas 1962, 1963). These experiments have been conducted in the Institute of the Biology of the Southern Seas and at the Department of the Physiology of Higher Nervous Activity in Moscow University. The Karas experiments showed that conditioned reflexes to visual, tactual, and vibrational stimuli when reinforced with food are elaborated easily (less than 30 trials) in Carcinus maenas. However, their latencies are variable, and "intertrial reactions" do not disappear during the course of the experiments. There have been attempts to elaborate inhibitory conditioned reflexes (extinction, discrimination, and conditioned inhibition), but the responses were neither reliable nor stable. If either complex or chain conditioned stimuli were used, the conditioned reflexes did appear quickly; however, discrimination between a complex stimulus and its single components was impossible, as was the discrimination of the temporal relations between the stimuli in the chain (at least, after 100 trials). It has been concluded that innate, reflexive behavioral components are more important for crabs than are learned ones. Many more behavioral studies have been made on the freshwater crayfish Decapoda. It should be noted that these ancient animals settled in rivers and lakes in the territory of the former USSR thousands of years ago. Historically, crayfish were an ordinary and inexpensive food favored by different social groups (there are many examples in Russian fiction). For many years, the crayfish fishery in the former USSR was a significant part of the world catch (Ivanov 1955; Malinovskaya 1984). The long history between native people and this animal is represented in the identity of its name in all Slavic languages (Russian, Ukrainian, Belorussian, Polish, Czech, etc.). Its etymology is likely derived from the catching behavior of the claw (in the romano-germanic languages the name of this animal additionally reflects its way of moving at the bottom, distinguishing it from most aquatic animals) (Shuranova and Burmistrov 1988).

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The detailed description of native crayfish (which is found in the European part of this country; genus Astacus) and its distribution was accomplished in the last century (Kessler 1875) and was reinvestigated (Birshtein and Vinogradov 1934). The increasing interest in this animal among Russian biologists stems back to a translation (1900) of the famous book written by T. H. Huxley (1880). Since the turn of the century, then, the crayfish has been used extensively for teaching (Berkos 1905). In the 1930s a little book was published (it is extremely rare now) devoted to crayfish behavior in the natural environment and to its artificial cultivation (Podyapolsky and Podyapolskaya 1933). This book is based on long-term observations of the crayfish in its natural habitat. After World War II, the intensive study of these animals, both in the field and in the laboratory, began in Lithuania (Tsukerzis 1970, 1989). This venture included a comparative analysis along a multiplicity of indices (including behavioral) between different native species (Astacus astacus and Astacus leptodactylus), as well as with the American crayfish Pacifastacus leniusculus (Dana), which was introduced to Lithuanian lakes. A general revival in social and scientific interests that took place in the USSR at the end of the 1950s further influenced the investigation of crayfish physiology and behavior. At the Far-East Scientific Center, a special group of multidisciplinary investigators (from such areas as neurophysiology, neuromorphology, and neurocybernetics) pursued the goal of not only describing but also imitating some simple crayfish movements to be eventually used in the development of robotics. It seems that, over the course of this investigation, there was increasing interest in the behavior of the whole animal (Yakovlev and Kan 1974). This was reflected in many publications that appeared even after the dissolution of this group (e.g., Baraniuk 1983; Belianin, Doroshenko, and Stepushkina 1980; Baraniuk, Shishlov & Yakovlev 1983; Belianin, Kan, and Stepushkina 1980; Belianin and Ogayants 1978; Kan 1980; Sergeev, Gracheva, Deryabin, and Puchkova 1980). In these papers and in closely related papers of other authors (Burtyka 1975; Doroshenko 1983; Doroshenko and Burba 1983; Doroshenko and Shashtokas 1975; Kamenev and Persiyanov 1978; Korneeva 1975; Sologub, Belianin, and Kan 1981; Tsukersis 1983, 1986, 1989; Tsukersis & Doroshenko 1975; Tsukersis, Shashtokas, and Burba 1978) different kinds of crayfish behavior were investigated, including spatial orientation, role of different sensory systems, searching of preferred room or food location, elaboration of conditioned reflexes with positive or negative reward, locomotion and feeding behavior in ontogenesis, and crayfish migrations in nature. These papers were all based on the ethological and cybernetic approaches supported by the original research group. The main conclusion of the aforementioned collaborative study may best be expressed by the words of the head of the research group: The crayfish has rather perfect behavior based on its hereditary mechanisms and on its ability for individual learning. The elaborated adaptive responses in the crayfish are true conditioned reflexes. They are specialized, and arise only to conditioned stimuli; they appear at a rate which is close to that of vertebrates. Additionally, the marked ability to train the crayfish's excitatory and inhibitory processes permits behavioral


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classification of the crayfish among invertebrates possessing a highly developed central nervous system (Kan 1980:4). It is remarkable, however, that during this same period, some authors doubted if the elaborated reactions of the crayfish were "true" conditioned reflexes (Baraniuk 1983). Moreover, another opinion surfaced in the literature, stating that "though the crayfish is the classical invertebrate living in our basins, its behavior has not been investigated completely" (Tsukerzis 1983:117). HIGHER CRUSTACEANS AS AN OBJECT FOR COMPARATIVE-PHYSIOLOGICAL ANALYSIS OF INDIVIDUAL BEHAVIOR Recently the term "individual behavior" has come to refer to behaviors directed toward the maintenance of the status quo of the organism. The antithesis to "individual" seems to be the term "social," forcing a distinction between these terms based upon interactions of the animal with its environment or conspecifics. From the physiological point of view, however, "individual" means a set of behavioral traits that belong to this particular individual and which differentiate it from conspecifics. For Pavlov, who studied conditioned reflexes in dogs, "individual" was close in meaning to "acquired" and had the opposite meaning to "inherited." It is obvious that the delineation between acquired and inherited behavioral forms is not so clear now as it seemed to be in the first half of this century. On the other hand, it is known that even in humans it is not easy to determine individual features in behavior (Teplov 1985). Keeping in mind the problem of definition, we would like nevertheless to consider individual differences in regard to the behavior of some higher invertebrates. Main Data Concerning the Organization of the CNS and Sensory-Motor Structures of Decapoda The order Decapoda, which includes most well-known crustaceans (crabs, shrimps, lobsters, and crayfish), are located in a high position on the phylogenetic scale among the invertebrates (other such species being the social insects and the cephalopods). They all possess highly developed sensory systems. Most of them have complicated eyes capable of detecting small changes in their surroundings and are sensitive not only to visible light but also to polarized rays. It is further possible that some decapods can discriminate color. Additionally, in the crayfish there is evidence of extraocular receptors that can control the illumination level of the environment. The surface of the body is covered with a large number of mechanosensory receptors, which are multiformous. They allow sensation of light touch and detection of small local vibrations of water around the animal. There are also highly developed chemoreceptors, located mainly at the antennules and the distal joints of the forelegs, that orient the animal to chemical signals. There are also a number of

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internal mechanoreceptors that control the position of different joints in the appendages, parts of the exoskeleton, and other internal and external receptors. It should be noted that sensory systems in decapods are fairly well understood (Atwood and Sandeman 1982). This is true for their motor system as well. For example, it is well known that the crayfish possess not only four pairs of walking legs but also a pair of manipulatory organs (the claws) and several pairs of abdominal appendages ("swimmerets"). The abdomen itself can function as a motor organ, though it is used mainly in dangerous situations. The legs allow the crayfish to move on a substrate in both forward and backward directions; it can also climb very well up and down on a vertical surface. The walking legs can also be used for grooming, touching various objects, and searching for food. The abdomen serves to ventilate the gill chamber and is especially important in sexual and parental behavior of the female. All muscles in Decapoda consist of striated fibers (they lack smooth muscle). The ultrastructural and contractile characteristics of single muscular fibers may be different, depending on the motor neurons (fast or slow) that supply them. The muscles are innervated usually by a small number of different motor axons. The multiterminal innervation is a favorable adaptation for modification of the membrane potential along the entire muscle fiber. This is essential for triggering the contractile mechanism because the action potentials are generated only in some (especially fast) muscles (gradual postsynaptic potentials develop in most muscular fibers). The strength of muscle stress depends directly on the shift of the membrane potential. Many muscle fibers are innervated by means of excitatory and inhibitory axons; the latter have their terminals not only at the muscle fibers but also at the presynaptic endings of the excitatory motor neurons (the phenomenon of "presynaptic inhibition"). Gradual muscle electrogenesis, together with a high degree of plasticity in synaptic transmission and the development of peripheral inhibition, provide the subtle gradation of muscle contractions (Atwood 1982). The nervous system of the crayfish consists mainly of abdominal, thoracic, and cerebral ganglia, interconnected by nerve fibers. Recently there has been identification of structural and functional activity of many neurons (mostly motor neurons and, to a lesser extent, interneurons) in the ventral nerve cord, especially in the abdominal ganglia. The number of central neurons in these ganglia is rather small (about 300 pairs of neurons in one abdominal ganglion). Approximately 600 neurons were identified in the abdominal nerve cord of the crayfish by means of intracellular dye injection, together with electrophysiological investigation (Skinner 1985). It is known that a single walking leg of a typical decapod is innervated by about 100 motor neurons, while the movements of every abdominal segment are controlled by 50 motor neurons (Laverack 1988). The identification of these neuronal functions facilitated marked progress in studying the neuronal mechanisms of many actions that often require one or several ganglia. The cerebral ganglia are divided into three structures: The trito-, deuto-, and protocerebrum. These receive inputs from many different sensory receptors, in-


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eluding those of complex eyes, antennulae, and antennae. These structures are additionally responsible for the organization of integrated output. The structural and physiological peculiarities of the cerebral ganglia neurons have not been investigated as fully as the motor neurons because of similar problems confronting research in the vertebrate brain (the small size of single neurons and neuronal processes, the great number of neuronal elements interwoven with each other, etc.). It is important to emphasize, however, that the brain of decapods has a very sophisticated structure that is comparable with that of higher insects (Mellon and Alones 1993; Sandeman 1982; Sandeman, Sandeman, Derby, and Schmidt 1992; Titova 1985; Tsvileneva 1970; Tsvileneva and Titova 1985). Procambarus cubensis: Its Benefit for Studying Individual Behaviors It has been noted that different decapods, including freshwater crayfish, are ideal animals for the physiological analysis of many behavioral manifestations. On the other hand, most of them, especially the European crayfish Astacus, are aquatic animals that are nocturnal. Therefore, they would require special conditions to investigate them in a standard laboratory. Because it is necessary for the physiological analysis of behavior to use a laboratory-adapted animal, we chose the freshwater crayfish Procambarus cubensis, which was introduced to Moscow aquaria at the end of the 1970s. Procambarus cubensis belongs to the family Cambaridae, primarily indigenous to North America, and to the subgenus Austrocambarus (Hobbs 1984, 1989), which live in shallow ponds in Cuba. From the physiological and neurophysiological points of view, Procambarus cubensis is very similar to the European crayfish Astacus. However, it has some features which favor its use in experimentation over Astacus (Shuranova and Burmistrov 1990). First, P. cubensis differ from Astacus in their requirements for water quality and temperature. They live well at room temperature (18°-23°C) and can survive after brief rises and falls of temperature between 35° and 50°C. They do not require artificial oxygenation while living in shallow tanks; if the water level is as high as 30 cm, oxygenation is desirable, but not necessary. These animals, whose size may be controlled by the volume of the aquarium, can live for many months in individual shallow tanks (17 cm in diameter containing about 0.5 1 of water). If the bottom of the tank is covered with sand, it is only necessary to change the water rarely (once a month or even every several months). The adults can be fed with various foods such as mosquito larvae, small turbiform worms, peas, some greenery, and cereals. Feeding periods can be regular or irregular, with a feeding interval of several days or even longer (it seems that in aquaria they never die from hunger, see, for instance, Huxley, 1880). Another feature supporting its use as a laboratory animal is the ease and rapidity with which Procambarus cubensis reaches the final stage of molting. Though the growth rate depends on many factors, in general these animals grow much faster than European crayfish. They become mature at an earlier age than Astacus, which

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favors their rapid cultivation the laboratory. Additionally, the reproductive cycle is not dependent on seasonal cycles, as is characteristic of Astacus. The copulatory behavior in Procambarus cubensis is similar to that of other crayfish. At one to two weeks post-copulation, the female produces the spawn, situated at its swimmerets under the abdomen. During hatching, the female tirelessly moves its swimmerets simultaneously in a forward-backward and side-toside direction. Then it remains in a preselected shelter, where it can remain without food. The juveniles begin to separate from the mother three to four weeks later; their length (from rostrum to telson) is approximately 5-6 mm. At the time of separation, they try to be near the mother, climbing to her swimmerets or other body parts whenever real or perceived danger threatens. The female's behavior during this period is intriguing; she moves slowly and smoothly, usually with closed claws, and does not demonstrate any aggressive reactions to her offspring, even when they are on her mechanoreceptor-rich antennae or inside the claw. After a week many juveniles become independent of the mother; they are feeding and molting frequently. After two weeks they double in size. After three to six weeks, some of them have reached 16-25 mm in length. If the juveniles live communally in a large aquarium or individually in small tanks, it is possible to obtain up to 70 young crayfish from one female. One may conclude that the freshwater crayfish Procambarus cubensis, which has lived for many generations in aquarial conditions, has many features that favor its use as a model animal for physiological experimentation over the native crayfish Astacus. These include its relatively small size, the preference for warm water, the lack of requirements for environmental conditions, a rather short life cycle, and ease of cultivation in the laboratory. These considerations are especially important in Russia because there are few large decapods. It should also be noted that Procambarus cubensis can be used in the same neurophysiological investigations as Astacus. Additionally, they are suited for solving some tasks that cannot be conducted on "wild" animals. This species of crayfish also seems ideal for ontogenetic studies of different functions as well as for genetic and neurogenetic research. Using Procambarus cubensis permits investigation of an invertebrate living in "quasi-natural" conditions with durable contacts with humans. These animals are suitable for daily observations and for mutual comparisons, from birth throughout its life cycle, and in a variety of natural and experimental situations. This species provides a unique opportunity for studying not only species-specific but also individual features of behavior in one of the most highly developed invertebrates. THE BEHAVIOR OF PROCAMBARUS CUBENSIS IN QUASI-NATURAL CONDITIONS Procambarus cubensis adapts easily to high levels of illumination, whether naturally or artificially produced. This characteristic enabled us to take photo-


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graphs and to film these animals in different behavioral situations. From these observations, we produced a film (16mm) that demonstrated multiple forms of crayfish behavior under conditions close to those found in nature. The Behavior of the Adult Crayfish Living in a Separate Vessel It is known that, under natural conditions, crayfish prefer to live in individual shelters. Therefore, confining the animal to an individual container in the laboratory seems rather ideal. An initial noticeable feature of crayfish is its extremely low degree of free movement activity. With an appropriate shelter, the crayfish can stay inside for many hours. Without a shelter, it prefers to be near a shadowed corner where it can remain motionless, providing that no obvious environmental changes occur. Feeding Behavior After some food is placed in its home water, the crayfish demonstrates general agitation before actually searching for the food. This agitation is characterized by enhancement of motor activity of some appendages, initially the large and small antennas and scaphognathites (sometimes simultaneously with swimmerets). Following these behaviors, locomotor movements directed toward signals associated with the food source appear. The "food signals" likely have vibrational (placing food in the water) and chemical characteristics; thus they are detected by the receptors situated on the large and small antennae, as well as by some parts of the second and third walking legs. The animal then moves toward the food location, touching the sand with brief and frequent movements of the small "claws." The mode of movement toward the food source requires some special consideration. Even in an animal that has not been fed for a long period of time, the beginning of the walking movement is delayed (up to several minutes) from the moment of food detection because of the stereotypical general agitation response. The movement itself is slow, consisting of some short forward pushes, followed by long pauses. Additionally, on the way to the food source, the crayfish may stop and return to the shelter several times, greatly increasing the interval between food detection and food ingestion. The method used in catching food depends strongly on the type of food. Small moving food sources (mosquito larvae, turbiform worms) are caught extremely quickly by the claws at the first or second thoracic legs (if there were several worms, a hungry crayfish will catch them with several claws at both sides of its body simultaneously). Nonmoving food is caught slowly, usually with the big claws working together (for instance, in the case of capturing a large pea). Under natural conditions, crayfish are omnivorous; they like green and meaty food, alive or dead in the case of the latter. In our laboratory they often lived communally with various types of fish. It was never noted that the crayfish succeeded in catching a fish; however, we did not specifically observe such attempts. The crayfish will furiously attack an earthworm much bigger than itself; gradually

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it overrides the worm's resistance and begins to eat it, piece by piece (this topic with other examples of feeding behavior is represented in our movie). Shelter Long-term observation of several crayfish permit us to see many individual features in their feeding behavior. Individual differences are even more marked when concerning shelter selection and behavior. In general, the crayfish prefers to be in a shelter if one is available. The process of adapting to a shelter placed in an aquarium requires a long time. First there are general signs of agitation: an increase in scaphognathite and swimmeret beating, together with a decrease in other visible movements. Then the animal goes slowly and carefully toward the shelter and "surveys" it from a distance in an apparent attempt to observe its interior. After several such attempts, the crayfish penetrates the shelter (always headfirst). After entering the shelter, the crayfish might remain inside for most of the remainder of its life. The behavior of some individuals closely resembled that of their hermit crab relatives. However, other animals preferred to be near but outside the shelter for many months. It may be also that the desirability of the shelter changed over time, such that the outside became preferable. It is clear that this behavior requires a much more detailed investigation. Using Thoracic Legs It has been described above how the crayfish uses its legs in feeding behavior. It is obvious that the main function of these legs is to coordinate activity necessary for walking. However, different legs have specialized functions and can work independently to a large degree. The most multipurpose leg is the first one, used for digging sand and for bringing sand and small stones over long distances. While such activity has been observed sometimes in males, it is more common for a "gravid" female. Strong, sporadic digging activity may be seen in a highly excited male upon placement with a female. In this situation it is possible that digging represents a kind of "substitutional" activity known in many vertebrates (Manning 1979). The increase of digging has also been noted in experiments with the elaboration of conditioned reflexes in crayfish (Shuranova 1991). Another function of an individual thoracic leg is grooming various body parts. Most often one may see grooming of the telson by means of the 4th-5th pairs, of the rostrum and the eyes by means of the 2nd-3nd pairs, and of the mouth appendages by means of the 2nd pair. It should be noted that sometimes the crayfish is moving several legs independently. This activity seems to be very important for cleaning the body surface. Interrelations Between Crayfish Living Together for a Long Time One portion of our movie is devoted to the behavior of several crayfish from the same litter, living together in a large aquarium filled with water, plants, and snails. In comparison with the above-described animals living alone, these animals were


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much more active. In fact, most of the time they moved on the bottom, especially along the aquarium walls, even in the absence of external influences. Sometimes they climbed the plants and "rested" there. The placement of food in the aquarium evoked very strong agitation of all animals, which began to catch the food immediately after finding it and ate it at the site where it was caught. When living together, these animals have continuous contacts with each other, though it is difficult to describe the nature of these contacts because there are rarely visible changes in behavior except for that of defensive reactions, which may appear in several forms. Thus, there are some remarkable behavioral differences between the crayfish living alone and those living in a group. One difference, which is not behavioral, lies in the general appearance of these animals. Group maintenance of crayfish results in little or great loss of appendages in all members of the group. This loss of or damage to appendages may be caused partly by the aggressive actions of the animals during the intermolt period (though we could not observe this), but mostly this fact probably reflects the "cannibalism" of newly molted animals noted in crayfish long ago (Huxley 1880). This behavioral phenomenon seems to be very complicated. It is clear that some appendages, the thoracic legs and large antennae, for instance, are lost more often than others. Under laboratory conditions it is possible to isolate such an animal, and it demonstrates many opportunities to recover after one or several molts. Even an animal that had lost almost all walking ability and had no righting response (probably because it did not put new sand in the statocyst) would recover if isolated. All it could do in the interim was eat food from the pincers; however, after one molt such animals could support normal posture and after two to three molts, all appendages would be restored. Obviously, the phenomenon of "cannibalism" should be rather rare under natural conditions because the crayfish can find shelter prior to molting. However, cannibalism is the cause of many problems that arise during artificial cultivation of these animals. Group living permits observation of many forms of social behavior in the crayfish. We demonstrate in our movie the aggressive relations arising between two or more animals of the same sex just after placing them in a small tank. If several animals are placed simultaneously in a big aquarium, they usually do not immediately interact with each other. Only after some delay one may see some hierarchical relations between them. We believe that the interrelations between different individuals, especially if they live together for many months, are in fact very elaborated and variable. A specific kind of social behavior is sexual behavior. We observed sexual behavior manu times just after placing two animals in a new tank or after placing male or female into the home tank of the animal of the other sex. More often, however, we watched the behavior of male and female crayfish living together over a long period of time. It is astonishing to see how quickly the behavior of a male in its home tank changes when a female appears. Even more amazing is the behavior of the female, which, at some distance from the male, changes its posture suddenly. It looks completely immobile, with all its body, including the claws,

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stretched forward as if relaxed. It appears to be dead. The male then tries to turn the female upside down. The female remains neutral during this procedure, not helping and not opposing the male, but occasionally she attempts to escape. A single copulatory act can last several minutes. It may be repeated several times on the initiative of the male. Though copulation itself is rather stereotypical, the preparatory period is extremely variable. It depends on many factors, including the relative size of the partners, male practice, and female intentions. Several times we observed the choice of the partner in the case of coexistence of two males with one female, and vice versa. Two crayfish of different sex can live together for a long time (many months at least), being sometimes so close to each other that they share one shelter. "Parental" Behavior and the Behavior of Juveniles The behavior of the female after laying eggs needs special consideration. First, one may observe the female's general agitation, which is evidenced by aggressive reactions to ordinary situations that previously did not cause visible responses. The female avoids observation, stays mostly in her shelter, and near the shelter builds a barrier of sand. Once inside, she constantly moves her swimmerets, ventilating the eggs. After about a month, single juveniles sitting at the swimmerets begin to be visible, identifiable by their big black eyes. Gradually, some of them leave the mother and move independently, but remain not far from her. The period of separating all the juveniles from the mother may last one to two weeks. Soon after that the female usually molts, and her behavior changes. Even after molting, however, she permits the juveniles to jump on her swimmerets or on her back. Over time, the juveniles become more and more independent of the mother. The juveniles have basically the same appearance as the adults. Some of their behavioral traits, such as searching for food and eating it, are similar to that of adult crayfish. However, their manner of movement is different. Because they are very small and light, they prefer not to walk but move by means of a set of "jumps," which are analogous to the "tail-flips" of adult crayfish. Additionally, they leave a vertical surface and return to the bottom of the aquarium by means of "gliding." They are more behaviorally active than the adults and do not try to hide in the shelter. One may say that they are similar to the juveniles of many other animals more familiar to humans. In summary, we may say that the crayfish Procambarus cubensis, like other crayfish living under natural conditions, has very complicated behavior that can be studied individually from birth to death. It would be interesting to compare its behavior with the behavior of other animals related to the same taxonomic group but differing in their body plan and ecology (crab, hermit-crab, shrimp, etc.). It would also be interesting to compare crayfish behavior with that of animals outside of its taxonomic group such as the vertebrates.


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BACKGROUND LOCOMOTOR ACTIVITY AS AN INDEX OF THE FUNCTIONAL STATE OF CRAYFISH For a more detailed investigation of the individual behavioral traits of Procambarus cubensis, we used various techniques for measuring physiological parameters. We attempted to control for experimenter bias and demand characteristics as much as possible. The first parameter measured was that of locomotor activity in the crayfish. To measure locomotion we used two noninvasive methods that complement each other. They permit characterization of "spontaneous" locomotor acts that appear in the absence of any external influences. Additionally, these techniques permit observation of long-lasting activity evoked by some changes in external stimuli (Sadauskas and Shuranova 1982). The first technique was the video recording of an animal placed in either a round tank identical to its home tank or in a plastic rectangular chamber ( 6 X 1 9 sq.cm). The second technique was an optical "gate" using the same rectangular chamber. These techniques can be used concurrently. The crayfish, with a body size of 3 to 4.5 cm, could move freely within. Crossing of the midline of the chamber was detected by an infrared diode. Shuttle movement between the two compartments was reflected in the shifting pen of a chart recorder. Structure of a Single Locomotor Act The gate technique, together with video recording, demonstrated that spontaneous locomotor movements of the crayfish, which are directed mostly forward, have a rather slow mean rate (0.3-3 cm/s). Going backwards, probably caused by some unintentional extrinsic factors, was rare. One remarkable feature of crayfish walking is its nonuniformity. Single locomotion represents a succession of several brief forward-pushes, intermingled with long pauses, during which the crayfish was completely immobile. In one example an animal was observed to confine its movements during the first 20 seconds to the end of the chamber. Activity stops, then increases again with the passage of time. It should be noted also that, when moving along the wall, the crayfish does not usually change its direction before it reaches the end of the chamber, where it stops. In the round tank the crayfish preferred to walk along the circumference using the same way of moving (the succession of forward-pushes and pauses) as in the rectangular chamber. Background Locomotor Activity We have studied the dynamics of the locomotor movements of Procambarus cubensis over a long period of time by means of the gate technique, during which the animal remained mostly in darkness without any intentional influences. The mean rate (number of shuttle responses per minute), the regularity of movement along the long wall of the experimental chamber, and the mean speed of move-

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ment were higher during the first 10-15 minutes after being placed in the apparatus than in later periods. Some animals run from one end of the chamber to another without stopping at the ends of the chamber. In spite of significant individual differences, the interval between the first 10 shuttle responses was rather stable except for the first one (the interval between placing animal inside and its first shuttle response) which in about 30% of the experiments was much longer than the second shuttle response. It was also observed that both the length of the inter-shuttle intervals and the variability increased. Therefore, for quantitative evaluation of the movement activity (MA) the number of locomotions for a particular time period (mostly 2, 5, or 60 min) was the dependent measure. In experiments of one or two days duration (N = 27 crayfish), the number of shuttles per hour and its change over time varied strongly. However, in all experiments MA persisted not less than 10 hours. In fact its average value during the tenth hour from the beginning of the experiment was about 80% of that for the first hour, when it had reached its maximum. Thus the "spontaneous" MA of the crayfish decreased very slowly under constant environmental conditions. The first hour in these experiments took place in the morning or evening. On average, the distribution of the above-mentioned index (shuttle rate) reached a maximum at night and a minimum during the morning period, though in different experiments the position of these points varied. Visual inspection of actograms and measurement of the number of movements in succeeding 5-minute periods revealed several kinds of spontaneous MA in these experiments. The most common was single movements along the chamber (whose duration was 10-30 seconds), alternating with long pauses often lasting minutes. The middle movement rate was about one per 2-5 min (individual values ranged from 0 to 11). It should be noted that the same value could be maintained during 2 to 6 subsequent 5-minute periods; thus the level of MA was kept constant over time. In other observations, there were brief (not more than 10-15 min) alternating periods of very rushed and regular walks. This was typical of the behavior at the very beginning of the experiment. Then the maximal rate of shuttles was reached (in some individuals), of up to 4-6 per minute. The third remarkable observation of spontaneous MA was its complete absence for long periods of time (up to 2-3 hours). This phenomenon has been observed only in experiments lasting not less than a day, mostly in morning or noon hours. The primary concern in these experiments was the long lasting maintenance of MA in constant experimental conditions. This activity was additionally evident in a separate experiment whose duration was more than 10 hours, as well as in subsequent experiments with 5-7 day intervals. This behavior differs from crayfish behavior in its "home" aquarium where it (without external disturbances) prefers to be immobile. It is obvious that this stable MA is analogous to the "background, or general, movement activity" of many different animals placed inside a restricted room, especially if the room's dimension is close to the animals size. Although in such experiments the animal has a "choice" to move or not to move


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and then "decides" to move, it is not clear to what degree its movements are "spontaneous" or "intentional." Early in observation, this MA is caused primarily by experimental conditions requiring adaptation to new external factors. The persistence of this MA after adaptation to the experimental situation reveals its avoidance character. Initially, it reflects the crayfish tendency to escape a "novel" situation, even if the situation is favorable. It is remarkable that this tendency decreases very slowly (in analogous experiments with rats, such movements soon cease; Piazza, Deminiere, and LeMoal 1989). In fact, this tendency for spontaneous MA never ceases even in Procambarus cubensis reared for many generations in aquaria. In the conditions of the experiments described above, this tendency for movement seemed quite strong. We believe that the study of MA will be useful in comparative-physiological investigations, especially in studies in which related species are compared under analogous conditions. Moreover, consideration of the background MA seems to be helpful for reducing the delineation between the physiological and ethological approaches to the study of animal behavior. Let us look in more detail at the dynamics of crayfish MA in long-term experiments. It is obvious that one may see at least three kinds of MA. Two type of movements last tens of minutes each. The third type of movement consists of extreme intensive movements that last no more than 10-15 minutes. We suggest that different kinds of MA corresponded to different levels of the functional state of the crayfish. The highest level of alertness takes place only in the very beginning of the experiment. This likely reflects the immediate reaction to placement in the experimental chamber, together with its investigatory activity. There were also long-lasting periods of complete immobility, which we suggest may coincide with a sleeplike state of the crayfish similar to that described in the scorpion (Tobler and Stalder 1988). During the main part of the experiment, the MA was rather low and irregular (but much more intensive than in the "home" container). It appeared to correspond to the different stages of "quiet wakefulness" of the crayfish. This period is variable and requires more detailed analysis. It is clear, however, that evaluating the functional state of the animal is necessary in providing a complete analysis of a behavioral task. We would like to emphasize the importance of this index in behavioral investigations in invertebrates and the possibility of its estimation by means of noninvasive recording of the background MA (Shuranova and Burmistrov 1994). CRAYFISH RESPONSES TO SUDDEN CHANGES IN THE ENVIRONMENT The external world of the crayfish is replete with constant changes that can have significant effects on the animal. Consequently, all animals possess highly elaborated mechanisms for detection of these external disturbances. It seems obvious that higher vertebrates, when detecting large changes in their surroundings, react behaviorally not to these changes as such but rather to their probable consequences. These consequences can be predicted from their inherited or individual

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knowledge or even from the animal's life experience. In fact such an animal "pays no attention" to many external changes that are "neutral" from the biological point of view. When describing behavioral responses of lower vertebrates and invertebrates (Mordue and Golds worthy 1980; Pardi and Papi 1961) to similar environmental changes, terms such as "taxis" and "tropism" are frequently used. These terms do not reflect the reaction evoked by a specific environmental change, but rather reflect an animal's preference for some stable, external condition. This difference results in the impression that "lower" animals differ principally from "higher" ones. They react to the environmental changes directly, because of the inherited knowledge about its biological importance. Yet, on the other hand, they do not notice the environmental modifications that are without immediate effect. In spite of the well-known evidence indicating the elaboration of conditioned reflexes in many invertebrates and demonstration of the changes of inherited preferences, the ordinary point of view often considers invertebrates as situated anywhere between the "true" animals (mammalians) and robots. Crayfish Responses to External Stimuli under "Natural" Conditions Observations of these animals over a 10-year period and our video records of the crayfish Procambarus cubensis indicate that, when in a constant environment, they remain mostly immobile. This means that during many minutes they do not walk, move their appendages, or even change their posture. It is possible, however, to discriminate between different states of an individual crayfish based on its appearance. A behavioral state which we call "quiet wakefulness." characteristic of a crayfish living in the individual aquarium, is evidenced by a more-or-less flexed abdomen (with the telson touching the bottom) and relaxed walking legs (the claw being almost closed). Moreover, the antennae and antennules, which are extremely movable, rarely move when the animal is in this state. In fact, it is not easy to differentiate this state from one resembling sleep. In the latter, the crayfish reaction to even the presentation of food is very slow. Many external stimuli (light, tapping the side of the tank, placing some small object in the water, removing objects from the water), which may be seen as "neutral" but are doubtless perceived by the crayfish, do not produce, as a rule, any immediate movement reactions (if such movements did appear the latency was very long). This immobility would cast doubts as to the abilities of crayfish to perceive external stimuli. However, even in this situation it is possible to observe some reactions indicating the general activation of the animal. These include postural changes, lifting the first thoracic legs and opening the claws. Additionally, after some brief external stimulation, it is possible to see an increase in the rhythmic activity of specialized appendages responsible for the active flow of water into the brachial chamber (scaphognathites) and also of the abdominal appendages (swimmerets), which take part in the same ventilation process


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(Shuranova and Burmistrov 1988). The Autonomic Reactions Our observation suggesting that the ventilator system of the crayfish can be used to indicate the perception of external disturbances led us to undertake experiments that include the recording of the electrical activity of the heart (ECG), and the scaphognathites (SGG) in the adult Astacus. We have performed similar experiments with Procambarus cubensis. Both species were placed in an apparatus that allowed them to walk freely. In experiments on Procambarus the conditions were very similar to its "home" container. The parameters of the ECG and SGG in the absence of any external influences on the crayfish would remain stable for several hours. However, "spontaneous" and brief changes in the intervals and amplitude of the ECG-deflections as well as diversified changes in the SGG were occasionally observed. These changes in electrical activity of the heart and of the muscles moving the scaphognathites usually appeared simultaneously (Shuranova and Burmistrov 1994). Similar changes have been found under conditions where external disturbances did not evoke any visible responses from the crayfish (e.g., a soft tap on the side of the tank, or on the table where the tank was situated; various sounds outside the chamber; the oscillations of the water caused by placing some small object in the tank; moving "shadows" outside the tank, etc. The autonomic reactions developed just after presentation of these stimuli. The reactions lasted approximately 5 to 10 seconds and were in the same direction in both the ECG and SGG. In the case of the SGG, however, they were much more variable in appearance and outlasted the changes in the ECG. The most common autonomic response is represented by an increase of the interval between subsequent deflections, which could be concurrent with changes in their amplitude in the ECG and also with various changes in the form of the deflections in the SGG The illumination of dark-adapted crayfish did not result in marked changes of the autonomic indices of those described above. Often the ECG and the SGG did not change at all. Sometimes in Astacus, but more often in Procambarus, a slowly developing acceleration could be observed in response to general illumination of the chamber or to the presentation of a localized bright spot of light directed outside or inside the tank. There was a remarkable activation in the ECG and SGG after illuminating the crayfish with a very brief (1 ms) bright flash of light. In this case, the activation response occurred immediately upon presentation. The autonomic responses in Procambarus have also been recorded while the animals were under the influence of a strong (10 mT), alternating (50 Hz) electromagnetic field. Autonomic responses to such stimuli appeared to be greatest after the stimulus was terminated. It should be noted that all of these autonomic responses (including responses lasting for long periods of time) occurred mostly in the absence of any visible movements. Only after illumination of the experimental chamber were there any noticeable movements. Even here, however, the move-

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ments were restricted to the separate walking legs, not extending to displacement of the entire body. We may conclude, then, that external stimulation that does not evoke some visible reactions (behaviorally "neutral" for the animal) are detected by its central nervous or neuroendocrine system. This is evident in the changes of the activity of main vital organs such as the heart and the ventilation system, which are, in decapods, under strong neuroendocrine control (Cooke and Sullivan 1982). It should be noted that, in spite of the obvious anatomical differences, there is a remarkable similarity in the central control of the heart and ventilation activity between crustaceans and vertebrates. This is not only evident from central control but also in the sophisticated control of the ventilation system. This seems to be clear from the biological point of view; the heart, supplying body tissues with oxygen and food materials, must function with more stability than the ventilator system. Locomotor Reactions It has been noted that a characteristic feature of crayfish is a lack of immediate locomotor reactions in response to sudden changes in the surroundings. It is possible, however, to examine the reactions of the CNS that are due to changes in autonomic arousal and background locomotion activities. This analysis has been undertaken in experiments on Procambarus cubensis, whose locomotions were recorded by means of the "gate" technique. This index allowed us to observe the reactions of crayfish to an artificial alternating electromagnetic field of middle (not more than 10 mT) intensity. The main finding of these experiments is that the crayfish sensed the field. This was reflected in the changes in background MA (Shuranova, Sadauskas, and Vekhov 1988). The reaction to the field depended strongly on the previous level of the background MA, and was expressed as a decrease or increase in activity. In a series of experiments in which the middle level of the background MA was extremely low (about one walk for 30 min), the activation of MA increases rather uniformly during the 5-minute application of the electromagnetic field. This increase occurred in 70% of the trials. It may be suggested that the true latency of the crayfish behavioral reaction is shorter than it appears; but because the first component of the response is inhibitory, short responses cannot be revealed in such an experiment. In a special session on the same animals, we tested them in the very beginning of the experiment when their MA was high and regular. Then brief (less than 1 minute) application of electromagnetic field resulted in the elongation of the interval between locomotions following its presentation. In general, the crayfish behavioral reaction to the application of external factors seems directed toward the restitution of the environmental conditions that had existed before. It has been suggested that the essential role in the detection of such a complex factor as an alternating electromagnetic field is to act on the mechanoreceptors, which are located in extremely large numbers on the surface of the body and the appendages of the crayfish (Bush and Laverack 1982; Laverack 1987).


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Several experimental sessions have been conducted on the same animals with the aim of investigating their motor reactions to a change in illumination. It seems obvious that this external factor is rather complex. The main changes that have taken place in the motor system of the crayfish in response to this stimulus are short-latency EMG-activation, caused by the excitatory reaction of the on-cells in the optic lobe, and long-latency EMG-activation due to the response of the socalled caudal photoreceptor located in the last abdominal ganglion (Galeano 1976; Kennedy 1958; Prosser 1934; Shuranova and Burmistrov 1988; Wilkens 1988). The activation of the motor system resembles in principle the processes taking place at different levels of the motor system in mammalians, caused by light stimuli. This phenomenon seems to reflect the increase in the "readiness" of the animal to counteract the environmental changes. For example, in the rabbit it is possible to record not only EMG-reactions to light arising in different skeletal muscles (Revina and Shuranova 1980, 1981) but also the movements of one or several limbs as well. In the crayfish, the main effect of brief (20-40 seconds) illumination accessible to observation was the complete cessation of any ongoing motor activity. The locomotor movement appeared often just after the end of the illumination (more than 50% of such movements developed during 30 seconds after the end). This probably reflects only the beginning of the long modifications in nervous activity caused by the reintroducing darkness. In fact, the most remarkable component of the behavioral reaction was the increase of the rate and regularity of crayfish walks over several minutes (up to 10-12 minutes) after a single presentation of light. The manifestation of this component of the behavioral response depended on the preexisting level of the background MA, but to a lesser degree than at the application of the alternating electromagnetic field. The activity changes in the background MA predominated after brief illumination of the crayfish. One may suggest that this external disturbance evoked the searching behavior of the crayfish to a greater degree than did the alternating electromagnetic field. The abrupt changes in the environment, which had no immediate effect on the behavior of the crayfish, caused not only a relatively short (seconds) shift in the functioning of the heart and ventilator systems but long-lasting (minutes) modifications in its preexisting background MA. This indicates essential changes in the integrative activity of crayfish CNS caused by external factors that may be characterized (arbitrarily) as neutral ones. THE PHYSIOLOGICAL NATURE OF CRAYFISH RESPONSES TO SUDDEN CHANGES IN ITS SURROUNDINGS The physiological nature of the above-described crayfish reactions to some external stimuli warrants special consideration. It has been noted that such types of invertebrate responses are usually characterized as purely motional, necessarily directed toward, or away from, the source of the stimuli. On the other hand, in papers devoted to vertebrates, especially to mammalians, there is a long tradition

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of looking at such reactions as "orienting" responses. The beginning of this tradition is in Pavlov's experiments with conditioned reflexes. He wrote in 1923: Everyday, or one may say every minute, we meet in our experiments with the positive, active animal reaction to each oscillation in its environment. This is the fatal reaction of the organism—a simple reflex which we call the orienting, or purposive reflex. At the appearance of a new agent in the surroundings, the animal orients its "detecting surfaces" according to this agent to have the best impression of it. Other ongoing activity must go away in the case of immediate requirements of the external world. This is the most bothersome, really non-omitted, non-overridden cause of the disturbance of our main event that is the conditioned reflex in our laboratories now. It is obvious that this event should be investigated thoroughly in all directions . . . but it represents a great obstacle for studying many facets of our primary phenomenon, (cited from Pavlov 1951: 133). Later, from the investigations of P. K. Anokhin (1958), E. N. Sokolov (1958), O. S. Vinogradova (1961), and others, it was shown that the orienting response was not only "impeding" but also essential for the elaboration of the conditioned reflex. It became obvious that the reaction, which seemed elementary at first, has a complicated structure. It involves many parts of the central and autonomous nervous systems and manifests itself in, for example, the modification of the EEG, in various changes of muscular, heart, and ventilator activities (Guselnikov 1958). Besides this generalized reaction, higher animals and humans demonstrate specific modifications in the activity of the appropriate "analyser" that facilitate the perception of a stimulus (Sokolov 1958). It may be noted that Russian contributions in studying the orienting response are well known and have been emphasized at a special international meeting devoted to the orienting reflex in humans (Kimmel 1979). It has also been demonstrated that this reaction, being "nonspecific," depends on many factors, including the relevant sensory system (Petelina 1958). Also very important is the kind of behavior that follows the orienting response. This factor hardly could be quantified in experiments on restrained animals. However, this factor becomes a very important consideration in experiments on freely moving animals (Valdman and Kozlovskaya 1972). There is a question as to at what phylogenetic stage such a phenomenon as an orienting reflex first appears. It was Birukov's (1958) opinion that this phenomenon should be studied using a comparative-physiological approach. However, "one may not consider it too broadly; different, and rather complex, reactions of paramecians, sea-urchins, sponges and so on, evoked by external stimuli should not be considered as orienting ones" (Birukov 1958:21). He goes on to say that "It is reasonable to consider the orienting response only in the case of the animals which can realize this reflex by means of appropriate sensory and motor structures" (Birukov 1958:21). A similar point of view has been expressed by Kostandov: "The true orienting reflex may be developed only in animals possessing the neocortex, characteristic in reptilians" (Kostandov 1970:211). It should be noted that there are no experimental or theoretical works devoted to the problem of the orienting reflex in higher invertebrates.


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Therefore, let us look in detail at the above-described locomotor and autonomic responses of the crayfish to sensory stimuli. The activation of many muscles in the walking legs and abdomen has been observed in Astacus sp. in response to general illumination involving the complex eyes and to local illumination of the caudal photoreceptor (Shuranova and Burmistrov 1991). This activation of a motor system corresponding to the change of the functional level of the brain seems to be similar to the muscular reaction of the rabbit (and other mammalians) (Revina and Shuranova 1980, 1981), which also coincides with the arousal in the electrical activity of the cortex. The state of "readiness" did not result in the development of true locomotion in the crayfish during illumination but manifested itself in the locomotor act just after the cessation of the light and also in the activation of the background MA during the next 10-12 minute period. It may be suggested that this reflects the enhancement of crayfish's searching activity in the darkness following illumination. It was noted earlier that autonomic responses during illumination were absent or consisted of rather slowly developing increases in the rate of ECGand SGG-deflections. Uncertainty of the autonomous reaction to illumination also took place in experiments with dogs (Petelina 1958). Much more remarkable autonomic responding was observed when we used a brief intense flash of light. We believe that the long lasting activation of the ECG and SGG corresponds to searching behavior of the crayfish. When we directly compared the effects of exposure to steady illumination with that of a brief flash, the results were similar, however, the flash evoked a stronger reaction. The delayed activation of background movement has also been observed in response to the application of an alternating electromagnetic field, which evoked activation changes in the ECG and SGG. To the contrary, many extrinsic stimuli that were ineffective in eliciting motor reactions in the crayfish resulted in a decrease in autonomous activity. It was shown that often the heart can completely stop for several seconds, confirming the data of Mislin (1966) (lobster heart can "shut off for at least 15 minutes: Wilkens 1976). It may be suggested that heart arrest is a "bypass product" of inhibition in the scaphognathite movements, which may influence the detection of sound or vibrational stimuli (in fact, there is a common central control of the heart and ventilator systems: Field and Larimer 1975). Let us compare these crayfish reactions with the orienting response of higher vertebrates investigated by many authors. It is obvious that there is no reason to tell about the orienting response "in the strict sense," that is, about the modifications in the functioning of the appropriate "analyser," because of many obvious anatomical differences between animals of such distant taxonomic groups. Yet, it is helpful to consider those components of the orienting response that reflect nonspecific changes caused by the "unexpectability" of the external factor, usually called its "novelty." Unfortunately, there are some problems in summarizing data of other authors when different experimental conditions are used. Additionally, in experiments with animals it is not easy to distinguish between the orienting response as such and the behavioral response following it (Sokolov 1958; Valdman and Kozlovskaya

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1972). It is important, too, to be sure that there is an orienting reflex as such, in the absence of any additional reactions (Pakovich 1985). Therefore, it is easier to compare our data with the data obtained in humans where the orienting response was not complicated by any behavioral manifestations (Graham 1979). F. K. Graham undertook an analysis of ECG changes to auditory stimuli and discriminated three components of the reaction caused by the complex nature of the stimulus. The abrupt onset of a stimulus can evoke a startle-reaction which coincides with the short-latency, brief acceleration in the ECG. The main result of the startle-reaction is very rapid interruption of the ongoing motor activity. The second component is the orienting response as such; it correlates with the slowing of the ECG. This component may be seen better in the case of weak stimulation, or after the cessation of a strong stimulus. The significance of the orienting response, according to Graham, resides in the increase in sensory input to the organism. The third component of the reaction is evoked by the "plateau" of the stimulus. It is represented by the delayed acceleration in the ECG and may be interpreted as a "defensive" response, depending greatly on the physical intensity of the stimulus. These components can be divided experimentally (for example, by means of the repetitive stimulation) but usually, especially in testing a single stimulus, they overlap each other. In accordance with these views, the above-described crayfish responses to different extrinsic factors, whose main component has been represented by the inhibition of the ongoing oscillations in the heart and ventilator systems, seems to be analogous to the orienting response in the strict sense. It has been observed mostly in response to brief stimuli acting on the mechanoreceptors of the crayfish, which have a common nature with the sound receptors of vertebrates. As noted above, the short-lasting inhibition in the activity of some inner organs could be helpful in the detection of stimulus location. It is not clear, however, in what way it is possible to consider the acceleration changes of the autonomous activity that took place in response to illumination or to the alternating electromagnetic field. They might have common features with defense reactions as defined by Graham, but they also depend on the physical nature of the external factor (light energy, first of all) and manifest in general agitation of the crayfish, which develops gradually and lasts for a long time. The primary conclusion here is that crayfish responses to "neutral" stimuli may be compared with reactions of the higher vertebrates (including humans) in the sense that they begin with manifestation of the orienting response. The function of this response is probably to permit the animal to concentrate on some sudden change in the environment that has no immediate effect on it and that produces uncertainty as to sequences brought on by the environmental change. Here, then, is an example of a situation in which it would be possible to expect the appearance of "emotional" reactions in the crayfish. It is not our aim to discuss here the extremely complex problem of emotions. However, we would like to emphasize some important points concerning their nature. First, emotions play a central role in the behavior of individuals (Simonov 1981) and of groups (Salzen 1991).


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Second, a requirement for situations likely to produce emotional reactions have "pragmatic uncertainty in regard to the actions which should be done" (Simonov 1981). "The term emotion was used to describe the process of monitoring the state of the world with regard to expectations based partly upon commands issued by motivational systems. Correspondence with, and deviations from, expectations are appraised and either positive or negative emotions generated" (Toates 1988:29). Third, it is known that emotions are a component of goal-directed (motivated) behavior. Among many motivations that govern animal behavior is, in our opinion, the tendency to keep the external world constant. In fact, every disturbance in the environment can result in different consequences for the animal, which may be evident only after some delay. Thus, when the "expectancy" for a stable environment disappears, it is possible to predict the appearance of emotional reaction in the animal. Exactly the same situation has been described above in the experiments with the crayfish. There are, however, problems concerning the phylogenetic view of emotions. Emotional reactions were not considered in some books about invertebrate behavior (e.g., Dethier and Stellar 1970; Dewsbury 1978; Kandel 1976). Additionally, there is no mention of invertebrates in many works specifically devoted to emotions (Salzen 1993; Simonov 1967, 1981; Toates 1988). It is known that Darwin (1872) suggested the existence of emotions even in crickets, but during that time invertebrate behavior was studied only sporadically. Later, Vagner, who was a great specialist in invertebrate behavior (especially spiders and bumblebees), concluded categorically that emotions are absent in all the invertebrates (Vagner 1913, 1925). He found, for instance, that the rate of heart contractions estimated visually in the spider changed only during animal movement (Vagner 1925). Add to this Shepherd's opinion, We must conclude that a complex innervation of the internal viscera, and a complex set of muscles which can independently signal autonomic and other internal states, are two components that are necessary for the expression of emotion in animals. These two components are almost entirely lacking in invertebrates and lower vertebrates. Therefore, although these animals can express behavioral states through whole body actions, they lack the ability to express emotions, as we generally understand this term. (Shepherd 1983:531) On the other hand, some authors consider emotions as a fundamental behavioral mechanism existing in every animal, independently of its phylogenetic position (Fonberg 1986; Plutchik 1980), but this view is not substantiated with experimental evidence regarding the invertebrates. In accordance with Shepherd's opinion, the decapods have highly elaborated internal organs controlled by autonomic neuronal nets whose activity is modified by a set of neuroactive substances and neurohormones released from the endocrine glands (Fingerman 1987), as well as by inhibitory and excitatory neuromediators produced in cerebral ganglion (Cooke and Sullivan 1982). This ability (together with large body size) distinguishes the decapods from most insects, whose small

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size and specific construction of the heart and tracheal respiratory system prevent study of their autonomic reactions. As for the means of expressing different internal states, including emotional ones, they are evidently in existence in many arthropods. In decapods, internal states may be reflected not only in specific body postures but also in movements of appendages such as the antennae, scaphognathites, and swimmerets. Thus decapods are highly suitable for the investigation of objective indices of emotional reactions such as "expressive movements" and "autonomic responses." We consider our conclusion about the existence of emotional events in crayfish as preliminary and certainly requiring additional investigation in different situations and with the aid of different indices. However, in the above-described experiments there was both the required uncertainty of the situation produced by some unexpected external factor and the marked autonomic reactions not correlated with animal movement. This permits us to suggest that there is an ability to achieve emotional estimation of the external events in crayfish, as well as in other higher crustaceans. Thus, the delineation between behavioral organization in invertebrates and vertebrates seems to be situated not merely in the animals' ability to generate overt emotional responses. How the ganglionic nervous system can produce these reactions, which require many highly elaborated brain structures in mammalians, should be investigated specifically in the future. CONCLUSION Using Decapoda as an example, one may see that the progress in neurophysiology (the progress that was a dream of T. H. Huxley over a hundred years ago: see Huxley 1880) had rather little influence on studying the integrative behavior of these animals. The development of the neurophysiological investigations chiefly resulted in many discoveries concerning neuronal mechanisms of different reflex reactions, from rather simple (claw closing and opening, eye withdrawal), to the very complex (coordinated swimmeret movements, coordinated movements of the walking legs, coordinated actions of many muscular groups involved in the "tailflip") (Atwood and Sandeman, 1982; Burmistrov 1991, Shuranova 1991; Sandeman and Atwood 1982; Shuranova 1991; Shuranova and Burmistrov 1988). At the same time, knowledge of the integrative functions of the central nervous system, particularly the complicated ones that manifest themselves in individual adaptations of the animal, remain at a level not far from that at the beginning of the 1960s (Schone 1961, 1964). We are facing a situation in which many invertebrates serve as "models" in studying some problem, including learning. Still, the opinion prevails that calls for the division of all animals into two groups, the first consisting of "true" animals such as the higher mammalia and the second consisting of lower vertebrates and all invertebrates. The behavior of the latter is thought to resemble that of robots. It is obvious that there are many reasons for this division. First of all, there are great anatomical differences between the humans and invertebrates. There is also an ab-


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sence of domesticated invertebrates ("pets"). It should also be noted that there are many dangerous invertebrates that surround and force humankind to confront them. It should further be noted that many data indicate highly developed learning abilities in insects. Bees and ants can solve many complicated tasks in a similar fashion to some higher vertebrates (see Kartsev, this volume; see also Udalova and Karas, this volume). Such research suggests that some insects have an ability to form abstractions, to generate comprehension, and so on. This fact seems surprising; how is it possible to attribute such abilities to the extremely small piece of nervous tissue that makes up an ant's or bee's brain? On the other hand, it is known that modern microcomputers can do many things that are inaccessible to the human brain; however, there is a large gap between humankind and computer. The same may be true for the higher mammalians and higher invertebrates. It is not obvious beforehand that they solve even comparable tasks using the same principles. Therefore, it is necessary to compare invertebrate behavior with that of higher mammalians to a greater degree. This ultimate goal spawned the choice of our experimental animal. The crayfish in the world of invertebrates holds approximately the same position as the dog or the rat among vertebrates. It is possible to compare them using many parameters. For instance, comparison of the crayfish and the rat (whose behavior has been investigated by many authors and has been reviewed many times: Barnett 1963; Kotenkova, Meshkova, and Shutova 1989; Silverman 1978) indicates that their behaviors have many common features. It is clear that these crustaceans may better be compared with the mammalians than with insects that possess mostly very small body size and a short life span and whose development includes metamorphosis. It appears important, too, that there be opportunities to conduct behavioral research on decapods (crayfish, crab, shrimp) that have lived for several generations in freshwater aquaria and are adapted to everyday "contacts" with humans. Animals such as ours, whose "natural history" and individual behavior can be traced throughout their life span, are suitable for the evaluation of individual features of their behavior. Pavlov considered the analysis of individual behavior crucial in his experiments with dogs. As with humans, understanding these individual differences will require time and sophisticated testing procedures (Teplov 1985). It seems evident that, for evaluating individual features in animal behavior, it is necessary (1) to know perfectly the peculiarities of species behavior and (2) to investigate thoroughly the behavior of at least one representative individual. In this respect humankind has great experience with domesticated animals, especially as they have lived together during all of their respective cultural histories. It is interesting to note that other mammalians (all of them close to humans in their constitution) living in the wild are much less known. There are different ways to study their individual behavior (first of all, by domestication of some individuals), but there are also problems at every turn. These problems are not comparable, however, with those confronting us if we try to investigate the individual features of invertebrate behavior. There are no invertebrates similar to the cat or to the dog;

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moreover, only very few invertebrates are welcome in the daily life of humans. Many invertebrates that share their habitat with man live nevertheless in "another world." As some invertebrates domesticated by man (honeybees, for example) do not differ from their wild relatives, the benefits of domestication seems questionable (Vagner 1913). Domestication of invertebrates is known only in anecdotal form. Therefore, many factors strongly interfere with studying individual features in invertebrate behavior. The simplest "solution" to the aforementioned problem is to conclude that these mostly small creatures possessing negligible numbers of nerve cells are not capable of modifying essentially inherited features as a function of life experience. Therefore, it is not too far-fetched to say that they do not have any individuality, namely, that all of them have "the same face" and that their differences are completely within the range of "intraspecies variability." This point of view has been expressed many times by Vagner (1913, 1925), who had determined invertebrate psychology as the "segmentary psychology" (Vagner 1914). It should be emphasized, however, that there is no substantial experimental evidence for such a conclusion. It may be noted that physiology (and comparative psychology) is suited to the investigation of individual characteristics of animal behavior to a greater degree than ethology. Indeed the physiological approach permits measurement of the functional activity of different organs and systems. This allows us to characterize this activity (including behavioral) by means of many indices and to compare different individuals using many parameters. Additionally, this approach presupposes studying all the parameters on the same time scale, that is, dependent on the ongoing state of the animal and on the existing environmental conditions. The data obtained in our work with the crayfish Procambarus cubensis allow us to suggest that these animals possess marked individual behavioral features that manifest themselves in the course of long observation of their "natural" behaviors. Due to experimental investigation of the background locomotor responses and autonomic activity recorded in freely moving crayfish, it has been suggested that the crayfish possess some signs of individuality in their behavior in common with that of higher mammalians. Crayfish reveal a wide spectrum of functional states, which are determined to a great degree by external influences. The crayfish is continuously "monitoring" the state of the environment and reacts immediately to the smallest changes. It is possible that in their responses to sudden changes in the external world, which seem to be analogous with the orienting response of mammalians, crayfish can show emotional manifestations that are individual estimations of the situation. It seems evident that crayfish can contribute directly to the study of the orientation response and emotional expressions by multiplying the number of indices and broadening the set of experimental situations needed for a comparative analysis. This could provide the prerequisites essential for studying individually acquired reactions in these invertebrates at a level comparable with that of the higher vertebrates.


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Chapter Six Learning, Memory, and Motivation in Ants Galina P. Udalova and Anna Ja. Karas

Ants possess one of the most complicated social organizations in the order Hymyenoptera. This complexity manifests itself in a wonderfully diverse form of intraspecific and interspecific relations. Much of this complexity is derived from the behavior of individual colony members. The complicated forms of individual behavior and the principles of ant social organization suggest that there are common traits between ants and vertebrates. Comparative questions about the evolution of ant behavior and the ecological importance of ants have attracted many specialists to study these important and interesting animals. In Russian myrmecology there are several main directions. The first includes the study of ant biology, which comprises the investigation of the ecology, ethology, and physiology of ants. These studies are concerned with comparative questions among species and the role of ants in the ecosystem (Arnoldi 1970; Dlussky 1967, 1975, 1981a, 1984; Kipyatkov 1974, 1981, 1986, 1991; Marikovsy 1979; Reznikova 1971, 1975, 1983, 1990; Ruzsky 1905, 1907; Tarbinsky 1976; Vagner 1913; Zakharov 1972, 1974, 1981, 1991). The second direction involves the practical utilization of ants in forest defense. The problem was recognized in the 1960s and stimulated interest in the investigation of ants. Attention was given to such problems as the ecology of various ant species, methods of rearing, and their relationships with plants and other animals, particularly vertebrates. During this period, great importance was attached to investigations concerned with developing methods of maintaining ant populations under both field and laboratory conditions. Investigations were also carried out testing the possibility of using ants as indicators of different natural breaches in biological monitoring. These aspects of ant investigation are reflected in the collections of studies, Ants and Forest Defense. These books reflect the proceedings of the All-Union Myrme-

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cological Symposia. The first was held in 1963 (see also Sejma 1979). The third direction involves the study of individual and group behavior, particularly as it bears upon the problem of colony organization. It includes investigations of phenomenology and the mechanisms of communication and orientation in ants. As to the problem of orientation the following aspects are considered: the sensory stimuli needed for spatial analysis, the strategies and tactics of movements, and the orientation mechanisms involved in learning and memory (Elizarov 1970; Eriyshev, Kaul, and Legkostupova 1991; Frantsevich and Zolotov 1986; Kaul 1977a-c, 1983, 1985, 1987, 1991; Kaul and Kharlamova 1987; Kaul and Kopteva 1982; Kaul and Korosteleva 1990; Kupressova 1984; Kupressova and Plekhanov 1979, 1981; Reznikova 1979, 1983; Sulkhanov 1979a, 1979b, 1980, 1986, 1989, 1991). In addition to research reports, there are monographs (e.g., Frantsevich 1986) and many articles and communications appearing in such special collections as Ants and Forest Defense (1963-1991) and the Proceedings of the Colloquia of the Section on Social Insects of the Ail-Union Entomological Society (1990). Among the studies investigating the problem of ant communication are Wassmann (1906), Zabelin (1979), Kaul and Eryischev (1991), and Sulkhanov (1991). Special attention should also be given to the work of G. M. Dlussky (1981a, 1981b). Dlussky showed the importance of learning in the communication system of for aging ants. It is essential to investigate the ability to learn and remember in ants because of their complicated forms of behavior and structure of their brain. Therefore, it is understandable why particular interest has been shown by Russian workers in the influence of various biological and ecological factors on learning and memory in ants. We consider the study of learning and memory the fourth direction in Russian investigations of ant behavior. In this chapter we will provide a review of the literature and present data from our own investigations. In our experiments we have studied such problems as the peculiarities of ant learning and memory under different types and levels of motivation, the regularities of spatial orientation, and evaluating the behavioral plasticity of ants. The primary technique used in these experiments was a multipleunit maze. This technique allowed us to closely approximate the conditions found in the ants' natural environment. In addition, the maze technique, because it deals with foraging behavior, allows us to compare the ability of ants with that of animals from other phylogenetic groups. INVESTIGATION OF LEARNING AND MEMORY IN ANTS Memory is a capacity to keep and reproduce information about the inner and external environment, including information related to behavior. Much of the behavior of animals is based on a "genetic" memory. Some behavior, especially that acquired by learning, is due to individual memory. The traditional view that some type of behavior is due solely to instinctive reactions and that other types are due solely to experience has been replaced by a position that stresses the

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interaction between them. The importance of this interaction can be seen in studies in which learning is modified by inborn mechanisms and those in which inborn reactions are modified by experience. This "interactionist" position has been based, for the most part, on experimental work performed with insect behavior. The Russian investigator G. A. MazochinPorshnyakov (1975, 1980), for instance, described two forms of behavior: (1) programmed behavior (inborn, instinctive) with a genetic makeup that sets into motion a succession of operations retained in species memory and (2) modified (learning) behavior based on individual skills and memory. Having noted the relativity of this division, Dlussky (1984) suggested that both innate and acquired behaviors include a genetic component that determines the predisposition of the nervous system to engage in behavior. It is obvious that every behavioral reaction is determined by the information encoded in the species and individual memory of the organism and is provoked by the current sensory stimulation. Thus, the general idea, well known before the classic work of von Frisch (1965), regarding the instinctive nature of all insect behavior became outdated after it was found that insects, especially the highly organized species, are capable of learning. Moreover, we believe that the capacity to learn in some insect species, particularly ants, is not inferior to that of vertebrates. This conclusion is confirmed by the results of numerous Russian investigations (see, for example, Lobashov 1951; Lopatina 1971). There is every reason to believe that the learning ability of insects, such as bees and wasps, can be expressed not only in "simple learning" situations but also in situations requiring abstract solutions (Mazokhin-Porshnyakov 1968, 1969, 1974, 1975, 1980, 1981; Mazokhin-Porshnyakov, Semenova, Kartzev, and Rabinovich 1987; Semenova 1982). In contrast to the study of conditioned reflexes, the ability of ants to form "representations" has been studied to a lesser degree. One can state, however, that ants are able to exhibit simple and complicated forms of learning during the solution of sensory, instrumental, and spatial tasks (Dlussky 1981b, 1984; Reznikova 1983; Zakharov 1974). It is accepted that there are two kinds of ant learning: that reflecting knowledge of the individual and that reflecting knowledge of the group. Group learning has been investigated more then individual learning because ants are social animals. Individual learning has been considered mainly during the investigation of visual recognition and maze behavior. The simplest example of ant learning is observing individuals rushing to the place where earlier they have found prey. This is seen when the passive forager Monomorium kuznezowi, being mobilized to some food source, continues coming to the location of that source for several days, though the food has disappeared (Zakharov 1972). All data on learning in ants may be divided into two parts: (1) elaboration of reflexes to the stimuli of different modalities (i.e., visual, acoustic) and (2) forming a maze habit.

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Conditioned Reflexes to Visual and Acoustic Stimuli in Ants Vision plays an important part in the life of many ant species (Dlussky 1975; Dlussky, Volcit, and Sulkhanov 1978). In natural conditions, ants of different species have been shown to orient visually according to the contours of ground objects as well as to small visual objects (Kaul 1977a-c, 1983; Kaul and Kharlamova 1987). Hence, it follows that it is possible to elaborate in ants a conditioned reflex to visual stimuli. On the basis of morphological and electrophysiological investigations of ant vision (Mazokhin-Porshnyakov and Trenn 1972), it has been found that their visual discrimination is essentially lower than that of vertebrates. Nevertheless, the architecture of the compound ant eye and the location of the visual organs on their head allows them to see the entire surrounding hemisphere. The ability of ants to form color discriminations was investigated in the ant Formica sanguine (Mazokhin-Porshnyakov and Murzin 1975, 1977; Murzin 1976, 1977), using the method of the conditioned reflex. It turned out that only 5-7 conditioned stimulus (CS) - unconditioned stimulus (US) (food) pairs were enough to form the reflex to a color-conditioned stimulus. After this procedure, ants preferred to follow the color previously paired with food independent of location and brightness. Thus, the idea that ants have the capacity to discriminate colors was supported, and some suggestion was made as to how ants might utilize color information in their foraging behavior. The capacity of forming a food conditioned reflex to color stimuli was also demonstrated in the ant Formica rufa (Kaul 1977a). In this experiment 7 CS-US combinations established a stable conditioned reflex that did not extinguish after an interval of 14 days. A special question arises concerning the type of visual signals recognized by ants as landmarks. To answer this question, foragers of two ant species, Formica sanguinea and Catagliypnis setipes, were placed in a situation in which their preferences for different types of visual stimuli as well as the elaboration of conditioned reflexes (with food reinforcement) could be studied (MazokhinPorshnyakov and Murzin 1977; Murzin 1976, 1977). The foragers of both species were taught to select the correct path to food according to the shape and color of the visual stimuli (independent of their size). These reflexes were elaborated faster than those formed with color only. Visual stimuli differing in shape and pattern can also be differentiated by ants Myrmica sanguinea. Ants preferred more "dismembered" figures to less dismembered ones and horizontal shading over vertical shading. They can also be taught to discriminate a triangle from a rectangle in an invariant manner, independent of the projection transformation. The latter result suggests that ants appear to be able to form abstractions. Foragers of Cataglyphis setipes turcomanica succeeded in learning to discriminate a circle, a triangle, and a square. One could conclude that the color and the shape of the visual orientators are important determinants for foraging ants (Mazokhin-Porshnyakov and Murzin 1977). The data indicate a hierarchical organization of visual signs for landmarks signalling food. There is an opinion that


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the number and type of angles are of greater importance than the concrete shape or size of a geometrical figure. The ant Lasius niger L. is also able to form a conditioned reflex to color (Inozemtsev and Ostrovenko 1986). Rather than food, as in many of the previous experiments, the reward was larvae from the colony. The ants received a reward if they ran to a yellow color or received a light mechanical blow if they ran to a blue color. Within 8 trials the reflex was elaborated and was remembered for at least 3 days. It could be extinguished after 5 nonreinforcments. The data we have just reviewed and those obtained in other countries indicate that ants can perceive a large spectrum of visual stimuli and have a high capacity to use them in the formation of conditioned reflexes. The ability of ants to develop conditioned reflexes to sound stimuli has been studied to a lesser extent. Esk'ov (1973) demonstrated that the ant Formica rufa could find the location of food by pairing food with a sound signal (1000 Hz). Five trials were sufficient to produce an orientating reaction to food caused by a sound alone. According to Kaul (Kaul 1977; Plekhanov and Kaul 1975), Formica rufa can elaborate a conditioned reflex to sound stimuli at the same rate as that needed to demonstrate such conditioning with visual stimuli. Maze Learning in Ants The elaboration of motor skills in mazes of various types is probably one of the most important techniques available for the study of learning in animals. When used for the study of ant behavior, the maze method can reveal some regularities of ant spatial orientation. This method also provides data that enable us to compare learning abilities in animals belonging to different levels of phylogenesis. Maze learning in ants has been studied for a long time in Western countries. It appears that ants can learn to negotiate a maze faster than other insects and perhaps quicker than fish, frogs, and tortoises (Thorpe 1964). This is in agreement with data on their high capacity for spatial orientation that have been obtained by foreign and Russian researchers (Dlussky 1967; 1981b; Elizarov 1970; Frantsevich 1986; Frantsevich and Zolotov 1986; Kaul 1987; Kaul and Kharlaova 1987; Kaul and Kopteva 1982; Kupresova 1984; Kupresova and Plekhanov 1979, 1981; Reznikova 1983; Sulkhanov 1979a, 1979b, 1980, 1986; Vagner 1913; Zabelin 1979). Food reward is most often used in the elaboration of the maze habit. In the Russian literature there is only one study employing a defensive irritant (electrical shock) as reinforcement in a T-maze (Vasilyeva and Vasilyev, 1987). The typical criteria used for estimating the capacity of ants Formica sanguinea and Formica cunicularia, for example, to negotiate a maze are as follows: (1) the rate of forming an active and passive avoidance reflex and (2) the number of correct and incorrect reactions. S. I. Zabelin (1979), using a cross-shaped maze, studied the transfer of directional information by transmission of a tactile code to nest mates in the ants

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Tapinoma simrothi caravaevi. In the first variant of his experiments, food was located in one of the maze arms. After the food had been transferred to another arm of the maze, foragers orientated themselves to the new direction. In this case the new choice was facilitated by tactile contact between the individual foragers (see also Reznikova 1987). In a second variant of the experiment, food was located in both arms of the maze simultaneously. An ant that was entering the maze was observed to select an arm, not randomly as would be expected, but one that had just been visited by a nest mate. This occurred after an exchange of antennae strokes. These data provide evidence that ants can communicate by some type of "antennal code." Various maze designs for the investigation of ant learning and spatial orientation were developed by Zh. I. Reznikova (1983). In some species of ants the formation of the maze habit is a function of the type of colony and ecological conditions. Mazes of various design were used in these studies. For instance, a maze might consist of several cylinders, one placed inside the other in such a manner that their outlets face in opposite directions. The food bait is located in the inner cylinder. In the beginning of an experiment, ants were allowed to eat in the area near the foraging zone of the periphery in a single cylinder (the maze of the first degree of complexity). It was found that during the first hour of observation about 70% of Formica uralensis succeeded in penetrating the maze of the first type. As the experiment, continued the maze was made more difficult by adding more cylinders. Under these conditions, fewer ants were successful. However, the ants Cataglyphis turcomanica, when faced with the same problem (i.e., increasing complexity), were much more successful in negotiating the complex mazes. These differences were explained by the fact that C. turcomanica forages in groups. Species differences were also observed among Formica pratensis and Formica cunicularia, with the latter species exhibiting better performance (Reznikova 1971, 1975). Additional data indicated that the radial arm maze can be used to reveal species differences in ant learning and suggest that some species can master the logic structure of a problem and apply the experience to a new situation (Reznikova 1979, 1983, 1987). In other experiments, mazes were constructed not in the shape of cyclinders but in the form of fans. One such experiment was performed on a laboratory nest of Formica polyctena. A radial maze was constructed with a vertical bar. The top of this bar contained 11 compact paper strips in the shape of a fan (each strip was at a 15° angle). The reward, a small drop of sugar syrup, was placed on the the upper strip. Then, every 10 minutes, the drop was moved to other strips, with the angle between the original position and subsequent positions increasing. Dependent variables included total search time, the number of visits to the "correct" strip, and velocity of the movement to the lure. The results indicated that ants can quickly guess the strip on which the lure will next appear and that the memory of the task can last for 10 days. The data described above suggest that some species of ants can accumulate information on the direction of a food source, the number of food items, repond to


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temporal regularities present in the situation, and transfer and receive this information to and from other ants. This has also been demonstrated (Reznikova and Ryabko 1989) in experiments employing "binary tree" mazes. In such mazes, ants had to transfer information about the succession of turns on the way to the lure to other ants that did not visit the maze. On the basis of such findings the conclusion can be drawn that ants can find the algorithm necessary for a solution of a task and have the ability to extrapolate. This ability is present in honey bees and in ants (Mazokhin-Porshnyakov 1981). The results reported thus far indicate that social insects can use several logical operations, perhaps on the same level as that used by higher vertebrates (Reznikova 1983). The analysis of ant behavior using the maze method reveals many possibilities for studying various aspects of orientation, learning (both group and individual), and spatial memory. To properly estimate the biological ability to learn, however, it is important to take into consideration the role of motivation. The role of motivation forms an important part of the analysis of vertebrate behavior, but its role in invertebrate behavior is not generally accepted. There are few experimental studies on insect motivation, and the interpretation of their results is controversial. Our investigation was concerned mostly with two issues: (1) specific characteristics of ant behavior using various types and levels of motivators and (2) the importance of motivation in the expression of learning. THE ROLE OF MOTIVATION IN LEARNING AND MEMORY IN INSECTS The concept of motivation, "motive," and "motivational state" has been discussed in the Western literature. Motivation is considered to be an energizing component that may also direct behavior. We will not discuss this question here but rather present our own data and those published in the literature on the following topics: goal-directed behavior, inherited as well as acquired behavior, the influence of specific drives (food, defense, etc.), and behavior produced by the development of motivational excitation. Apparently, drive is the main measure of the strength of motivation. The physiological mechanism of motivation consists of activating memory traces of an external object and presenting the conditions that are needed to satisfy this particular drive. The development of motivation is based on the processes of habituation, sensitization, and associative learning. The motivational elements of a situation and the effect of reinforcement strongly influence the formation of conditioned reflex and adaptive behavior. Investigating the role of motivation in the learning and memory of insects is faced with the difficulty of identifying the animal's motivational state. The term "motivation" often is not used, but only implied. In ants, motivation is considered one of the components of the so-called inner state of the organism. This inner state influences locomotor activity, and the magnitude of such activity might be a one way to assess motivation (Hodgson 1955; Hubert 1962).

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It has been demonstrated in experiments with Myrmica rubra (predators with a group type of foraging strategy, for example, scout ants recruit ants that are not able to conduct a search for food) that mobilization efficiency depends on the degree of the scouts' excitation. Three degrees were revealed: (1) scout ants move slowly, without stridulation, and the sting trace is continuous; (2) the movement velocity is higher, with sharp stridulation, and the sting trace is interrupted; and (3) the movement velocity is very high, stridulation is continuous, and there is no sting trace. The number of recruited workers is in proportion to the degree of scout ant excitation (Dlussky, Voltsit, and Sulkhanov 1978). Reznikova (1983), who analyzed this fact, wrote about the "mental state" of ants. In the study of peculiarities in the recognition of honey bees, motivation was considered as an inner incentive state with a definite biological drive that has to be satisfied (Semenova, Mazokhin-Porshnyakov and Lubarsky 1980). Plekhanov and Kupresova (1984) consider motivation or motives as inner incentives underlying animal behavior, such as the states of being hungry, thirsty, and sexually excitated. They divided all varieties of motives into five basic groups: (1) choice of the optimal environment, (2) search for food, (3) search for a sexual partner, (4) care about offspring and, (5) game playing with knowledge elements. The authors emphasize that insects do not have all of the motives that vertebrates have. In regard to insects, some motives are more expressed than others. Many investigators deny the existence of game playing in insects, although games might be revealed in ants and bees. Motivation does play a role in food or sexual activity of insects and serves as the basis for orientation and movement. In this case, the insect can move into an area with potentially dangerous conditions, if there is an attractive object (i.e., one that it is "motivated" to obtain) (Kupresova 1984). The above-mentioned behavioral motives rarely appear as isolated phenomena. Many act simultaneously, but one is usually dominant. Nepomnyashchikh (1984) suggested that the interaction between various motivations provides the physiological mechanism that induces a change in behavior. He defined biological motivation as a nonobservable variable whose increase, above some threshold, triggers the corresponding behavioral act. Many studies investigating the behavior of bees, wasps, and ants demonstrate that the perception of environmental stimuli and the type of behavioral responses elicted or emitted depend on the dominating motivation. Any behavioral act in insects needs an appropriate incentive. In the central nervous system the motivation is compared with the information provided by the environment. Only when the motivation is in place does a motor act follow (Plekhanov and Kupresova 1984). The incentive state (i.e., motivation) can influence the frequency and intensity of the response even if the environmental conditions are constant. Experiments with crickets (Rheinleander, Shuvalov, Kalmring, and Popov 1981) confirm this. The nature of the female reaction to a call from a male depends on the motivation dominating in the colony. Insects will ignore environmental stimuli if there is a discrepancy between the stimuli and the incentives at a given moment. The perception of external stimuli by insects depends upon the dominant


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motivation at the time. Studies of the behavior of honey bees (Jacobson-Jessen 1959), wasps (Tinbergen 1952), and ants (Jander and Voss 1963) have demonstrated that Hymenoptera insects can select different characteristics of visual orienters, depending on the current motivation state. It seems reasonable, therefore, to distinguish between informational and motivational characteristics of signals (Popov 1985). The amount of information perceived by insects also depends on the type of motivation (Kartzev and Mazokhin-Porshnyakov 1987; Semenova, MazokhinPorshnyakov and Lubarsky 1980; Semenova, Kartzev, and MazokhinPorshnyakov 1989). It has been shown that insects, such as honey bees and wasps, can search for food or the entrance of a hive, or nest more or less efficiently, depending on the type of visual signal (Kartsev, Semenova, and Mazokhin-Porshnyakov 1987; see also Kartsev's chapter in this book). The performance of bees, for example, in discriminating figures larger than 2 cm is improved if the reward is returning to its nest rather than food. During foraging, the ant Cataglyphis setipes can remember the geometric characteristics of signals (i.e., shape, size) faster if these signals served as orienters to the nest rather than serving as markers for food (Mazokhin-Porshnyakov and Murzin 1977). Mazokhin-Porshnyakov considers these data as providing evidence for motivationally mediated blocking of the mechanism of conditioned reflex and individual memory. It can be seen from even our brief discussion that the interaction among learning, memory, and motivation has been studied insufficiently in insects. Investigations of insect behavior are too often conducted without taking into account the biology of the species and, in particular, the motivation underlying the behavior. The inability to consider the motives underlying behavior can result in serious mistakes in experimental design and in the interpretation of results. It is necessary to develop methods to assess the motivational states of individuals and of the entire colony when studying social insects. This is the rationale behind the studies performed in our laboratory in which different levels and types of motivators are explored (Dashevsky, Karas, and Udalova 1987, 1989, 1990; Karas and Dlussky 1982; Karas, Udalova, and Zagoraeva 1986; Karas, Udalova, and Dashevsky 1990a; 1990b; Karas, Udalova, Dashevsky, and Zhukovskaya 1991). THE ROLE OF MOTIVATION IN MAZE LEARNING IN THE ANT MYRMICA RUBRA The Method of Investigation A number of studies on the role of motivation in learning and memory of ants have been performed in the Department of Higher Nervous Activity at Moscow University and at the Biological Institute at Leningrad University. The experiments were conducted with the ant Myrmica rubra, whose biology has been well studied

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(Arnoldi 1970; Dlussky 1975a; 1975b; 1981; Dlussky, Voltsit, and Sulkhanov 1978; Kipyatkov 1974, 1979; Reznikova 1983; Zakharov 1972). They are predatory insects that live in the litter of the forest and have a structured system of foraging. That is, scouts recruit worker ants that are not able to search for a food source themselves. The feeding area of M. rubra has no guarded territory, except for a small zone (approximately 0.25 m) close to the nest. The scouts search for food over an area of about 2 m and prefer zones where they can easily find prey. The ants move by making complicated loops and are often observed making "shuttle" movements on their return to the nest. During the search for food, scouts primarily use their sense of smell. In well-known territory, they return to the nest by the shortest way and leave odor traces. Such traces play a crucial role in the orientation of scouts and activation of foragers only in low levels of daylight. When light is available, ants use visual orienters when they search for food. The scouts can carry small prey themselves. When the scout cannot carry the entire "bounty" back to the nest, it can mobilize foragers. Information from the scout ants about the food source is passed to other members of the colony in various ways. These include an olfactory trace (either interrupted or continuous), changes in intensity of stridulation, and/or the rate of movement and intensity of tactile contacts in the nest. The activation of animals in the nest usually proceeds by means of tactile stimuli. The communication systems used in group foraging appear to be formed by a process of learning (Dlussky, 1981b). Our experiments were performed with colonies of ants taken not far from the towns of Pushchino or Zvenigorod (Moscow region) or Old Petergof (Leningrad region). Ant colonies consisting of approximatly 200-700 individuals (workers, queens, and brood) were placed in artificial nests constructed from earthenware pots. Figure 6.1 provides a diagram of the apparatus. Ants were placed in the laboratory nests 2-4 weeks prior to conducting an experiment. The pot was placed on a tray filled with water to provide the necessary humidity. Covering the opening of the pot was a round arena with a diameter of 26 cm. The arena served as the foraging area, and a hole permitted access to it from within the nest. The colony was supplied with food and water. The conditions within the colony such as temperature and humidity were optimal for Myrmica rubra (Kipyatkov 1974). In these investigations we used the maze technique developed by Karas (Karas and Dlussky 1982; Karas et al. 1985, 1986). In the arena a maze was affixed to a metal stand 7 cm high. The stand and the walls of the arena were smeared with vaseline oil to prevent the ants from escaping the confines of the arena. The maze consisted of two symmetrical halves and was contructed as a series of paper "bridges." The maze was 13 cm long and 5 cm wide. An incline from the arena to the maze served as a "starting ramp." When an animal climbed this ramp to the start box, the ramp was removed, preventing retracing. The right and left exits (T and R) were connected to the arena by permanent bridges. The right (A) and left (G) target areas (or goal boxes) were removable and made of foil. In these areas an experimenter placed a


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Figure 6.1 The scheme of the experimental apparatus: I: the earthenware pot with ant nest. II: the tray. Ill: the screen. IV: the arena. V: the maze. 1: uprights. 2: entrance to nest. 3: the central bridge. 4: the side bridges. 5: the starting zone. 6: removable bridges. O: entrance to maze. A and G: target areas. B and V: symmetrical areas: T and P exits: K,L,E,C,D and Y,Q,J,U: other sections of right and left halves of maze, respectively.

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drop of sugar syrup or a pupa or larva, depending on the experiment. The maze was placed at a distance 50 cm from the laboratory window. Different illumination conditions were used: natural and artificial light. When artificial light was used, the source was an electric 40-60 watt bulb of frosted glass placed 40-50 cm from the center of the maze. In some tests, a paper screen was placed around the apparatus. When this screen was used, light was uniform and outside visual stimuli were eliminated. In order to train the ants to run the maze, it was first necessary to train the ants to visit the starting area. This was done by placing a reward there. The maze habit was then developed in the most active individuals, which were tagged with nitropolish or with metal rings having a diameter of 80 microns. After an ant passed the starting area and reached the top of the ramp where the "start box" was located, the ramp was removed, preventing a return to the nest. At this point the ant had to complete its run through the maze. Moving along the maze, the ant was able to approach the target area to find, depending on the experiment, food, brood, or both. The subject could exit the maze by entering a ramp that led back down to the arena and the nest opening. It is important to remember that there are two such exit ramps, corresponding to the left and right halves. The trajectory of moving from the entrance of the maze to its exit was called a "complete cycle." In each cycle the ant could receive reward in several locations. After each cycle, all parts of the maze were changed to prevent the ants from orienting by means of olfaction. The experiments typically lasted one or two days. The recording of responses and the movement of an ant into the various segments of the maze were performed visually. A system was developed whereby letters were used to identify the paths taken by an individual ant. The method of free choice was used throughout the experiments. The frequency of visits to the maze, the trajectory, and the time needed to make a movement were all under control of the individual ant. Unlike an ordinary maze with a blind alley, our symmetrical multichoice maze allowed ants to realize a great number of alternative decisions. The correct solution to the maze was considered be the shortest number of paths, that is, the elimination of entering uneccessary paths. The correct solutions were divided into two main types: optimal solutions had trajectories of different lengths but without repeated runs to previous traveled maze segments. There were 16 solutions of this kind, for example, 0,I,K,A,D,S,T or 0,I,K,A,M,J,G,Z,V,P (the letters identify individual segments). The other correct solutions were referred to as surplus ones, in which the ant would retrace its steps (for example, 0,I,K,A,D,D,A,K,I,0,0,Z,B,Z,Z,E,T.). In order to evaluate the learning of ants in this situation we used as dependent variables the number of movements and the duration of each cycle. The duration of each cycle was further broken down into three stages: (1) from entering the maze to the first approach to the rewarded target area, (2) from the first target area to the last visited target area, and (3) from the last visited target area to the exit. We also determined the first correct cycle, the total number of correct solutions, stabilization of the habit, that is, ten noninterrupted correct cycles, and some other


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criteria. The experiments were performed in July-September over several years. The brood was maintained at temperature of 3° to 7° C. We made 7 series of experiments with 140 ants trained to transport offsprings, and 5 series with food motivation. Also, we carried out experiments in which the level of food motivation was changed from high to low (15 ants) and vice versa (8 ants). We also conducted experiments in which the type of motivation was altered (28 ants). The Peculiarities of Ant Learning in a Multi-Choice Maze with Different Motivation Two kinds of social behavior were studied in scout ants: (1) foraging, which is under the control of food motivation, and (2) offspring defense. The characteristics of learning will be illustrated by the data obtained with two samples from a single ant colony. This colony was kept with the regime of unrestricted feeding, when sugar syrup was available at any time. Twelve ants acquired the foraging habit in the maze. Learning took 20-34 (mean value was 22.3±1.3) trials. Ant behavior was very variable and unstable. The insects used a number of tactics to investigate both halves of the maze and engaged in multiple visits to the same parts of the maze. In other words, the "surplus" solution prevailed. Very frequently, ants approached the target areas of the maze but did not take the syrup. More ants approached the syrup than actually consumed it. The first stage of the maze cycle was shorter than the third one in terms of the number of moves and duration. The second stage occurred in 25% of the cases and had more moves than the third one, but there was no difference between them in terms of duration. The number of moves and duration of the stages, especially of the first one, declined by the end of the learning session, but these changes were small or insignificant. An absolutely different behavior was observed when ants defended and took care of their offspring. Fifteen ants were taught to bring pupae from the maze to the nest during 19-41 trials (26.7±1.7). Surplus solutions occurred within 3-8 trials. After that, the solutions became optimal and a stereotypical movement developed. In most cases, the movement trajectories were asymmetrical (e.g., ants moved only on one side of the maze). The number of moves and especially the duration of the first and second stages as well as of the whole cycle decreased during the first 8 trials. Then these characteristics stabilized. The first 8 trials as a period of dramatic change in the behavior of ants was also noted by other researchers (Inozemtsev and Ostrovenko 1986; Plekhanov and Kaul, 1976; Schneirla 1946). It is important to emphasize that all quantitative characteristics of the behavior under offspring defense motivation were better than those under food motivation, with the exception of the number of correct solutions. The characteristics of the maze habit described above were also revealed in experiments with ant groups taken from other colonies and tested under the same experimental conditions. On the basis of all characteristics, it may be concluded

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that learning with offspring defense motivation manifested itself more clearly than learning associated with food motivation. For example, for all ants, with offspring defense motivation, the first correct solution appeared after 1.6+/-0.03 trials and stablized after 3.56±0.14 trials. Moreover, the percentage of correct solutions was 93.8%±1.8%. In the case of food motivation, the same characteristics were achieved after 2.56±0.62 and 4.83±0.55 trials, respectively, and the percentage of correct solutions achieved 75.9±16.9%. To summerize the results thus far, under conditions of unrestricted feeding of the colony, ants developed the maze habit much faster if offspring defense served as motivation rather than food motivation. Optimization of the habit involved a decrease in the number of moves and cycle duration and in the type of optimal solution observed. It should also be noted that the ants exhibited remarkable stamina in their ability to carry brood for 6-10 hours without a break. No such behavior was observed under conditions of food motivation. When food served as a reinforcement, the learning efficiency was weak or seemed absent. It looked as if the ants were exploring the maze for the first time and did not use any of their previous experience. Hence, two types of strategy for correct performance were revealed. During defense motivation the maze habit developed after the first several trials and consisted of 1-2 minimized stereotyped behavioral programs (i.e., the correct solutions repeated without changes from trial to trial). The second type of strategy was revealed with food reinforcement and consisted of variable solutions and, as a rule, surplus solutions. These results suggest that the strategy and efficiency of ant learning depend on the kind of motivation. Schneirla (1953) was the first to note that ants learn quicker and with a smaller number of errors if ant larva are used as reward. Analyzing the foraging behavior of ants in our maze suggested to us that the behavioral components associated with food and the orienting-investigating motivation can be reciprocal or synergic to each other. In the natural environment, foraging ant scouts display clear and continuous exploratory activity (Dlussky et al. 1978; Zakharov 1972). We believe that this type of activity is responsible for the variability in behavior we see in the feeding area (Reznikova 1990). Apparently, this activity does not extinguish in the course of an ant's learning a maze. It should be noted that this strategy is accompanied by spatial-motor asymmetry in individuals, but not in the entire sample (Udalova and Karas 1986, 1989). In contrast to food motivation, social defense motivation requires extremely quick solutions of a spatial task. In this case, the orienting-investigating activity extinguishes very quickly, and the behavioral program becomes restricted and produces a stereotypical behavioral pattern. The behavioral optimization manifests itself in acquisition of asymmetrical trajectories in individuals and in the entire sample as well (Udalova and Karas 1985, 1989). Thus, the results suggest that the strategy and efficiency of learning in ants results from the competitive interaction between exploratory activity and specific dominant motivation. Social dominant motivation is one of the most important factors controlling the strategy of learning a multi-choice maze.


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Schneirla (1933) divided the process of maze learning in ants into two stages: from the start box to the goal box and from the goal box to the nest. He suggested that at each stage a different motivation controls behavior. He showed that ants move faster when they go to the feeding area than on the return trip back to the nest. In our experiments, the first stage of every trial (from the maze entrance to the first reinforcement) optimized more quickly than the rest of the trajectory during both food and defense motivation. This is due not only to the maze structure but also to faster extinction of the exploratory activity on the way to the target area. It seems reasonable to assume that the two stages of maze performance have different significance regarding the dominant and orienting-exploring motivations. It has been shown in many experiments that changes in stimuli also affect the orientation system of ant foragers (Chauvin 1964; Vowles 1954). When an ant leaves the nest, the system of orientation and memory of the path are functioning. When food has been found, the systems controlling exploratory behavior and food gathering are activated. Again, during transportation of food to the nest, the orientation system becomes dominant. In our experiments, individual differences were found in in the elaboration of the maze habit from animals taken within the same colony. Individual differences were also observed under identical types and levels of motivation. These conditions seem to predetermine group homogeneity and, hence, behavioral individualization is scarcely probable. It should be emphasized that the problem of individual diversity in ant behavior is the subject of wide speculation. Individual differences in locomotory activity in the arena, in pupa transportation, in leaving the nest, and in aggression have all been described (Bogatyreva and Bogatyrev 1987; Le Roux and Le Roux 1979; Verron 1974). Even in small, functionally homogeneous groups, one can distinguish ants with good memory who stimulate other individuals and organize them for some activity. It is considered (Reznikova 1990) that the behavioral peculiarities of the outnest workers are determined by their functional use. Some authors attribute the causes of individual diversity to its efficiency in memory, learning, or sensory perception rather than the age or size of the individual (Bernstein & Bernstein, 1969). The peculiarities of individual behavior can be demonstrated on the basis of an ant's psychophysiological characteristics, such as aggressiveness and method of orientation (Reznikova 1983, 1990; Sulkhanov 1986). Individual differences in the learning ability of ants (Abramson, 1981; Maier and Schneirla 1964) can be explained by the fact that the capacity of learning in workers correlates with their social rank (Zakharov 1981). Under the conditions of our experiment, the individual peculiarities of learning manifested themselves in terms of the different frequency of maze visits, different number of movements and cycle duration, and the character and variability of the trajectories. Profound qualitative differences in the mode of maze habit formation, that is, in the mode of processing and memorizing environmental information, were shown. Thus, in a group of 25 ants from a single colony transporting their brood in the maze, most of the individuals (72%) solved this problem by gradual

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but rather rapid learning: Unnecessary movements were reduced, and by the 8th-11th cycle the optimum strategy was formed. Twelve percent of the ants demonstrated an imprinting-like learning mechanism: the task was solved in an optimum mode, or close to it, in only 1-2 cycles. Later, with further exposure to the maze, the behavior of such ants became almost stereotyped. About 16% of the ants demonstrated latent learning capacities: during the first 2-3 cycles they only investigated the maze but did not take the brood. When these ants finally seized the brood, they returned to the nest with the minimum of errors. The methods used by individual foragers during the formation of the maze habit were studied in 30 ants from 3 colonies. The colonies were kept under conditions of unlimited feeding. Because the strategy in such conditions is characterized by a greater variability of decisions and the lack of distinct optimization, data analysis was based on the number of errors and their distribution during the learning period. Four basic modes of task solving were revealed. The first was recorded in 13.3% of the insects and consisted of a gradual and rather rapid learning: 1-4 errors were observed in the first 5 cycles, after which there were no errors at all. The second mode was demonstrated in 10% of the ants. The decision was already correct in the first cycle, and there were no subsequent errors. The third pattern occurred in 36.7% of the individuals. They, too, had no more than 4 errors, but the errors appeared random. The fourth mode (40% of the ants) consisted of random alternations of correct and incorrect cycles. But only one type of cycle (correct or incorrect) dominated statistically in each half of this group. The individual differences in ant behavior are closely connected with colony peculiarities. The differences in the level of learning in colonies of the same species are shown to be determined by the proportion of "capable" and "incapable" individuals. This seems to depend upon the number of colony members and its territorial organization. Thus, a small and large colony of Formica cunicularia were taught to look for a lure. The ability to solve this task and find the lure appeared to be greater in the smaller colony. This phenomenon was explained by a higher portion of ants specialized as foragers. Moreover, ants in such a colony, relative to those living in larger ones, appear to have a richer behavioral repertoire (Reznikova 1983). In our experiments it has been found that there are differences in the learning indices of seperate colonies that were taught under identical conditions. These differences were more distinct under conditions associated with food motivation than with brood protection. The State of the Maze Habit When the Level of Food Motivation is Changed Neurohumoral motivational factors can inhibit and excite the central nervous system, which influences the response of an animal to environmental stimuli or situations. The physiological response also depends on the level of motivation. For example, Musca domestica taste receptors are activated when exposed to sucrose, whereas the behavioral reaction appears in hungry individuals only (Dethier 1966).


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We have assumed that the behavior strategy of ants negotiating a maze depends on the level of the current dominant motivation (Karas and Dlussky 1982; Udalova and Karas 1986). The theoretical basis for this assumption is the notion that the development of motivational excitation in the central nervous system occurs according to the principle of the dominant. To solve the problem regarding the level of motivation in ant learning, special experiments were conducted with ant foragers under the conditions of controlled access to food. Thirty-six hours of food deprivation produces an increase in food motivation in our colonies. It has been found that after food deprivation the efficiency of learning is increased in comparison to ants that have had unrestricted access to food. In "hungry" ants, a short period of variable surplus solutions (5-7 trials) were observed, followed by optimally correct solutions and stabilization of learning. This optimization process and the rapid appearance of the first correct solution resembled that found under larval or pupa motivation. However, much more variability of solutions was associated with food motivation than with defense motivation. Our results suggest that increased food motivation promotes the process of optimization of the maze habit in foragers. The effect of the level of food motivation has been demonstrated even more clearly in experiments where the same ant colony underwent transition from a "satisfied" state into a "hungry" state and vice versa (8 and 15 individual ants, respectively). During the transition from a low to a high level of food motivation, the stabilization of 1-2 correct solutions and optimization of the quantitative characteristics of the maze habit proceeded more quickly. Hence, the maze habit was realized more efficiently. During the high level of food motivation, a rapid and distinct development of the optimized maze habit was revealed. In the case of unrestricted access to food, the extinguished exploratory activity increased again during food deprivation. This was expressed as an increase in the number of moves, especially during the first trials. The duration of the trials increased, as did the variability of solutions and the number of errors. Optimization of the habit was slow. In these experiments it has been found that the characteristics of the maze habit of an individual forager depends on the history of acquisition. The performance of ants, for example, who learned only under a high level of food motivation differed from ants who experienced the transition from low to high. After the transition, ants showed an increase in the number of moves, exploratory approaches and incorrect solutions. These ants, however, did not engage in many of the wasteful movement schemes observed in ants who were trained only when "hungry." During the transition from a hungry to a satiated state distinct optimization of behavior was observed, especially in the first stage: the number of exploratory approaches decreased as well as the number of movement schemes, in comparison with learning in the "satiated" colony. Apparently, learning under one level of food motvation influences the strategy of foragers when the level of food motivation is switched. Thus, the initial level of food motivation in forager ants determines the learning

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characteristics in the maze and the ability to transfer the learning or to switch the behavior from one program to another. It should be noted that the level of food motivation in an ant colony can be affected by factors other than food deprivation. These include temperature, season, quantity and developmental stage of the brood and colony size. The State of the Maze Habit When the Type of Motivation Is Changed Special studies have been undertaken to elucidate ant behavior under conditions in which the motivation is abruptly changed. Consideration was given to the possibility of studying the learning of ants established under one type of motivational state and replacing it with another. This is one aspect of insect behavioral plasticity that has not been sufficiently studied and remains controversial up to now (Kartsev and Mazokhin-Porshnyakov 1987; Kartsev, Semenova, and MazokinPorshnyakov 1987). For example, when ants Formica schaufussi were taught to pass without errors through a maze for a food reward, they appeared to be unable to find the correct path in the same maze for the return home (Weiss and Schneirla 1967). Dlussky (1967) showed that, after an alteration of visual orientators, ants Formica pratensis and F. exsecta needed two or three days to find the right way to the feeder. Such behavioral "inertia" was noticed by Lubbock (1898). We have studied the ability of ants to form the maze habit when the defence motivation was replaced with food motivation (model 1) and vice versa (model 2). Two types of experiments were carried out with the same and with different functional states, namely the levels of food motivation. In the case of model 1, 18 ants from the "replete" colony were taught to transport their brood in the maze. Then, after 36 hours of food deprivation, the insects were tested using syrup reinforcement. Under defense motivation, the typically good pattern of learning was observed. When the new reinforcement was administered, the reaction manifested itself as an initial abrupt increase in the number of movements in the cycle, the duration of a cycle also increased, and there was much variability in performance. With extended training, the quantitative indicators decreased and the ant behavior optimized. It is important to note that the learning and test indicators differed in the first stage rather slightly, that is, the exploratory reaction appeared only after reinforcement. The second type of model involved 10 individuals whose learning and testing were performed at a similar high level of food motivation resulting from food deprivation and then switched to a defense motivation. The animals in this group showed some interesting differences. There were more movements in the cycle, and the cycle itself appeared to last longer because of the surplus correct decisions. The first two cycles proved to be very short, with no optimization process revealed. The first stage of the cycle minimized very quickly, however. Seven ants used only one out of four possible travelling patterns. Such maze habit peculiarities, with the defense motivation in a "hungry" colony,


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can be explained by the fact that at the high level of food motivation exploratory activity was not depressed and overshadowed the motivation pattern characteristic of defense. In this case, the competitive interaction of food and defense motivations clearly manifested itself. With a change in the reinforcement, a weak exploratory reaction was registered. Such reactions were studied in additional experiments. In the first type of experiment, with the food motivation replaced by the protective one, seven ants from a "hungry" colony were initially taught with food reinforcement. Then the colony was fed, and the brood transportation test was conducted. The maze habit typical for the high level of food motivation was found. Differences in performance were observed when the motivation was changed. Four ants stopped entering the maze after several visits to it, that is, they were unable to realize the habit. Such "refusals" can be explained either by the frustration caused by the necessity of taking a new type of reinforcement or by the fact that those individuals were specialized in foraging, but not in parental care. Three ants succeeded in realizing the habit under reinforcement. A distinct exploratory reaction appeared in these individuals during the first cycles. The insects were faced with great difficulties in the first stage of the cycle. This is reflected in the difficulty they had picking up the new reinforcement and in the number of movements when searching for the target area and picking up the brood. This is also indicated by the large number of movements. As training continued, the optimal strategy was revealed. The second type of this model involved 10 ants who were taught at a level of high food motivation. Under these conditions, the typical learning strategy was demonstrated. The test was conducted at the same level of food motivation. The first replacement of syrup reinforcement by pupae caused a relatively weak exploratory reaction. The number of movements increased both during the entire cycle and in the first stage. Then the quantitative indicators recovered. To summarize these experiements, ants demonstrated the ability to revert back to the previously formed maze habit following immediate changes in reinforcement, with replacement of the food motivation with a defensive motivation or vice versa. The characteristics of these behavioral changes usually involve, first, a vitalization of exploratory reaction, which gradually extinguishes, followed by the optimization of the maze habit. The exploratory reaction was most evident when both the type of reinforcement and the colony motivation for food were changed, that is, when both of the biological factors were manipulated. More abrupt violations of the elaborated habit were found with the transition from food motivation to the defensive motivation. In this case, the exploratory reaction was vitalized in the first stage of the cycle. This phenomenon was not observed when the defenisve motivation was replaced by food motivation. Significance of Motivation Factors for Estimating the Plasticity of Ant Behavior The following conclusions can be made regarding the results of experiments in

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which the level or type of motivation was varied. The readiness of the animal to execute a certain type of activity, the way to activate the sensory and motor programs, and the possibility of forming adequate reactions—all these processes depend in a large part on motivation. A hierarchy of motivations exists in ants and in other insects. The type and level of the original dominant motivation influence the rate and efficiency of learning in ants. In our experimental conditions, it has been shown that there is some interaction between food and defensive motivations and search-exploratory activity. When ants were taught in a symmetrical multichoice maze, with the care of offspring as motivation or with a high level of food motivation, the learning indices bore a resemblance to each other. This can be explained by the necessity to preserve the brood, in the first case, or by the necessity to reduce the colony food needs, in the second case. By contrast, if ants were taught at a low level of food motivation, the search-exploratory activity was strongly expressed during the entire learning period. If the functional state of individuals is changed by food regulation necessary for the colony or by transfer from food reinforcement to that of brood transportation, the search-exploratory activity that was extinguished in the period of learning becomes prominent once again. Disinhibition of search exploratory activity appears to be a universal way for ants to respond to changes in their "inner state." Probably, the gradual extinction of the exploratory activity coincides with the choice and the formation of a new behavioral strategy that is appropriate to the new physiological state of the insect. Under these conditions, some ants were observed following paths that were used previously, while other ants alternated old and new trajectories. Seldom did transfer occur. But in this case, the trajectories developed earlier were not always adequate under the new situation. With all modes of maze behavior, the importance of memory traces is quite evident. It should be mentioned that the role of short- and long-term memory in the learning of ants has already been noted in many studies (Chauvin 1964; Dobrzanska 1958; Plekhanov and Kaul 1976; Rozengren 1971; Schneirla 1961). At the same time it is clear that the mode in which the individual reacts after a change in the type or level of motivation depends not only on the preservation of the previous learning traces but also on the ability to form a new traces suitable to the new functional state. It should be emphasized that, when the type of motivation was altered, individual differences in displaying the maze habit increased. The most illustrative example is when ants refused to solve the problem, that is, they refused to enter the maze situation. Apparently, in these cases we were observing a "neurotic state" in ants. Presumably this state was caused by the impossibility of adequately solving the task after an alteration in the inner functional state (i.e., type or level of motivation) of the individual. However, such ants were few. On the basis of this fact, one can conclude that, as a whole, the scout ants of Myrmica rubra show a rather high level of behavioral plasticity.


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THE PLASTICITY OF ORIENTATION BEHAVIOR IN ANTS Behavioral plasticity in animals is based on the ability to reconstruct earlier developed systems of temporal connections in accordance with altered environmental conditions. The plasticity is determined by the individual peculiarities of the nervous processes, the age of the animal, and the degree of its training. The problem of behavioral plasticity in ants and other insects has not been intensively studied in the past but is receiving new attention. In the opinion of Polyakov (1964), insects, unlike mammals, cannot use individually acquired experience in a new situation. On the other hand, investigation of the signal activity of the honey bee colony suggests a well-expressed plasticity in higher insects (Lopatina 1971). It is considered (Reznikova 1990) that the degree of behavioral plasticity of social insects is not clear at the colony level and at the level of intercolonial and interspecific relations. The question of the balance between the specialties of individuals in the colony and their behavioral plasticity is not clear either. It has been shown that ants have various behavioral strategies relative to different forms of activity and at different levels of their social organization (Dlussky 1967; Reznikova 1979; Reznikova et al. 1977). Dlussky (1967) has described the behavioral plasticity of Formica sanguinea when they learned to follow landmarks on the way to the nest. When the landmarks were changed, the ants made the necessary corrections. Having studied the behavioral plasticity of ants of the Formica genus with respect to spatial learning, Schneirla (1953, 1961) demonstrated their ability to alter a habit when the structure of the maze or the location of food reinforcement had changed. At the same time, based on other tests, Schneirla concluded that ants have a low level of behavioral plasticity in comparison with vertebrates. In his opinion, ants are not able to transfer experience to a new situation. We have found that ants use different methods to form optimal strategies during learning and are able to express the developed habit when the level or type of motivation is changed. Moreover, ants demonstrate the ability to adequately change their behavior under new experimental conditions. These findings, however, did not allow us to determine the limits of behavioral plasticity in Myrmica rubra. In order to solve this problem, special experiments were conducted to study the ant's ability to alter the developed maze habit when faced with new conditions. By changing the structure of the maze or the location of reinforcement, we could determine the limits of the ant's spatial memory and its "cognitive" map (Dashevsky et al. 1987 1989; Karas et al., 1990a; 1990b; Udalova et al. 1991). In all experiments, Myrmica rubra formed a habit in the multichoice maze of using the collection of brood as motivation. In the initial two tests, the ability of ants to reorient with respect to the longitudinal maze axis (along the right-left direction or vice versa) was studied. The tests were made with individuals that showed a marked preference for one of the target areas during learning. In the first test, the entrance into the maze was asymmetrical; it was connected to the exit by a removable bridge on the side opposite to the preferred target area.

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The alterations were noted by all 12 ants. In the new conditions, an insignificant disorientation of ants was observed. During the initial 1-2 cycles the ants found the entrance with difficulty. After finding it, however, the ants could quickly identify the optimal trajectories, usually on the side of the maze connected with the entrance. In this test the ants demonstrated the ability to extend the developed habit from the preferred side of the maze to the nonpreferred one. In the second test, the location of the reinforcement was changed by placing it only on the target area that had not been preferred during learning. In this case, the symmetrical structure of the maze was disrupted and the probability of making a correct choice decreased to 0.5. A cycle was considered correct if the ant took the reinforcement from the previously nonpreferred target area without previously visiting the symmetrical area. The criteria of alteration were a sequence of, at least, 10 uninterrupted correct cycles and a reliable dominance of such cycles. A new maze habit developed in 14 of 16 individuals (87.5%). The initial cycles were usually wrong. Having found no brood on the previously preferred target area, the ants made a large number of searching movements, returning often to the previously rewarded target area and inspecting it. At the same time, some behavioral reactions were observed: The ants lifted the front part of the body, moved their antennae, and hung over the sides of the maze. However, they spotted the reinforcement and found the correct optimum solution. Accordingly, the total number of movements in the cycle and at the first stage of the cycle increased greatly at the beginning and then decreased. This parameter reached the significant level only after the 20th cycle, but even at that time it had not stabilized. All of the main indices of the maze habit were worse than those during the initial learning. Development of a new habit was accompanied by a change in the initial spatial-motor asymmetry. Therefore, the test alternating the lateralization of reinforcement was more difficult than the asymmetrical entrance test. Nevertheless, most of the ants were able to change the choice of the target areas, that is, they demonstrated the ability to transfer the habit from one side of the maze to the other (Mazokhin-Porshnyakov, Semenova, and Milevskaja 1979; Mazokhin-Porshnyakov, Taimova, Frolova, and Shamukhamedova 1971). By means of this model, the ability of ants to form multiple reconstructions of the maze habit was investigated (Udalova et al. 1991). The reconstructions consisted in alternating the location of reinforcement from the right to the left target area or vice versa, using the nonpreferred target area during trials 1, 3, 5, 7 and the preferred target area during trials 2, 4, 6, 8. A group of 21 ants from two colonies demonstrated the ability to carry out the series of 8 reconstructions over the course of 1-2 days. This can be seen in the case of 10 ants from one colony. During the first alteration, the behavior of the ants changed as described above. That is, an exploratory-search pattern appeared and slowly extinguished. During the second alteration, when the reinforcement was placed on the target area preferred during learning, the dynamics of the indices of the maze habit resembled that of the first alteration, but a statistically reliable stabilization of the habit was observed. In the


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alternations that followed, the behavioral changes were, in principle, like those during the first two alternations. The performance of the ants were better on even alternations than odd alternations. We should emphasize that these experiments and others performed in our laboratory indicate that ants can alter a maze habit. In another experiment investigating the ability of ants to apply their knowledge to a new situation, brood was placed on two "false" areas. The developed habit was considered to be altered if new correct solutions were formed, which consisted of the ants' seizing the reinforcement without approaching the formally correct target areas. This experiment was more complex than the preceding one. The alternation was noted by 15 ants (62% of the sample). In the beginning, the ants made a great number of seeking movements. They had been examining the previously reinforced areas for a long time, passing from one area to the other. Finding no reinforcement there, they started moving along both halves of the maze, making many "false" and repeated movements. Even in the initial cycles, however, the ants usually approached the correct area and took the brood back to the colony. Subsequently, correct solutions appeared that became fixed, and optimal trajectories developed. The number of movements in the cycle and its duration decreased. Simultaneously, the number of approaches to the now correct area increased. The reorientation of the transverse axis of the maze was accompanied by a strengthening of the lateral spatial-motor asymmetry. The relative complexity of this task was evidently due to the fact that it was necessary for the ants to learn to take the brood at areas originally not connected with obtaining the reinforcement. Another reason might consist in the necessity to move, not forward from the entrance of the maze, but backward. CONCLUSION This chapter reviewed the Russian literature on learning and memory in ants and presented the results of several maze experiments performed in this laboratory on the role of motivation in learning. In the comparative-physiological investigations of the learning process, it is of great importance to use techniques that resemble those found in nature. The complex maze of the type used in our experiments is but one example. Moreover, it is necessary to take into account the biological and ecological peculiarities of the species when designing and interpreting the results of experiments. In our experiments we have made use of various types of motivation in combination with the method of free choice. The results of a series of investigations with Myrmica rubra indicate that performance is dependent on the type and level of motivation. Motivation can determine the readiness to execute a definite type of activity that is appropriate for a given situation. Using return of brood as the motivation, we have shown that ants can perform in a complicated maze situation at a level comparable with that of higher vertebrates. The level of performance depends upon the dominant level of motivation. Performance obtained with food motivation does not necessarily mimic performance found under brood motivation. Myrmica rubra has the ability to generalize

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information present in spatial cues to enable them to modify their maze habit when motivation is changed. In this regard they also demonstrate reversal learning with a level of performance that is also vertebrate-like. Summarizing the results of our investigations and the other Russian literature on learning and memory in ants indicates that ants and other higher Hymenoptera (i.e., bees and wasps) possess good spatial memory and orientation behavior, which enable them to learn quite complicated behavior. There are also individual differences in the ability to acquire such complex learning (see the chapter in this book by Burmistrov and Shuranova on individual behavior in crayfish). The learning of ants enables individual members and entire colonies to react appropriately to changes in external and internal conditions. Despite differences in morphology, the sophisticated learning ability and well-developed communication system found in the higher social insects are very close to those found with vertebrates and in some cases surpass vertebrate performance on analogous tasks.

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714. . 1989a. Asymmetry of movement direction in ants Myrmica rubra (in Russian). Zhurnal Evolyutsionnoy Biokhimii i Fiziologii 25:21-31. . 1989b. Spatial-motor asymmetry in ants (in Russian). In: Neyrobiologiya tserebralnoy lateralizatsii (Neurobiology of cerebral lateralization), 173-198. Leningrad: Leningradsky Gosudarstvenny Universitet. Udalova, G. P., Zhukovskaya, M. I., and Karas, A. Ja. 1991. The ability of ants to adjust to alterations of the maze habit (in Russian). Zhurnal Vysshey Nervnoy Deyatelnosti 47:1154-1162. Vagner, V. A. 1913. Biologicheskiye Osnovy Sravnitelnoy Psikhologii. Tom 2 (Biological principles of comparative psychology, vol. 2). Saint-Peterburg: Tovarishchestvo M. O. Wolf. Vasilyeva, L. L., and Vasilyev, V. V. 1987. Imitation of learning processes in T-maze using electrodefensive technique (in Russian). In: Muravyi i Zashchita Lesa (Ants and forest defense), 216-219. Tezisy Dokladov 8-go Vsesoyuznogo Mirmekologicheskogo Simpoziuma (Proceedings of the 8th All-Union Myrmicological Symposium). Novosibirsk: Nauka. Verron, H. 1974. Note sur l'activite' locomotrice des ouvrieres de Lasius niger (Hymenoptera, Formicidae). Compt. Rend. Acad. Sci. (France) 75:2361-2363. von Frisch, K. 1965. Tanzsprache und Orientirung derBienen. Berlin: Springer Verlag. Vowles, D. M. 1954. The orientation of ants. 1. Substitution of stimuli. /. Exp. Biol. 57:341-375. Wassman, E. 1906. Itogi Sravnitelnoy Psikhologii. Chast 1. Instinkt i Razumnost v mire Zhivotnykh. Chast 2. Sravnitelniye Issledovaniya Dushevnoy Zhizni Muravyev i Vysshikh Zhivotnykh (Some results in comparative psychology. Part 1. Instinct and intelligence in the animal World. Part 2. Comparative study of psychical life in ants and higher animals). Kiyev: Imperatorsky Universitet Svyatogo Vladimira. Weiss, B. A., and Schneirla, T. C. 1967. Inter-situational transfer in the ants Formica shaufussi as tested in a two-phase single choice-point maze. Behaviour 28:269-219. Zabelin, S. I. 1979. Transfer of information about direction of movement toward the lure by a tactile code in ant Tapinoma simroth karavaievi (in Russian). Zoologichesky Zhurnal 55:1168-1172. Zakharov, A. A. 1972. Vnutrividoviye Otnosheniya u Muravyev (Intraspecific relations in ants). Moskva: Nauka. . 1974. Interaction between individual workers in ants colony (in Russian). In: Ekologicheskie i Evolyutsionnye Aspekty Povedeniya Zhivotnykh (Ecological and evolutionary aspects of animal behavior), 119-126. Moskva: Nauka. . 1981. Behavior organization in ant colonies (in Russian). In: Chteniya Pamyati N. A. Kholodkovskogo (Readings in memory of N. A. Kholodkovsky), 34-58. Leningrad: Nauka. . 1991. Organizatsiya Soobshchestv u Muravyev (Organization of ant societies). Moskva: Nauka.


Chapter Seven Local Orientation and Learning in Insects Vladimir M. Kartsev

THE STUDY OF ENTOMOLOGY AND EARLY INVESTIGATIONS OF INSECT BEHAVIOR IN RUSSIA The study of insects began in Russia at the end of the seventeenth century. For a long time, its primary directions were taxonomic and faunistical descriptions of insect species in primary areas of the Russian Empire. In 1859, the Russian Entomological Society was founded, now one of the oldest societies in Europe (at present it includes a section devoted to social insects, where social and other aspects of insect behavior are regularly discussed). Near the turn of the century, applied entomology began developing. At first agricultural, forest, and medical branches were formed, followed by ecology (V. N. Beklemishev and G. A. Victorov), and then physiology (N. Ju. Kuznetsov and A. S. Danilevsky). The oldest entomological departments in Russia were founded by N. M. Kulagin (1860-1940) at Moscow University (in 1925) and by M. N. Rimsky-Korsakov (1873-1951) at St. Petersburg University. The first study on the nervous system of insects was performed by E. K. Brandt (1839-1891); this direction was then successfully developed by A. A. Zavarzin. A pioneer in the investigation of insect and spider behavior was V. A. Vagner (see Shuranova, this book). Although he did not believe in the learning ability of insects, many materials contained in his monograph (Vagner 1913) are of interest today. He stressed adaptivity of insect instincts and considered the rapid recall of landmarks by insects to be a type of primitive learning comparable with learning in vertebrates. In 1926, the world-famous academician I. P. Pavlov, in the preface to the book by B. N. Shvanvich, Insects and Flowers, agreed with the positive opinion about the existence of conditioning in invertebrates, particularly in honey bees. Subsequently, great similarities in conditioning in insects and in vertebrates has been

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shown, as will be discussed later. Shortly before the end of his life, Pavlov concluded that the behavior of higher animals is based not only on reflexes but also on some comprehension of laws of logic or probability. Developing this consideration further, L. V. Krushinsky (1977) created a theory about elementary intelligent activity (in vertebrates). In 1968, G. A. Mazokhin-Porshnyakov designed original experiments with honey bees and demonstrated evidence of their ability to use some logical operations, though the methods differed from those outlined by Krushinsky. The investigation of insect intelligence was preceded by the investigation of their vision; a unique book for its time, Insect Vision was published in the United States and translated into English (Mazokhin-Porshnyakov 1969a). At present, insect behavior and sensory systems are studied in several academic locales in Russia and the surrounding republics. The largest center of insect study is at the entomology department of Moscow University. Here the schools of bioacoustics and of chemical reception and biorhythmology were founded. Some monographs should be noted. In Bioacoustics of Insects, written by R. D. Zhantiev (1981), many original results concerning acoustic communication in insects (primarily Orthoptera) as well as the neuronal mechanisms of detection and localization of sources of sounds were included. In the field of bioacoustics, the discovery of echolocation in noctuid moths was made (Lapshin, Fyodorova, and Zhantiev (1992). In Chemical Reception of Insects by Ju. A. Elizarov (1978) (published posthumously), the structure of chemoreceptors, ways of coding information in the nervous system and movements of insects toward sources of chemical attractants were reviewed. In Circadian Rhythms of Activities in Insects by V. B. Tshernyshev (1984), the endogenous basis of rhythms, their physiology, and their role in behavior were reviewed. Biorhythms were also investigated by V. M. Afonina and V. A. Zotov. In the research group of G. A. Mazokhin-Porshnyakov, in addition to the learning of honey bees and wasps and nest orientation of bumblebees (P. M Filimonov), the discrimination of parasitized and nonparasitized hosts in egg parasitoids (V. M. Kartsev and A. V. Timokhov) and the territorial behavior of dragonfly larvae (G. I. Rjasanova) are studied. Many investigations of insect behavior are carried out in laboratories in St. Petersburg. N. G. Lopatina (Pavlov Institute of Physiology of the Russian Academy of Sciences), who previously studied conditioning in bees in the laboratory headed by M. E. Lobashov, now focuses her attention on nervous processes in mutant bees. In the Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences (RAS), there are laboratories (headed by F. G Gribakin and A. V. Popov) that deal with insect vision and hearing. In Mechanisms of Photoreception in Insects (Gribakin 1981), data concerning the structure of photoreceptors, their sensivity, and their evolution have been presented. In Acoustic Behavior and Hearing in Insects (Popov 1985), patterns of acoustic communication in crickets and cicadas, nervous mechanisms of hearing and detection of signals, phonotropism, and other questions are reviewed.

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Some other researchers in Russia should also be noted: V. B. Bejko (behavior and orientation of Hymenoptera, especially of solitary bees, at the Institute of Evolutionary Animal Morphology and Ecology of RAS, Moscow); V. V. Buleza (pheromone communication in insects, at the same institute); T. M. Vishnevskaja (insect vision, at the Institute for Problems of Information Transmission of RAS, Moscow); G. I. Rozhkova (insect hearing, at the same institute), E. K. Es'kov (acoustic signals of Hymenoptera, biology of honey bees; two monographs, 1979, 1981, at the Rjasanian Pedagogical Institute (Rjasan'); A. Ju. Haritonov (orientation of dragonflies at the Biological Institute of the Sibirian branch of RAS, Novosibirsk); and N. R. Bogatyrev (social and foraging behavior of bumblebees, at the same institute). There are scientific schools of sensory physiology and insect behavior in the countries derived from the USSR. In Ukraine, researchers include, at the Schmalhausen Institute of Zoology, I. A. Levchenko (bee dances and social interactions in bees, one monograph, 1976); P. A. Mokrushov (visual behavior of adult dragonflies); V. V. Zolotov (visual behavior and orientation of insects; sensory systems); and V. B. Pichka (morphology of sensory organs), working in the laboratory headed by L. I. Frantsevich. In Visual Analysis of Space in Insects (Frantsevich 1980), neuronal and optical mechanisms of vision as well as behavior based on visual information were reviewed, adopting mathematical approaches for analysis. In Lithuania, we find A. V. Skirkevicius with colleagues (Institute of Zoology of Lithuanian Academy of Sciences, or LAS, Vilnius) and V. Buda (Institute of Ecology of LAS) studying pheromone communication in insects. Here, we have outlined the primary source of Russian entomology as it is associated with insect behavior (excluding myrmecology, which is reviewed in another chapter in this book). The remainder of this chapter will review visual behavior and individual learning in social Hymenoptera. Learning in Insects Many, if not all, aspects of animal bionomics are associated with behavior. In some of the biological sciences, "behavior" may be understood in different ways, with an exact definition of this term generally absent from monographs. In general, behavior is the modus operandi leading to some goal. This general definition may be applied to descriptions of the experiments reviewed below. The study of insect behavior during the past few decades resulted in an important conclusion about convergent similarities in many behavioral features among insects and in vertebrates (Bitterman 1988; Lobashov 1955; Lopatina 1971; Mazokhin-Porshnyakov 1981). Additionally, it was found that almost all insect reactions require individual learning (Kartsev and Mazokhin-Porshnyakov 1989; Mazokhin-Porshnyakov and Kartzev 1984) and are not manifested by social learning per se. We consider learning as the change in the probability of performance of a definite adaptive action, in response to previous performance of this or another action (or, in agreement with Thorndike 1911, learning is an ability to re-

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peat successful actions). For example, even the reflexive reaction of proboscis extension in response to contact with sugar water may be suppresed in bees by aversive conditioning (Abramson 1986; Smith, Abramson, and Tobin 1991). Learning is also included in more complicated species-specific activities such as bee dances (Levchenko 1976; Lopatina 1971) or in the perception of species-specific songs by crickets (Popov 1985). An important biological prerequisite to the evolutionary development of learning ability is the ability of animals to orientate themselves in unstandard and unpredictible environmental conditions. Because all free-living animals face this problem, learning capacities may be considered to be determined not by the position of the given animal species in the phylogenetic tree, but by the successful variability of its natural behavioral tasks. Though it is wonderful in the name of diversity that biological differences between insects and vertebrates are obvious (in insects, for example, the absence of education of progeny by parents, relatively short life span, and differences in the structure of the nervous system), principal differences between the behavior of both groups are not revealed. We believe, as will be discussed later, that peculiarities of insect behavior are concerned not with learning abilities or parameters of conditioning, but with situational specificity (what we call "stage dismemberment") in their behavior. The objective study of behavior is possible through the classical conditioning method developed by Pavlov. We have tried to adapt the traditional language of higher nervous activity to the description of experiments reported here, especially if such terminology was used in the original work. But in the study of more complicated behavioral activities, reflex theory is less useful. It poorly explains situations, for example, with many interrelated conditioned stimuli. Additionally, not all reactions may be considered reflexes; imprinting or insight may be noted. Behavior changes that are not reflexive, though appearing to be examples of learning, were also found in some hymenopteran parasitoids that discriminate between parasitized and nonparasitized hosts by means of pheromone markers left at or in parasitized host individuals (Klomp 1980; Lenteren 1976). We have studied this phenomenon in egg parasitoids (Kartsev 1985; Kartsev, Timokhov, and Mazokhin-Porshnyakov 1993). Much of the data obtained with insects suggests that we should move away from a generally reflexive definition of learning in insects. Our aim is to report experimental data obtained in Russia that might be unfamiliar to the Western reader, but we have not attempted to classify these data in agreement with different scientific approaches (such as behaviorism or ethology). In the following section single conditioned stimulus-single reward experiments are reported. Next, we report single conditioned stimulus-two reward scenarios. Then we report experiments with multiple conditioned stimuli-single reward paradigm variations. We next report experimental situations wherein receipt of reward was dependent on use of simple laws of logic and probability. Finally, we examine the relative roles of innate and acquired elements of behavior through


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means of statistical analyses. SINGLE CONDITIONING The discovery of learning capacities in bees made by Karl von Frisch (1977) was followed by a great number of works that confirmed and completed previous results. In all of the examples described below, one conditioned stimulus was paired with a definite reward, the diversity of which was derived from the variation in the kind of conditioned stimuli and reward. Two types of conditioning may be distinguished: In classical conditioning using one conditioned stimulus, the strength of conditioned stimulus-unconditioned stimulus pairings is assessed on test trials in which the conditioned stimulus is presented without reward. In the second type of conditioning, the animal receives two conditioned stimuli, only one of which is reinforced. This is known as differential conditioning. Experiments of both types have been conducted in Russia since the 1950s and are still of interest today. Experiments have also been conducted using methods similar to those developed by von Frisch. In the field, a free-flying bee was offered a choice between rewarded (with sucrose) and unrewarded targets. These targets can differ along various dimensions such as odor, color, and/or shape. The appearance and strength of conditioning was estimated statistically through the ratio of choices between both targets. In other experiments, local food and defensive responses were investigated in a small chamber linked to the hive by a glass tube. The Role of Stimulus Modality Faster conditioning was observed if odors were used as conditioned stimuli, with rate of conditioning depending on the kind of odor. Odors of the bees' favorite flowers (red clover, sunflower, hoary madwort) were learned after one conditioning experience. For example, on the second training trial, 95% to 100% of the bees landed at the correct target. The odor of yarrow (Achillea sp.), however, which is unfamiliar to bees, required six to ten trials before reliable conditioning was exhibited (Lopatina 1971). The speed of conditioning also depends on the salience of conditioned and appetitive stimuli. A larger colored square ( 8 X 8 sq. cm) is learned about twice as fast as a smaller square ( 2 X 2 sq. cm) (Nikitina 1959). Increasing the concentration of mint in the sugar-water reward also resulted in accelerated conditioning (Nikitina 1965). When the amount of appetitive stimuli is varied by manipulating the sucrose concentration, performance is better the more concentrated the sucrose solution (Nikitina 1965). Discrimination of Left and Right Position preferences may be conditioned as well. The ability of insects to

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orientate themselves in mazes seems to mean that insects are able to discriminate between left and right turns irrespective of external markers, as do dogs (Fless 1958) and other vertebrates. But in experiments without maze rotation, the influence of external markers (various gradients including the Earth's magnetic field) cannot be excluded. Additionally, walking orientation may differ from orientation in three-dimensional flight. In the experiments of Masokhin-Porshnyakov (Masokhin-Porshnyakov, Semenova, Kartsev, and Rabinivich 1987), a bee or a wasp (Paravespula vulgaris or P. germanica) was trained to enter a nontransparent cylinder (diameter 70 cm) opened from above. A horizontal mount with two visually identical feeders (at a distance of 5 cm one from another) was positioned on an internal wall. While in the cylinder, the insect always reached the mount from one side. There was sugar water in one feeder and concentrated sodium chloride solution in the other one. The position of the mount was changed after every insect visit. The experimental cylinder was periodically moved to another place at a distance of some meters. Landmarks were excluded, with the only criterion remaining being the choice of left or right position. Conditioning (significant preference for the rewarded side) was successfully revealed in 13 out of 18 bees examined and in all 9 wasps examined; 5 bees did not learn even after a dozen visits. Consequently, it seemed that bees and wasps are able to perceive "left" and "right." Learning a position task seems to be more difficult than learning the place, odor, or color of a food source. A comparison of the performance of bees and wasps in the previous experiment indicates that wasps are able to learn a position task better than bees. In the section below we shall see that this is not the only difference between bees and wasps. Some individual data from the previous experiment should be noted. One wasp chose the rewarded feeder every visit without mistake when the feeder was placed in a southwestern position. This wasp may be successfully combining position and celestial orientation. There is also the case of a bee who persistently preferred the right feeder (the incorrect one), even though the left feeder was always rewarded. Such a position preference has no explanation at this time. Aversive Conditioning Conditioning with negative reward may be performed as well as with positive reward. In a number of experiments (Lopatina 1971; Voskresenskaja and Lopatina 1953) various aversive stimuli were tested: a 50% solution of calcium chloride (reaction: the refusal of feeding), drastic temperature increase in the experimental chamber (reaction: wing ventilation), and slight electric shock (reaction: sting extension, buzzing). Conditioning was considered successful if the aversive reaction was demonstrated by the bee in response to a conditioned stimulus. In all cases, conditioning was successful. The authors did not find principal differences between the speed of aversive conditioning and of appetitive conditioning. Thus, an odor was learned after 1-2 combinations with calcium chloride.


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Delayed Conditioning An important property of the nervous systems of "higher" animals is their ability to remember stimulus traces. This ability was demonstrated in bees, using a Tmaze. In the stem of the maze, a bee was exposed to a green or blue light for 15 seconds. After removal of light, the bee was captured in the stem and confined for a specific period of time. It was then released, and it proceeded to the decision point of the maze. The position (left or right) of the food reward depended on the color signal presented in the single arm. Conditioning was considered successful if the experimental animal selected the rewarded arm 10 times in succession. If the delay did not exceed 30-45 seconds, conditioning occurred after 6-12 repetitions. Delays longer than 45 seconds produced no conditioning (Nikitina 1959). Inhibition of Conditioned Connections An important peculiarity of conditioning is its temporal instability. It has been found that conditioned connections extinguish when bees are given another task and when the animal encounters a familiar stimulus that is no longer rewarded. The general conclusion from a number of experiments—mostly performed in Germany—suggests that in bees new information replaces previous information (although some conditioned reflexes may exist simultaneously). Thus, a foraging bee caged in its hive can remember a conditioned color up to two weeks, but upon its being released, the connection will extinguish rapidly after unreinforced exposure (Lopatina 1971). The rate of extinction, of course, depends upon the modality of the conditioned stimulus as well as on the amount of previous learning. Characteristics difficult for bees to recognize, such as the shape and size of figures, position (left vs. right), and the pattern or alternating positions of reward, are rapidly forgotten (Mazokhin-Porshnyakov, Lubarsky, and Semenova 1987; Mazokhin-Porshnyakov, Semenova, Kartsev, and Rabinivich 1987). Conditioned connections that are extinguished may be preserved in a latent state, because they spontaneously recover after 1 to 2 trials (Voskresenskaja and Lopatina 1952). In differential conditioning there are two possible ways of stimulus presentation: simultaneous and successive. It has been shown under simultaneous conditions of stimulus presentation that extinction proceeds much more rapidly than if the stimuli are presented successively (Lopatina 1971). Delayed Inhibition Bees visiting a feeder with a sucrose reward can be trained to wait for at least 60 seconds before they are permitted access to the feeding site. At the beginning of the delay the bee can be observed making "anticipatory" reactions. It vigorously begins to test the feeding station every two to three seconds. When the feeder is presented after the delay, the bee does not immediately consume the reward. Moreover, upon filling up it does not immediately leave the training site. It is also

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interesting to note that upon its return to the hive it does not immediately return to the feeding site after unloading. Rather it can be observed walking and cleaning comb. After some time, it will return to the feeding site. This behavior was observed for four or five trials, after which the bee would return rapidly to the feeding site. After arriving back at the site, it hovered above the floor and landed in order to test the feeding locus some time after the 60 second interval. This behavior was observed in 20 individuals (Lopatina 1971). "Delayed inhibition" was considered to be determined by "a change in excitability of some nervous centers." However, we would like to take the opportunity to note that physiological terminology does not help explain behavior if the actual physiological mechanism remains unknown. From our point of view terms such as "conditioned timing" or "delayed conditioning" might be used as well as "delayed inhibition." No matter what term is used they all mean that a bee can be trained to wait for some period before obtaining reward. Conditioned Inhibition This term is used to describe tasks in which a rewarded conditioned stimulus is no longer rewarded when placed in combination with a second conditioned stimulus. In experiments with two visual stimuli, the stimuli in combination began to inhibit the food reaction after 10 to 12 trials. Combinations of visual and olfactory stimuli were learned after 16 to 18 trials (Lopatina 1971). In fact, the ability of the bees to select a conditioned stimulus when presented alone and to reject the same stimulus when presented in a compound (i.e., the conditioned inhibitor) suggests that bees have the ability to perceive the elementary logical law of object relations (we will discuss this further below). In more complicated experiments, bees were conditioned to two stimuli— amylol and butanol odors. The inhibitory combination consisted of amylol odor and a red color (this color was likely perceived by bees as black, because bees do not see red; Mazokhin-Porshnyakov 1969). After inhibition to this combination was established, the combination of red and butanol was presented. The new combination was found to be inhibitory as well. Thus, the bees generalized red as signalling the absence of food. Plasticity of Nervous Activity One of the common criteria of nervous system plasticity is the ability to alter a learned reaction following a change in the rewarded stimulus. This ability was demonstrated in bees. Bees were conditioned to the odor of 50% amylol, while the unrewarded stimulus was 100% amylol. When the bees began to choose 50% amylol unmistakably, the reward was shifted to the 100% amylol. Different individual methods of reaction to the new situation were observed: Some individuals did not alter their reaction at all during a 3-day period, others changed their reaction after only 7 to 15 trials. A few individuals altered the reaction after just 1 to 2 visits. The same


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results were also obtained in an experimental variant, in which the alteration of stimulus significance was accompanied by pairing the previously rewarded stimulus with a distasteful calcium chloride solution (Lopatina 1971). Repeated alterations of stimulus significance were also investigated, with different colors used as conditioned stimuli. After the first alternation, 5 experimental bees relearned with an average of 16 trials, After the second and the third alternations, relearning required between 8 and 12 trials. The acceleration of relearning seems to be statistically significant. The Role of Mushroom Bodies in Conditioning The intensive investigation of behavioral aspects of conditioning in Russia did not historically correlate with the investigation of nervous processes. There are only a few works in this area. The role of the mushroom bodies in the elaboration of conditioned reflexes was demonstrated by A. K. Voskresenskaja (1957). Different degrees and localizations of extirpation in the mushroom bodies resulted in partial or complete destruction of previous conditioning. Unilateral (left or right) extirpation of mushroom body tissue, combined with extirpation of optic lobe tissue on the same side, did not result in destruction of color or odor conditioning (in 9 bees). Contralateral extirpation of mushroom body and optic lobe tissue resulted in destruction of color but not odor conditioning (in 4 bees). Partial bilateral extirpation of mushroom bodies revealed the same results (in 8 bees). Bilateral complete extirpation of mushroom bodies, with other structures of the supraoesophageal ganglion remaining intact, destroyed all conditioned connections, but not movement coordination or inborn color and odor reflexes (in 5 bees). Extirpation of various parts of the mushroom bodies, along with cutting the antennae, allow us to conclude that the nervous pathways of visual and olfactory conditioning are different. Each eye is unilaterally connected with its mushroom body; and there are more complicated bilateral connections between the olfactory lobes of the brain and the antennae. The Peculiarities of Conditioning to Barely Discriminable Stimuli After the learning abilities of insects were demonstrated, conditioning techniques were used for the investigation of insect sensory systems and processes. A series of experiments aimed at examining the ability of bees to discriminate shape and size of geometrical figures were carried out (Mazokhin-Porshnyakov, Semenova, and Milevskaya 1977, 1979). Unusual behavioral peculiarities demonstrated by bees in performing difficult tasks were found. Below, we shall see that bees are able to classify shapes by the number of angles, irrespective of other characters. But before examining this, simpler experiments should be reviewed. Traditional field experimental methods were used. A single free-flying bee was attracted to a small experimental table with a sweet lure located some meters from

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the hive. When the bee arrived at the experimental table, two different (in shape or size aspects) figures were offered. At the center of the rewarded figure, a little glass with sucrose was placed, the unrewarded target was similar except it contained concentrated sodium chloride solution. After every visit by the bee, the figures were arranged at the table in a new position. The glasses were regularly replaced with new ones in order to exclude Nasonov's gland odor left by the bee. As this method did not prevent quick reception of the reward following a mistake of salt testing, so lack of a discrimination could be considered an adaptive (although not optimal) behavioral strategy. However, the bees usually did not adopt this strategy; the bees tried to avoid the salt. Size and shape discriminations were subjectively difficult for bees, because they prefer to learn other characteristics of searched objects such as their position, color and/or odor. Only a small portion of the tested individuals solved these tasks, and learned preference of the rewarded figure disappeared with time (MazokhinPorshnyakov et al. 1979). Individual examples of discrimination to different kinds of visual stimuli are presented in Figure 7.1, which shows the performance of bees trained to discriminate various stimuli. The bees chose the rewarded figure during the entire day without exhaustion, if the discrimination criterion was the position or colors of the stimulus targets. However, bees find it difficult to discriminate shape, size, and shape/size combinations. Some of the consistent visits to the differentiated unrewarded figure are likely to be related to innate scouting activity. Other behavior was demonstrated by bees in difficult tasks. All trained individuals returned to random choices after a few dozen (50 to 70) visits. This behavior occurred often in the first half of the day, when physical exhaustion was unlikely. There were also individual variations in the extinction of conditioning. It is possible that, if a more stringent technique were used (for example, choice of the differentiated object might result in electric shock), the bees might not forget the experience or would cease to visit the experimental place at all. Bees were unable to solve this problem in mazes. A bee would repeat incorrect turns, though no reward was offered (Nikitina 1959). Sometimes conditioned bees reacted negatively to a colored marker (Chesnokova 1960). The described incorrect behavior may be designed to prevent exhaustion of the nervous system in difficult situations. Rejection of reward searching in difficult tasks, along with switching to random choices, is known in crows dolphins, and dogs (Krushinsky 1977; Prazdnikova 1970). The described phenomena confirm the earlier supposition about the similarity of conditioned connections in insects and in vertebrates (Lobashov 1955; Lopatina 1971; Voronin 1969). Reversion of Response This term denotes a phenomenon observed in a difficult task in response to additional complications of a previously solved task (Mazokhin-Porshnyakov et al. 1979). The bees were trained to discriminate between pairs of different-sized


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Figure 7.1 Discriminative abilities in bees. 1: shape discrimination, 2: color discrimination, 3: shape offigures,4: size offigures,5: shape and size simultaneously.

figures. The discrimination was found to depend on both the shape and absolute size of the figures: for circles, the minimum area ratio discriminable was 1:1.60; for crosses, it was 1:1.53; for stars, it was 1:1.17; figures with an area about 300 sq. mm were discriminated better than those with an area about 200 sq. mm. Bees spontaneously preferred larger figures. They can also be trained to choose a smaller, rewarded figure, if the difference between figures did not reach a minimum value. The gradual increase of similarity in the size of figures resulted in a switching of preference. The bees began to prefer the largest unrewarded figure, though they had to taste sodium chloride solution regularly. This was observed in experiments with two pairs of stars with sizes of 11 and 17, and 13 and 17 mm, respectively, and with a pair of crosses with sizes of about 17 and 29 mm. Further diminishing the difference in size resulted in an inability to discriminate. We ex-

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plain this phenomenon via reversion of a learned behavioral program to an inborn one. Evidently such reversion is an adaptation to revert to a secure nervous system pathway in difficult tasks. Therefore, the degree of learning directed against an inborn program is limited, but the possibility of such learning is interesting per se. STAGE "DISMEMBERMENT" OF BEHAVIOR IN INSECTS In previous sections, we have described many examples of conditioning in which one kind of reward was paired with one conditioned stimulus. In this section, the pairing of one stimulus with two kinds of reward will be reviewed. The problem under scrutiny is concerned with stage dismemberment of insect behavior. The dissection or separation of behavior processes into their constitute parts (for example, the construction of an ethogram) is to a great extent dependent on the personal aims of the investigator. It is difficult to avoid such subjectiveness. As an example of stage dismemberment, an experiment investigating nest construction in the caddis fly found that such behavior can be disrupted with mild electric shock. The degree of disruption depended on the stage of nest construction. This is an example of one objective criteria in which behavioral sequences can be dissected. We can suggest another criterion: the absence of transfer of individual experience (conditioning) from one situation (stage) to another (Sveshnikov, Fajdysh, and Filimonov, 1975). Discrimination between Square and Circle with Return to Nest or Food as Reward Experiments aimed to demonstrate the ability of bees to discriminate simple geometrical figures may be attributed to the phenomenon covered in the section on single conditioning. In the following experiments, some behavioral peculiarities were found, and therefore we deal with them separately. In the experiment described earlier, the ability of bees to discriminate between circle, square, and triangle was demonstrated (Mazokhin-Porshnyakov and Vishnevskaya 1965). Each test figure in these experiments consisted of many little similar figures, but the ability to discriminate single figures remains mysterious. Some individuals were trained to discriminate between single squares and circles using sucrose as the reward. In other experiments, bees were trained to stimuli associated with entering the nest (Mazokhin-Porshnyakov et al. 1978). The nest experiments were similar to the food experiments with the exception of the kind of reward. To conduct these experiments, the bee hive was fitted with a rotating horizontal deck. The deck contained two holes, which could be identified from a distance by the placement of various types of discriminative stimuli. Entering the correct hole resulted in a return to the nest, entering the incorrect hole did not. The results indicate that bees are able to discriminate simple figures in accordance with their static geometrical parameters, because circle and square are indis-


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tinguishable on the basis of physiological mechanisms, as shown in a number of experiments estimating flicker sequences (see literature cited in MazokhinPorshnyakov et al. 1978). The dependance of discrimination ability on behavioral motivation should be discussed. During food searching behavior, bees discriminate dark figures poorly and within restricted size limits (about 3 sq. cm). Contour figures were discriminated better, with a higher size optimum of about 7-20 sq. cm (additional experiments showed that contour figures were distinguishable from 0.8 sq. cm up to 50 sq. cm; multiple repetition of the contours inside the figure did not facilitate discrimination). These differences in the discrimination between dark and contour figures might be concerned with features of bee vision. However, in nest experiments, contrasting results were obtained. Dark figures are better learned when the reward is returning to the nest, while contour figures are better learned with a food reward. The differences in discrimination as dependent on behavioral motivation were found also in the experiments with a black dark circle (20 mm in diameter) and a 8-radial star of the same color (54 mm in diameter) (Kartzev et al. 1987). The percentage of discriminating and nondiscriminating bees was higher in a foraging situation than when nest orientation was used as reward. This is shown in Figure 7.2. There is one more peculiarity to note. Entrance-searching bees did not demonstrate extinction of conditioning. Bees trained with a food reward do, of course, extinguish. The dependence of the pattern of learning on internal behavioral motivation is a very important factor. A bee going to eat looks at its environment from a different perspective than a bee returning home does. Spontaneous preferences are known to be different too. Honey bees, bumblebees, and wasps preferred figures of different shapes, independently of behavioral motivation (Jacobs-Jessen 1959; Jander and Fabritius 1970). In our experiments, bees preferred a yellow color to light blue in a foraging test situation, but not when the task involved returning to the nest (Kartsev et al. 1987). Wasps preferred the shape of a figure but not its color when given tasks involving a return to the nest (Masokhin-Porshnjakow et al. 1976). This is not true when they were given tests involving food reward. The Absence of Relationships between Foraging and Nest Orientation Bees and wasps (Paravespula vulgaris) were trained to discriminate figures (by color or by shape) while foraging, and then similar figures were used to mark the entrances to the nest. The previously rewarded figure marked the open (i.e., rewarded) hole, while an unrewarded figure marked the closed (unrewarded) one. The control group visited an unmarked feeder with sugar water near the experimental table; this group had no training experience. During the first 10 visits after the beginning of nest training, the proportion of choices of open and closed entrances was 50:28 in 8 experienced bees, and it was 52:38 in 9 controls. These proportions were quite similar. In wasps (5 experienced

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Figure 7.2 Performance of two groups of bees discriminating stimuli that lead to food (curve 1) or to the nest entrance (curve 2), over the course of 80 visits.

and 15 controls), analogous proportions during 30 visits were found. Consequently, it seems that bees and wasps do not apply individual experience to the new situation. We found it surprising that insects could not identify familiar objects in a new situation. Transfer of training from nest to food orientation situations was not observed either. The bees that were able to discriminate between circle and star using nest search reward (only 4 individuals of 13) demonstrated the proportion of choices of food rewarded and unrewarded feeders of 20:20. In a group of 4 nestinexperienced bees, analogous proportions were found. There were no significant differences. The dismemberment of insect behavior (subdivision of behavior into separate stages) may be an important peculiarity of insect behavior. The preferences (i.e., instincts) and the patterns of learning are different in different behavioral situations. The only relationship between behavioral stages seems to be that of switch-


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ing from one stage to another. It is likely that there are more than two discrete stages in insect behavior. The subdivision into stages may be estimated by the absence of transfer of individual experience between stages. We tested the hypothesis that foraging for protein food (hunting) and foraging for carbohydrate food represent different behavioral stages in wasps. Wasps were trained to discriminate shapes while searching for small pieces of fish. One target contained fish, the other contained a synthetic material with fish odor. Then the rewarded and unrewarded targets were replaced with sucrose and sodium chloride solution, respectively. Conditioning was transferred in the new situation without any loss. Consequently, we concluded that foraging is a single behavioral stage, irrespective of the kind of food (Kartsev 1983). Although the dismemberment of conditioning is certain, some universality of perceptive experience can not be excluded. There were two bees not discriminating between circle and star in the entrance searching task and still another bee discriminating poorly. These bees, however, discriminated these same figures very well in the food search situation. It would be of interest to compare our results with those obtained by other authors. But we are not aware of other experiments dealing with the transfer of individual experience of bee under changing behavioral motivations. There are only a few relatively old works close to this problem (Kalmus 1937; Vagner 1913). Vagner noted that negotiation away from the nest is quite different from negotiation toward the nest, and these activities are remembered by bumblebees quite separately. He considered insect memory as a mosaic process. Some contrary results are available, however. In the experiments of Chesnokova (1959a), a bee was trained to reach a feeder in agreement with a sequence of three colored markers (see below, under "compound conditioning"). Then, an additional inverted sequence (which the bees saw on their return trip) was introduced. At the end of this additional sequence, reward was placed. The bees reached this reward but did not consume it; they then returned to the hive. This means that, during the flight away from the hive, the bee remembered the sequence of stimuli on its way back. It is possible, based on these experiments, to suggest that the pattern of dismemberment of learning is the main difference between insect (invertebrate) and vertebrate behavior. COMPOUND CONDITIONING In this section, combinations of reward with conditioned stimuli are reviewed. The experimental situations employed were similar to those found under natural conditions in which an animal learned not one, but many characteristics of the searched-for object and learned many landmarks along the way. There were two kinds of experiments: In the first, one stimulus characteristic was enough to detect the target. In the other type, all stimuli were necessary, and the insect had to learn the combination of stimuli and develop an ability to discriminate between it and other stimulus combinations.

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Spontaneous Preferences for Different Characters of Searched Object A traditional experimental pattern, which has been used by many authors, is as follows: An animal is trained to choose a target composed of several characters and then is tested with a target containing all the characters in combination with unrewarded characters. The following are some examples of such experiments carried out in Russia. Bumblebees Bombus agrorum and wasps Paravespula vulgaris were trained to discriminate between open and closed nest entrances (as described previously) by means of two figures differing in their color and shape (Mazokhin-Porshnyakov and Beiko 1976). Then, in a new pair of figures, the rewarded color was paired with an unrewarded (differentiated) shape, and vice versa. All four tested bumblebees significantly preferred color to shape. Of five wasps, two preferred shape and one preferred color (two wasps had no significant preference). These results, as well as those obtained in other experiments, allow us to conclude that color is more important for bumblebees and shape is more important for wasps. In bumblebees, such stimulus characteristics as volume (three-dimensionality) and color were studied. Bumblebees (5 individuals) were trained to choose a blue cube over a yellow two-dimenstional square. In the testing situation, where color and dimension were switched, bumblebees chose the blue square and yellow cube in the proportion of: 77:51. A slight preference for color over volume was evident. Interaction of Different Orientation Systems The ability of bees and wasps to discriminate left from right, irrespective of external markers, was discussed previously. In another experiment (Semenova, Kartsev, and Mazokhin-Porshnyakov 1989), a surplus externally colored marker was added to the left or right position. The technique described above was used, with the exception that a blue vertical stripe was placed near the rewarded feeder. Most insects learned to prefer the rewarded feeder, but two bees and one wasp (out of 19 bees and 7 wasps, respectively) did not. The task seems to be more difficult than choosing between two colored targets at the horizontal table (although the inability of some individuals to learn might be explained as due to seasonal peculiarities). After the training period, two types of test situations were carried out. First, the blue marker was either removed or moved to an unrewarded feeder. In between some of the test trials, some normal learning trials were continued in order to avoid relearning during the test. The results indicate that all three possible ways of using the two characteristics of the reward were observed in different individuals: learning the position based on "left vs. right" only, learning by using the external marker only, and learning both characteristics in combination. The last strategy was the most probable. For example, in bee No. 4, removing the blue mark-er did not disturb its orientation, and the proportion of choices of rewarded and unrewarded feeders remained 11:1.


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However, moving this marker to a false position produced a significant reduction in visits to the rewarded feeder, and the proportion of choices became 7:6. Consequently, it appears that this bee did remember both characteristics. Analysis of the proportions of insect choices at different feeder positions allows us to suggest that a few individuals attempt to use celestial markers, together with those markers explicitly provided by the experiment. Thus, in this experiment, insects appear simultaneously to use markers belonging to three orientation systems: (1) in relation to proper body axis, (2) in relation to landmarks, and (3) in relation to celestial markers. Synthesis of other characteristics of a target (color and odor) was observed by Lopatina (1971). Bees were trained to visit a blue table with the odor of octanol. Periodically, they were simultaneously offered a white table with the odor, a blue table without odor, and the familiar table with both characters. The author reported (without statistical analysis) that the bees initally visited all tables with equal frequency, but after several trials, a strong preference for the blue table with odor was observed. Elaboration of Stereotypes of Conditioned Reflexes The ability to comprehend relationships between conditioned stimuli is explained in physiology by "synthetical" nervous system activity or by the "elaboration of stereotypes of conditioned reflexes." For example, a moving bee should be able to learn not only the existence of some markers but also their consequences. This ability was studied in a maze (Chesnokova 1959a). A transparent maze was linked to the hive by a tunnel, which was further divided into two arms. The feeder was placed randomly in either the right or the left arm. The path in the rewarded arm was marked with a set of three colored circles located on the maze floor. Some individual bees were tested with a blue-green-red sequence, while other bees were tested with a violet-orange-green sequence. It was not surprising that the bees successfully learned to turn into the rewarded arm by remembering the color signals. Learning in this situation required an average of 120 visits and occurred over the course of 3 to 6 days. The main question, however, was whether the bees perceived all the colors and their consequences. An affirmative answer to this question was obtained in the test situations, in which a trained bee was offered either single colors or a changed sequence of all three (in a 2-3-1 configuration). In all 22 tests, bees turned back, or at least reduced their speed of walking to the feeder. In 5 other tests (with an inverse sequence of 3-2-1), normal walking to the feeder was observed three times. The inverse sequence may be more acceptable to bees, because they encounter this pattern on the return from the feeder. The author also reported (without statistical analysis), that one bee was successfully trained to turn left or right independent of the consequence associated with the colors (1-2-3 or 3-2-1), when the stimuli were located in the maze stem. In a maze with three arms, bees were trained to choose different arms marked

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either with different odors or with odors plus colors during consecutive visits (Nikitina 1959). The author concluded that the task was successfully learned, but the necessary statistics were not published. Unfortunately, the lack of statistical treatment also clouds the results of two further studies that are reviewed below. In field experiments, feeders were attached to three colored shields (0.75 m X 1.5 m) placed some distance from one another (1 m - 55 m) (Lopatina and Chesnokova 1962). When the bees recognized all shields, only one shield at a given moment remained rewarded, the rewarded position being regularly changed. First, the bees visited blue (1), then yellow (2), and then white (3), and so on. After the training period, the reward was removed completely. The bees searched for it, investigating the shields mainly in the sequence 1-2-3. Moving the shields to a new location did not disturb the sequence of investigation. Investigation was disturbed, however, by the substitution of the shields from colored to uncolored despite the fact that the positions of shields remained the same. In other experiments (Lopatina and Chesnokova 1965), bees were fed at a constant location on tables on which the color was changed regularly. There were three rewarded tables and one unrewarded (differentiated) table. After the training period, bees were offered all tables simultaneously without reward. Among 21 bees tested, 13 bees investigated the tables in the sequence determined during previous feeding on successive visits. Additional evidence that bees remember the sequence of colors was obtained by changing this sequence. When exposed to an unexpected color, trained bees significantly increased return and landing times. Discrimination of Two- and Three-Colored Combinations Bees and wasps were offered sets of 9 or 10 multicolored cards (MazokhinPorshnyakov and Graevskaya 1966). Only one card carried the rewarded combination of colors. Each card consisted of 36 squares (10 mm X 10 mm) of two or three colors of different intensities (this was done so that bees and wasps could not solve the problem by using intensity as a cue). The sets of cards were arranged so that reward was distinguishable only by the presence of two or three colors together (or by the absence of two or three other colors). For example, if the combination of blue + green was rewarded, differentiated combinations were blue + orange, blue + yellow, green + orange and green + yellow. If the combination blue + orange + green was rewarded, unrewarded combinations were blue + orange, green + orange, blue + green, blue + yellow + orange and green + yellow + orange. The positions of the cards were changed regularly. The rewarded card was sometimes replaced by similar cards with other patterns of little colored squares (in order to exclude this pattern as the sign of the reward). Each insect was studied separately. An individual was considered to have solved the task if it landed on the rewarded card in 80% of its visits (chance level was about 10%). This criterion was reached by the bees after an average of 15 visits while learning two-color combinations and after 25 visits while learning three-


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color combinations. Wasps (Paravespula vulgaris, Dolichovespula saxonica), however, did not solve the task. Their maximal level of visits to the rewarded card did not exceed 20%. It was found that wasps poorly distinguish colors in general. Even with monocolored yellow and blue cards, learning required 20 visits (bees were able to solve this in 10 visits). In other unpublished experiments, wasps could not discriminate between single green and blue figures, the differences in stimulus properties being obvious from the human point of view. In conclusion, we would like to note that the ability to learn a sequence of stimuli or to identify a combination of characters among others is actually the ability to solve logical tasks. To describe such abilities using the language of the conditioned reflex is complicated, if at all possible. However, all of the tasks reviewed above could be solved with the help of definite constant characters. This allowed us to avoid such terms as "intelligence" or supposition about facilitation of solving such tasks by inborn capacities SOLVING LOGICAL TASKS We consider "intelligence" to be the ability of individuals to comprehend laws governing relationships of elements in the environment. A second criterion of intelligence might be the ability to plan the behavior (to form modus operandi) in order to reach some definite goal. We do not know of positive examples of such ability in insects. A third criterion for intelligence might be the ability to make nonstandard decisions. This criterion is discussed in the next division. Avoiding philosophical discussions about the independence of these criteria, we describe below a number of examples of intelligent behavior, based on the first criterion. Estimating the degree of individuality in animal behavior is difficult. For example, a bee that rounded a mountain on the way to a food source and then gave information about the location through its dance may or may not lead to individual interpretations of the behavior. A bee's actions on foraging trips suggests that it can create "maps" and is able to follow such maps. However, we do not consider these actions or those of bee dances as signs of intelligence, because we suppose that such actions are, to a great extent, innate. Yet to the contrary, the ability to comprehend relationships of stimuli in an artificial experimental situation may be considered as evidence of intelligence. Thus, when discussing the intelligence of an animal, we must always have in our mind not only the question "what?" but also "how?" does the animal do something. The question of "how" is omitted in the experiments described below, because the artificiality of the experimental tasks do not provide an explanation in terms of innate predispositions for solving such tasks. These tasks may be classified as objectively difficult. Bees have been learning such tasks for a long time, and many individuals do succeed. The question "how" will draw our attention in the next division of this article. There is another approach to the estimation of animal intelligence, one that excludes previous learning. Insight (Thorpe 1964) or extrapolation (Krushinsky 1977) refers to an instant comprehension of a problem. In one of the experiments

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of Krushinsky, a vertebrate animal could see a feeder through a small screen. The screen prevented the animal from directly reaching the food. When the feeder began to move through a tunnel (thereby preventing the animal from seeing the food), some species of vertebrates were found to be waiting for the food on the other side. Extrapolation is the ability to predict the position of the invisible feeder on the basis of initial information about its motion. If bees are able to extrapolate, we should expect them to perform this task. We note two questionable points in this approach: (1) The previous experience of an animal is not taken into consideration. An animal without any relevant experience might be expected to demonstrate "pathological" behavior only. (2) The innate predisposition for extrapolation is not estimated prior to these types of experiments. For example, dogs (but not frogs) appear to have an ability for extrapolation as measured by the "tunnel" or "object permanence" task described above. It is possible that this task is quite natural for dogs but that it is unnatural for frogs, which never follow their prey. In other words, the subjective difficulty of this task is different for different animal species. Spiders, which usually follow their prey, were shown to accurately predict (extrapolate) the appearance of their prey (Heil, 1936). Extrapolation may be considered by many an unnatural quality of bee behavior. Therefore, it was a surprise to many bee researchers when a report appeared demonstrating extrapolation (Shekshuev and Gurevich 1983). A bee was situated at a feeder that was placed inside a tube (the tube substituting for the screen that is used in analogous vertebrate experiments). There was a small window in the middle of the tube. From the external side of the tube, the bee could reached the feeder with its proboscis. Then feeding was disrupted by moving the feeder to the left or to the right. The majority of bees flew away in response to this procedure. Some bees, however, began to move along the tube in the correct direction (the proportion of bees choosing the correct and incorrect directions was 78:45). These results were obtained if the middle part of the tube was transparent and bees could see the moving feeder before it became invisible, disappearing in the nontransparent distal part of the tube. In control experiment with a completely nontransparent tube, the proportion of choices in either direction was random. We can try, however, to explain these results without making the assumption of bee intelligence. When the feeder began to move, the bee wanted to reach it directly, so the bee moved in the direction of the feeder. When the feeder was out of sight the bee simply continued its motion, perhaps in part, because of its momentum. Thus, we conclude that animal intelligence may be better estimated in tasks using gradual and controlled learning. A great diversity of original logical tasks are described below. Comprehension of Common Abstract Character in Different Objects (Generalization of Visual Stimuli) The experiments (Mazokhin-Porshnyakov 1968, 1969a, 1969b, 1970) were


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aimed at demonstrating the ability of bees to comprehend classes of objects with a common abstract character. Thus, though there were no definite conditioned stimuli, they were replaced by the law of distribution of reward. Discrimination of Triangles and Quadrangles by Means of Number of Angles Irrespective of Other Characters During the first stage of the experiment bees were trained to discriminate one definite shape out of a pair consisting of a triangle and a quadrangle, as described above. After this, the first test was carried out. Each bee was offered a new pair of the same shapes with new sizes and new values of the angles, with neither figure containing reward (in order to additionally exclude recognition of feeders by stimuli associated with sucrose). Each bee was examined only a few times in order to avoid relearning in the absence of the reward; examination visits were alternated with learning visits to the first pair of figures. The results of the tests were analyzed for the entire group. After the first test, a second learning sequence was carried out with a second pair of figures, the same figures used in the first test. After this training, the second test was carried out with a third pair of figures, and so on. Just as in many of the experiments described earlier, the number of proboscis, antennae, and/or tarsi contacts were recorded as the bee contacted a test figure during each visit. The initial response to a target was considered to represent the bees choice. The proportion of choices of the triangle and the quadrangle was 33:34 in the first test (9 subjects), the absence of preference being obvious. However, comprehension of common characters of a class of figures is not possible by learning only one pair of stimuli. In the second test, preference became significant, and the proportion of choices was 44:14. In the third test, preference became even more obvious (49:7). Thus, when offered new pairs of figures, bees were able to identify the shape to be rewarded by comprehension of the number of angles in the figures. This conclusion is confirmed by other experiments dealing with the recognition of triangles and quadrangles. Bees were trained to discriminate triangle and square without changing the positions of the figures in relation to each other. In the test, they were offered a triangle and a square of new sizes and in a new position. The results of the first test were positive (44:4) and were confirmed in a second test (37:0). Bees, which were trained to discriminate dark triangles and squares, discriminated the same figures constructed by points, as counter figures, and even outlined by dotted lines. But they did not identify figures of familiar shapes after the color of the shapes were changed. This was shown in an experiment in which 6 bees were trained to discriminate between an orange triangle and square on a blue background. In the first test they were offered blue figures against the orange background. The proportion of choice was at chance levels (28:22), though the shapes and sizes of the figures remained constant. The loss of recognition may be

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explained by some type of emotional reaction in response to changing the colors, because these characteristics are very important for bees. In the second test (green figures on yellow background), the bees preferred the figure of the rewarded shape (38:6). This was confirmed in the next two tests with new combinations of colors of and background. Various distractors such as lines or spots partially masking familiar figures usually did not disturb recognition. This may be considered as an example of extrapolation of bees, which predicted invisible parts of the figures. But in the case of partitional application of discriminated figures, one upon another, bees did not discriminate them separately. The lack of discrimination obtained in two tested groups of bees does not exclude the possibility of the presence of individuals (or bee colonies) that can solve such tasks. Generally, bees consider overlapping figures as a single figure. This fact touches upon the principal question of whether bees divide their environment into separate objects. Success of learning in bees through many of these experiments means that human and bee divisions are usually similar. The example with overlapping figures is the only exception. Comprehension of the Number of Objects Bees were trained to discriminate a card with two black circles from cards with one circle and with three circles irrespective of their sizes and positions. During learning, several sets of cards were used. These sets were changed after each 4 or 5 visits. There were 21 cards (7 cards with one circle, 7 cards with two circles, and 7 cards with three circles); these cards were combined in 4 sets. The proportion of choices of two-circled and other cards was 69:32 in tested bees. Thus, bees showed a remarkable ability to perform calculation-like operations. These results agree with those of the previous experiment, where bees "calculated" the number of angles in geometrical figures. The ability of bees to calculate circles on the cards means that bees considered these cards to be different objects, in agreement with the human designation in similar tasks. Generalization of Figures by the Characteristic of Being "Bicolored" Bees were trained to discriminate bicolored figures from monocolored ones irrespective of their colors, sizes, or shapes. In the first test, the proportion of choices of the bicolored figure and of monocolored ones in 11 bees was 44:35:31, showing that a common character was not yet comprehended. In the second test, however, a strong preference for the bicolored figure was observed, the proportion of choices being 81:15:16. Generalization of Figures by Characteristic Black Circle Presented at the End of a Chain of Contour Circles Here, bees were trained to discriminate chains of circles. Chains with a black circle at the end (figures of class A) were rewarded, and those with the black circle in the middle (figures of class B) were unrewarded.


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In one (unsuccessful) variant of the experiment, bees were offered a set of three figures: one A-figure and two B-figures. There were 6 learning stages using 6 different sets, with each learning stage followed by a test. However, even in the sixth test, the choices made by the bees were random. We supposed that a richer set of figures may facilitate comprehension of a common character of the rewarded figures. This is because each set contains more information and because accidental receipt of the reward during the training period is less probable. In a new variant, a set of figures consisting of one A-figure and seven B-figures was used. Learning sets differed by the A-figure, with the Bfigures remaining constant. After each learning period, tests were carried out with one examination set (with a new B-figure and an A-figure that was not used in the original learning). The experimental pattern was as follows: The learning at set N 1, test at set N 2 (examination set); learning at set N 3, test at set N 2; learning at set N 4, test at set N 2; and so on for 6 presentations. The results of the experiment indicate that in the first test, the proportion of choices was random; but in the second test preference for the A-figure became significant and increased gradually over the course of the tests. Generalization of Figures by the Characteristic Black Square Outside the Contour of a Figure The experimental pattern was similar to the previous one with the exception of using not one but two examination sets of figures. Bees were able to solve this task, too. The proportions of choices during the tests were: (1) 10:50 (did not differ from random ratio 1:7, (2) 30:22, (3) 35:22, (4)44:5, and (5) 24:0. Transformation of Experience to a New Situation We have mentioned previously that there is an absence of transfer between foraging and nest experience. However, a transfer of experience to a new situation within the limits of foraging activity was observed. Bees were studied in situations in which previous experience could not be used directly, leaving some elements of this experience as useful or harmful (Mazokhin-Porshnyakov, 1981). The ability to use not the whole experience but only its elements may be considered a sign of intelligence. Three groups of bees with neutral, useful, or harmful experience were offered one test task. There were 4 figures: a blue square, a blue rectangle constructed of separate stripes, and the same two figures of a yellow color. Only the blue-striped rectangle was rewarded. In order to receive the reward on the first attempt, a bee had to remember both the shape and the color of the figure. The first group of bees were trained to visit a black circle; this was the control group with neutral experience. A second group of bees were trained to a greenstriped rectangle, differentiating it from a green square. This group obtained useful experience in shape discrimination. A third group was trained to visit the green square, differentiating it from the green-striped rectangle. This was the group with

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harmful previous experience. Each group consisted of 8 individuals. In the test, the proportions of choices of rewarded and unrewarded figures were (1) 57:23, (2) 71:9, (3) 38:36. The differences between each pair of groups were significant. The group with useful experience learned better, while the group with harmful experience performed worse than the control group. A similar experiment was repeated with color and size as discriminative stimuli. In the test, the bigger figure was rewarded. The first (control) group visited a black circle. The second group was trained to choose the figure in the pair by the abstract characteristic "bigger"; it was trained successively in three tasks differing in details. The third group was trained in three contrary tasks. The results of learning in the test task were (1) 57:32, (2) 73:12, (3) 34:36. The differences between groups were significant. This means that bees are able to combine a familiar abstract characteristic, "bigger," with new and necessary information about definite color. Comprehension of Regularity in Alternations of Feeding Places In the initial experiment, bees were offered two similar feeders placed on a table, with only one feeder being rewarded (Mazokhin-Porshnyakov et al. 1987). The position of the reward was regularly altered in agreement with the law "left, then right" (in relation to the direction between the nest and the table). Individuals were considered to have solved the task when, after several visits, they preferred the rewarded position. In other words, they did not return to the previously rewarded position, but predicted the new position of the reward. Out of 41 bees, 27 solved the task, as did all 13 wasps (Paravespula vulgaris and P. germanica). Similar results were obtained in a modified experiment with vertical placement of the feeder. The position of the reward was changed by the law of "lower, then higher," and the bees could observe both feeders. Six bees among 11 tested and all 8 wasps succeeded in solving the task. Thus, the ability to comprehend the rhythm of alternation of feeding location does not depend on peculiarities of visual information obtained by a bee (different parts of the eye are known to receive different visual information: Labhart 1980; MazokhinPorshnyakov and Taimova 1973). The second experiment was similar to the first, with the exception of the placement of the pair of feeders along the direction of nest to table, with the law of alternation being "nearer then farther." It was found that no insects (8 bees and 5 wasps) comprehended the law of alteration in this case because of strong preference for the nearer position. Here, an innate predisposition was in conflict with the logic of the task. In order to entice the insects to go against their innate preferences, the task "nearer, then farther" was given to individuals who previously solved the task "left, then right." In 12 such previously trained bees 9 solved the task "nearer, then farther," while in wasps this proportion was 9:6. Thus, bees and wasps are not only able to comprehend the law of alternation of positions of the reward, but they


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can also transfer their experience to a new situation. In the last, more complicated experiment, bees (wasps were not studied) were offered both tasks (nearer vs. farther; left vs. right) simultaneously, with the alternation made in two directions. Changes of directions were made after every two visits. For the first visit following this change, the position of the reward was unpredictable, while in the next visit, the opposite position was rewarded. Thus it is easy to calculate, that in the absence of predictable mistakes, the level of correct choices which may be expected would be 75%, with the random level of correct choices remaining 50%. An example of the change in rewarded positions might be: nearer then farther, right then left, farther then nearer, and so on. Predictable positions were stressed, and the choices of the insects were analyzed in these positions only. The algorithm of searching was, "Remember the position of the reward and search for it in the opposite position, if the direction of the location of feeders was not changed." Three bees among 12 tested solved this difficult task. These results were obtained after several days of previous training in the tasks "left vs. right" and "nearer vs. farther" (the animals were trained on these tasks separately). Evidently, solving this task demonstrates the highest level of bee logical capacities. SOLVING MULTIDECISION TASKS In the previous section, tasks were reviewed that, a priori, were considered solvable by primarily individual learning. However, even in those artificial tasks, the question of inborn predispositions to learning was not entirely avoided. For example, alternation of feeding places, "left vs. right" and "nearer vs. farther," were found to be different tasks for bees and wasps. How should we estimate the relative roles of inborn and acquired elements in animal behavior? We touch on this traditional question in the following experiments, using a mock foraging situations (Kartsev and Mazokhin-Porshnyakov 1982; 1989; MazokhinPorshnyakov and Kartsev 1979). There are many equally useful ways of visiting feeders. When the more standard ways were chosen by different individuals, the more important the role of inborn program was considered to be. In field experiments some flower-like feeders were placed on a table (40 cm X 40 cm or 50 cm X 50 cm). A feeder consisted of a colored (blue) paper star in which was placed a small glass containing a precise amount of sucrose. The task was to visit "flowers" without repeated investigations of already emptied feeders (sucrose was poured into the feeders in between bee visits). Similar tasks have been used in the optimal foraging literature (for example, Heinrich 1983; Pyke 1978), but our primary interest was in behavioral decisions, not in energetic adaptiveness of behavior. An insect was considered to have solved the task if the proportion of visits without reinvestigations of the feeders significantly exceeded chance.

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Visiting Four Flower-like Feeders Arranged at the Corners of the Experimental Table In order to satisfy itself (by filling its crop) an insect had to gather sucrose from all 4 feeders. It was found that bees and wasps (but not bumblebees) are unable to discriminate rewarded and previously emptied feeders from a distance. Avoiding feeders previously investigated (and now empty) could occur only by remembering each visited feeder (in relation to external markers) or by means of an algorithm of visiting. It is easy to calculate, that the probability of choosing four feeders successively is (1 x 1 x 2/3) x 1/3 = 2/9, or about 22%. The calculation is made in accordance with the consideration that an insect always goes from one feeder to one of the three remaining feeders). In this experiment, the visits of 6 bees and 6 wasps were registered. The mean percentage of correct visits in bees and in wasps was about 50%. This level is significantly higher than chance. The predominance of correct visits was also significant at the individual level in the majority of insects. Analysis of the dynamics of the number of correct visits provided the evidence for learning. There was great individual variability in these dynamics, but in general the proportion of correct visits increased in 11 of 12 tested individuals. The proportion of correct and incorrect (with one or more reinvestigations) visits for the group of bees was 45:75 during the first 20 visits, while in the remaining visits it was significantly higher, 190:173. Thus, as bees and wasps solved the task, learning occurred. This result allows us go to the main question of the experiment, which involves the relative roles of inborn and acquired elements in behavioral decision. It is easy to calculate that 4 objects (i.e., feeders) may be chosen by bees in 24 different sequences. Eight sequences result in trajectories around the perimeter of the experimental table. These trajectories are shown in Figure 7.3. Trajectories a and c were most often used, b and d were rarely selected. Consequently, the chance ratio of circle and broken line trajectories may be expected to be 8/24:16/24 = 1:2. In all individuals (both bees and wasps), the circular means exceeded the random level. The preference for the circular route cannot be explained by their energetic advantage. Evidently, the real reason for this preference is based on an initial (and likely innate) predisposition for searching and remembering circular routes for approaching a food source. In a more detailed analysis than is possible here, these reasons should be reviewed separately, but here both are considered as a single learning predisposition. This predisposition does not determine the behavioral decision completely, but reveals itself statistically. Still, the possibility of using noncircular routes remains. In the majority of individuals, the circular routes were preferred, and the proportion of other routes was often lower than chance. Some individuals preferred unidirectional circles, for example, bee N 6 moved 50 times counterclockwise and used no other methods for achieving correct visits. One wasp preferred a "broken line-like" route, which


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Figure 7.3 Four possible trajectories around the perimeter of the experimental table containing four flower-like feeders; a and c were preferred flight paths, and b and d were rarely chosen.

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did exceed the random level. No individual always used one definite method. The ratio of inborn and acquired elements in solving some task may be estimated at the species level by the percentage of individuals choosing a preferred decision, with the other decisions being equally adaptive. In other words, the ratio in question may be estimated by the degree of "standardization" of behavior in different individuals, as long as this standard is not determinated by the structure of the task. Standardization, in turn, may be estimated statistically. Groups of 6 bees and 6 wasps are not sufficient for such estimation. But comparison of one group with another is possible. In bees, the total proportion of circle and broken line-like routes in correct visits was 223:7 (97% were circle routes); in wasps, the proportion was 95:29 (77% used a circle route). The difference between bees and wasps in taking a circle route was significant. These results mean that the behavior of the bees was mainly managed by innate predispositions, and the role of learning was relatively less important than for wasps. Thus, although bees and wasps showed equal success (as measured by the percentage of correct visits) in solving the task, the mechanisms of the behavioral decision were different. One can suppose that the differences between the groups may be explained by general differences between bees and wasps. In order to verify this supposition, two other groups of insects were studied in a modified task. Visiting of Four Flower-like Feeders Arranged Linearly This task was similar to the previous one, with the exception that the position of feeders were arranged equidistantly along the diagonal of the table. Five bees and 5 wasps were tested. The insects solved this task, and the proportion of correct visits, about 50% in both groups, did not differ from that obtained in the previous experiment. The methods of visiting the feeders during correct visits were not randomly selected. Unidirectional routes for choosing all feeders sequentially (Figure 7.3c and 7.3d) were preferred. Random levels of unidirectional routes was estimated to be about 8%. The real proportions of unidirectional and returning routes were 97:26 (79% of unidirectional methods) in bees and 34:43 (44% of unidirectional routes) in wasps. The important conclusion in these experiments is that a preference for standard methods is stronger in bees than in wasps. This result confirms the supposition about differences in the mechanisms of elaboration of behavioral decisions in bees and in wasps. As for comparison of this with previous experiments, the differences between the probabilities of a random and a preferred route should be noted. In the first task, the probability of a circle route was 1/3; in this task, the probability is 4 times less (1/12). In the present experiment, the relative preference was stronger. This confirms the conclusion that insects use trajectories of motion in agreement with inborn predipositions. On the other hand, random choice and individual learning play a role too, and in wasps, this role is more important. Additionally, it is important to note that there is a piquant discrepancy in an


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insect modus operandi. In order to perform a preferred unidirectional route, it is necessary to begin the visit from the first or last feeder in the row, not from the middle. At the beginning of a visit, bees and wasps were observed at the lateral feeders. The proportion of first choices of lateral and middle feeders was 158:208 in bees (the preference of the middle feeder is significant!) and 112:99 in wasps. This means that insects did not learn the entire preferred route. However, if an "accidental" selection of a lateral feeder occurred on the first choice, the insect "automatically" moved to a circular route. This behavior resulted in a preference for unidirectional routes. This is a negative example of insects' ability to plan their actions and to predict results. The extrapolation experiment concerned with this question is doubtful, too, as discussed earlier. Visiting of Two Flower-like Rewarded Feeders Placed Between Two Similar Unrewarded Feeders The aim of this experiment was to design a task that must be solved against the influence of inborn predispositions (as in the task of alternation of the feeding places by the law "nearer, then farther"). Bees and wasps were allowed to choose all feeders one by one. In the row of 4 similar feeders, the first and the third were filled with 1/2 of a normal reward; the others contained the same volume of sodium chloride solution. Of course, an insect can learn the position of two constant feeding places, but the problem was to avoid similar unrewarded feeders. A correct visit was considered as having occurred when both rewarded feeders were chosen by an insect without any testing of the unrewarded feeders. The probability of accidental occurrence of a correct visit is 1/6 or about 17%. Five bees and 5 wasps were tested. For wasps, the mean proportion of correct visits was 36%; it is higher than chance. This percentage also exceeded chance for each individual, 3 of them exceeding significance. However, for the bees, the mean proportion of correct visits was only 5%; the bees visted the unrewarded feeders regularly. The proportion of correct visits fluctuated individually from 2% to 15%. Thus, there are obvious drastic differences between bees and wasps. The statistical method of estimating the relative roles of inborn and acquired elements in behavioral decision making may be considered useful. The Mechanism of Mistakes of Bees Based on Their Inborn Predisposition Bees did try to optimize their behavior and were able to learn to prefer the rewarded feeder on their first choice in each visit, the total proportion of choices of rewarded and unrewarded feeders being 216:152. The second feeder chosen, however, in agreement with the inborn predispositions of bees, was the adjacent one, which contained salt. These results do not mean that bees are unable to reject the rule "choose the adjacent similar object" always. The only undoubted

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conclusion is that wasps reject this rule more easily than bees. The task was difficult for wasps, too. During the first choice in each visit, they showed a strong preference for the rewarded feeder (227:82, with the proportion of first correct choices about 73%; this preference is stronger than in bees). Only about 50% of first correct choices were followed, however, by a second correct choice. In wasps, then, mistakes also occurred in the second choice, though the differences were slight in comparison to the bees. Discussion: Three Parameters of Animal Behavior In the description of insect behavior we used three parameters: (1) initial rules (predisposition) of behavior, (2) the relative roles of these rules and individual learning in the elaboration of a behavioral decision, and (3) the degree of how natural each task was for the given species. Each of these parameters is not new if reviewed separately, but our task is to interrelate all parameters together in order to create objective methods of describing behavior. Without achieving this task, descriptions of behavior remain, to a great extent, species specific and incomparable. The third parameter is estimated more speculatively. As an ideal, a "field of probable behavioral tasks" might be construct-ed for each animal species that reflected the variability and probabilities of all natural behavioral tasks that may be solved by an animal of a given species (Kartsev and Mazokhin-Porshnyakov 1982). In fact, this ideal field would be divided into subfields in agreement with stage dismemberment of insect behavior. Two tendencies of adaptations for solving various behavioral tasks may be supposed: (1) development of a great set of inborn behavioral programs (rules of behavior), the role of individual learning being relatively small, and (2) development of a high ability for operant learning, the role of inborn programs being relatively small. The latter tendency is the tendency to develop intelligence. Thus, from the biological point of view, intelligence is an ability to make useful actions that are unnatural. In other words, it is an ability to make nonstandard decisions. The aforementioned parameters of behavior cannot be measured in general today, but some success in the case of comparing bees and wasps has been reached. In many experiments, no data were obtained that would allow us to suppose that bees and wasps have different inborn rules (predisposition) for behavior. The rules in all reviewed situations may be considered to be similar. But the role of these unmodified rules for bee behavior is relatively more important than for wasps. Consequently, bees solve natural tasks (such as discrimination of colors) better, while wasps better succeed in solving "unnatural" problems. The differences between bees and wasps may be explained not by an accidental evolutionary choice of differing tendencies for the adaptation in solving behavioral tasks, but by differences in the variability of natural behavioral tasks. It is possible that learning ability, as related to the variability of natural tasks, is constant in different animal species. Bees usually forage on flowers only, while wasps also


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hunt and feed from carcasses. The study of the gathering of protein food by wasps did not reveal particular inborn rules for this activity, and the switch between foraging for carbohydrate and for protein food ocurred easily (Kartsev 1983). Thus, wasps may use the limited set of universal inborn rules in more variable tasks than do bees. Therefore, wasps possess higher learning ability. CONCLUSIONS The beginning of the study of insect behavior in Russia was based on the ideas of Karl von Frisch and I. P. Pavlov. In the 1950s the abilities of insects to achieve conditioning were vigorously investigated; the bee became the primary subject under investigation. Early successes in this field resulted in the establishment of general goals for such research. The primary aim was to prove the evidence of a similarity of conditioning processes in vertebrates and in invertebrates, despite drastic differences in the structure of their nervous systems. During that period, many new results concerning the formation and extinction of conditioned connections in bees and other insects were obtained. Some of them, such as the evidence for the ability of bees to identify and to learn three-colored combinations, as well as their ability to learn sequences of colors, do not lose their significance today (the only doubt may be related to the lack of published statistics in old works). But in the middle of the 1960s, the study of conditioning in Russia was abandoned, and now this direction is continued mainly by German and American scientific schools (Abramson 1994). When an important conclusion about convergent similarity in insect and vertebrate behavior was made, suddenly an opposite question about specificity in insect behavior arose. This specificity may be concerned with stage dismemberment of insect behavior. The bees or wasps trained to discriminate figures by their color or shape while searching for food did not identify these figures as markers of nest entrance, the spontaneous preference for landmarks for foraging and searching for the nest entrance being different. Thus, the only relation between dismembered stages is the switching from one stage to another. We do not know of such dismemberment in vertebrates. The quest for the limits of bee abilities led to the elaboration of new types of experimental tasks. In these tasks, the reward was not paired with one or more definite conditioned stimuli, but was arranged according to some law of logic. The bees were successfully trained to discriminate classes of objects by common abstract characters. For example, bees were trained to discriminate bicolored and monocolored figures irrespective of their colors, sizes, and shapes. Being trained with two different sets of figures, bees preferred the bicolored figure in the third set of new figures without additional learning, although they were seeing this third set of new figures for the first time. The bees were able to discriminate triangles and quadrangles irrespective of other characters and to solve many other abstract tasks. Not all individuals were able to do this, and unfortunate-ly the success of the experiments depended also on the peculiarities of the season. However, many

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examples of successful problem solving in bees are evident. We do not know how insects learn and what physiological mechanisms lead to those of their actions that are considered by us as "comprehension of a logic law." But they do act as if they comprehend the laws of logic as presented by the experimenter. An insect's ability to choose a new object on the basis of information about other objects of the same class can not be explained by learning definite characters of the reward (conditioning) or by the existence of an inborn behavioral program. Until the mechanisms of such behavior are known, it should be considered a sign of insect intelligence. The real difficulty of the task is concerned not only with its objective parameters (amount of information, logical structure), but also with the degree of how natural a task is for a given animal species. The more unnatural an action is for an animal, the more difficult this action is. Thus, from the biological point of view, intelligence might be defined as the ability to make unnatural actions in a situation where these actions are useful. From this point of view, logical tasks are unnatural for bees. In all tasks, however, both inborn and acquired components of behavior are closely interrelated. In order to estimate their relative roles, bees and wasps were studied in more natural tasks. Insects were offered some similar flower-like feeders with small portions of sucrose. The task was to avoid fruitless repeated investigations of depleted feeders. There were many equally adaptive trajectories of motion. But insects preferred some trajectories and rejected others. This preference, which was not determined by the structure of the task, was considered to be concurrent with inborn predispositions of behavior. Thus the degree of variability of individual decisions was used as a measure of the relative role of inborn predisposition of behavior. The behavior of bees was found to be more standard than that of wasps. Consequently, in bee behavior, inborn components were relatively more important, while learning was less important than in wasp behavior. This conclusion was confirmed in an experimental task in which inborn rules of visiting some objects were harmful. Wasps solved this task, while bees did not. These differences may be explained by differences in the variability of natural tasks in bees and in wasps. Evidently this variability is higher in wasps, because they not only visit flowers but also hunt, though no differences between inborn searching rules in bees and in wasps have been revealed. ACKNOWLEDGMENTS The author is greatly indebted to Professor G. A. Masokhin-Porshnyakov for taking part in this work and for supplying descriptions of his original results. The first part of the introduction is written by him. The author also sincerely thanks the editors of this work Zh. Shuranova, Yu. Burmistrov (Moscow), and Ch. Abramson (StiUwater, Oklahoma); without their help this work could never have been published.

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. 1970. Is insect behavior managed by instinct only? (in Russian). Priroda 5: 55-62. . 1981. About convergent resemblance in behavior of insects and vertebrates (in Russian). In: Voprosy Obshchey Entomologii, Trudy Vsesoyusnogo Entomologicheskogo Obshchestva, Tom 63 (Problems in general entomology, proceedings of the All-Union Entomological Society, vol. 63), 154-157. Mazokhin-Porshnyakov, G. A., and Beiko, V. B. 1976. Comparison of visual markers of the nest-entrance in bumblebees and wasps (in Russian). Izvestiya Akademii Nauk SSSR. Seriya Biologicheskoya 6:825-833. Mazokhin-Porshnyakov, G. A., and Grayevskaya, Ye. E. 1966. Identification of combinations of colors by insects (in Russian). Zhurnal Obshchey Biologii 27:112-116. Mazokhin-Porshnyakov, G. A., and Kartsev, V. M. 1979. A study of the sequence of flight by insects of several equal food choices (in Russian). Zoologichesky Zhurnal 65:1281-1289. . 1984. The peculiarities of searching behavior in social and parasitic hymenopterans (in Russian). In: B. P. Manteufel and A. A. Zakharov eds., Povedenie Nasekomykh (Behavior of insects), 64-79. Moskva: Nauka. Mazokhin-Porshnyakov, G. A., Lubarsky, G. Ju., and Semenova, S. A. 1987. On the ability of bees and wasps to alternate choices (in Russian). Bulleten Moskovskogo Obshchestva Ispytateley Prirody. Otdeleniye Biologii 92:63-69. Mazokhin-Porshnyakov, G. A., Semenova, S. A., Kartsev, V. M., and Rabinivich, A. Z. 1987. Insect ability to differentiate the space by the "right-left" character (in Russian). Zoologichesky Zhurnal 66:365-312. Mazokhin-Porshnyakov, G. A., Semenova, S. A., and Milevskaya, I. A. 1977. Characteristic features of the identification by the honey bee of the objects by their size (in Russian). Zhurnal Obshchey Biologii 35:855-862. . 1979. Similarities of behavior of insects and vertebrates while solving difficult visual tasks (in Russian). Zhurnal Vysshey Nervnoy Deyatelnosti 29:101-107. Mazokhin-Porshnyakov, G. A., Semenova, S. A., and Serdyukova, I. R. 1978. The mystery of the shape perception in the honey bee, Apis mellifera L. (Hymenoptera, Apidae) (in Russian). Entomologicheskoye Obozreniye 57: 722-730. Mazokhin-Porshnyakov, G. A., and Taimova, G. A. 1973. Visual discrimination of sizes by the honey bee (in Russian). Zhurnal Obshchey Biologii 6: 855-860. Mazokhin-Porshnyakov, G. A., Taimova, G. A., Frolova, A. I., and Shamukhamedova, L. Sh. 1971. Influence of learning upon behavior of bees in a new environment (in Russian). Zoologichesky Zhurnal 50:383-392. Mazokhin-Porshnyakov, G. A., and Vishnevskaya, T. M. 1965. The proof of the capacity of insects to discriminate the circle, triangle and other simple figures (in Russian). Zoologichesky Zhurnal 64:192-191. Nikitina, I. A. 1959. To the question on delayed conditioning in the honey bee (in Russian). Nauchnye Soobshcheniya Instituta fiziologii imeni IP. Pavlova Akademii Nauk SSSR 1:55-51. . 1965. The speed of conditioning in dependence on expression of conditioned and unconditioned stimuli (in Russian). Nauchnye Soobshcheniya Instituta Fiziologii imeni I. P. Pavlova Akademii Nauk SSSR J: 123-126. Popov, A. V. 1985. Akusticheskoe Povedenie i slukh Nasekomykh (Acoustic behavior and hearing in insects). Leningrad: Nauka. Prazdnikova, N. V. 1970. Differentiating visual patterns by their static parameters in dogs and fishes (in Russian). In: V. D. Glezer and A. Penchev eds., Issledovaniye

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Printsipov Pererabotki Informatsii v Zritelnoy Sisteme. (Research of information processing in visual system), 117-128. Leningrad: Nauka. Pyke, G. H. 1978. Optimal foraging in bumble bees and coevolution with their plants. Oecologia 36:281-293. Semenova, S. A., Kartzev, V. M., and Mazokhin-Porshnyakov, G. A. 1989. Interaction of different orientation systems during food searching in insects (in Russian). Zoologicheskiy Zhurnal 68:39-41. Shekshuev, A. Ya., and Gurevich, A. S. 1983. Extrapolation reactions of bees (in Russian). Pchelovodstvo 10:16. Smith, B. H., Abramson, C. I., and Tobin, T. R. 1991. Conditional withholding of proboscis extension in Honeybees (Apis mellifera) during discriminative punishment. J. Comp. Psych. 105:345-356. Sveshnikov, V. A., Fajdysh, E. A., and Filimonov, P. M. 1975. Usage of pain threshold for determining the stages in instinctive insect behavior (in Russian). Doklady Akademii Nauk SSSR, 223:411-419. Thorndike, E. L. 1911. Animal Intelligence. New York: Macmillan. Thorpe, W. H. 1964. Learning and Instinct in Animals. London: Methuen. Tshemyshev, W. B. 1984. Sutochnye Ritmy Aktivnosti u Nasekomykh (Diurnal rhythms of activity of insects). Moskva: Izdatelstvo Moskovskogo Universiteta. Vagner, V. A. 1913. Biologicheskiye Osnovy Sravnitelnoy Psikhologii (Biologicheskoya Psikhologiya), Tom 1 (Biological bases of comparative psychology (biological psychology), vol. 1). St. Petersburg, Moskva: Izdatelstvo Tovarishchestva Volf. von Frisch K. 1977. Aus dem Leben derBienen. Berlin: Springer-Verlag. Voronin, L. G. 1969. Phylogenesis of conditioned reflex (in Russian). Zhurnal Evolyutsionnoy Biokhimii i Fiziologii 5:191-197. Voskresenskaya, A. K. 1957. On the role of the mushroom bodies of the supraoesophageal ganglion in the closing conditioned reflexes in the honey bee (in Russian). Doklady Akademii Nauk SSSR 772:964-967. Voskresenskaya, A. K., and Lopatina, N. G. 1952. Differentiating between conditioned stimuli by their color and odor in honey bees (in Russian). Trudy Instituta Fiziologii imeni IP. Pavlova Akademii Nauk SSSR 7:141 -156. . 1953. Interrelationships between food and defensive conditioning in controlling the flight activity of honey bees (in Russian). Trudy Instituta Fiziologii imeni IP. Pavlova Akademii Nauk SSSR 2:542-561. Zhantiev, R. D. 1981. Bloakustika Nasekomykh (Bioacoustics of insects). Moskva: Izdatelstvo Moskouskogo Universiteta.

Appendix A Directory of Russian Scientists Engaged in Invertebrate Research

This appendix provides a partial list of Russian scientists engaged in invertebrate research. Many of the names will be familiar to you because their work is cited throughout this volume. We strongly encourage you to contact those individuals whose work interests you. Some of the most rewarding experiences in science have started off with a simple reprint request card. MOLLUSKS Arshavsky, Yu. I.: Laboratory of Bioinformatics, Institute of Information Transmission Problems, Russian Academy of Sciences, 19 Ermolovoy Ulitsa, 101447 Moscow, Russia (Clione, motor control). Balaban, P. M.: Laboratory of Neurobiology of Learning, Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, 5a Butlerova ulitsa, 117865 Moscow, Russia (Helix, plasticity). Gaynutdinov, Kh. L.: Kazan Institute of Physics and Technology, 10/7 Sibirsky Trakt, 420029 Kazan, Russia (Helix, behavior). Grinkevich, L. N.: Department of Medical Informatics and Electronics, Design-Technology Institute of Computing Machinery, Siberian Branch of Russian Academy of Sciences, 1 Universitetsky Prospect, 630090 Novosibirsk, Russia (Helix, neurochemistry). Panchin, Yu. V.: Laboratory of Bioinformatics, Institute of Information Transmission Problems, Russian Academy of Sciences, 19 Ermolovoy Ulitsa, 101447 Moscow, Russia (Helix, motor control). Shevelkin, A. V.: Anokhin Institute of Normal Physiology of the Russian Medical Academy, 6 Gertsena Ulitsa, 103009 Moscow, Russia (Helix, neuropeptides). Stepanov, I. I.: Laboratory of Neuropharmacology, Institute of Experimental Medicine, Russian Academy of Medical Sciences, 12 Academica Pavlova Ulitsa, 197376 St. Petersburg, Russia (Helix, Achatina, neuropharmacology).

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Zakharov, I. S.: Laboratory of Neurobiology of Learning, Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, 5a Butlerova Ulitsa, 117865 Moscow, Russia (Helix, development).

WORMS Inozemtsev, A. N.: Department of Higher Nervous Activity, Biological Faculty, Moscow State University, Leninskiye Gory, 119899 Moscow, Russia (Polychaeta). Khonicheva, N. M.: Laboratory of Brain Compensatory Functions, Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, 5a Butlerova Ulitsa, 117865 Moscow, Russia (Polychaeta). Sheiman, I. M: Research Group of Endogenous Neuroregulators, Institute of Cell Biophysics, Russian Academy of Sciences, 142292 Pushchino-na-Oke, Russia (planarians). Tiras, Kh. P.: Research Group of Endogenous Neuroregulators, Institute of Cell Biophysics, Russian Academy of Sciences, 142292 Pushchino-na-Oke, Russia (planarians).

CRUSTACEANS Burba, A. B.: Institute of Ecology of the Lithuanian Republic, K. Pojelos Street, Vilnius, Lithuania (shrimp, crayfish). Burmistrov, Yu. M.: Laboratory of Bioinformatics, Institute of Information Transmission Problems, Russian Academy of Sciences, 19 Ermolova Ulitsa, 101447 Moscow, Russia (crayfish). Butorina, L. G.: Institute of Freshwater Biology, Russian Academy of Sciences, Borok, Nekouz district, Yaroslavl Region 152742, Russia (Cladocera). Piontkovsky, S. A.: Institute of Southern Seas Biology, Ukrainian Academy of Sciences, 2 Nakhimova Prospect, 335000 Sebastopol, Crimea (Copepoda). Shuranova, Zh. P.: Laboratory of Biomagnetics, Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, 5a Butlerova Ulitsa, 117865 Moscow, Russia (crayfish). INSECTS Beiko, V. B.: Laboratory of the Structure and Dynamics of Communities, Institute of Evolutionary Animal Morphology and Ecology, Russian Academy of Sciences, 33 Leninsky Prospekt, 117071 Moscow, Russia (Hymenoptera). Bogatyrev, N. R.: Laboratory of Insect Ecology, Biological Institute of the Siberian Branch of Russian Academy of Sciences, 11 Frunze Ulitsa, 630091 Novosibirsk, Russia (bumblebees). Bogatyreva, O. A.: Laboratory of Insect Ecology, Institute of Biology, Siberian Branch of Russian Academy of Sciences, 11 Frunze Ulitsa, 630091 Novosibirsk, Russia (ants). Dlussky, G. M.: Department of Evolutionary Theory, Biological Faculty, Moscow State University, Leniskiye Gory, 119899 Moscow, Russia (ants). Es'kov, E. K.: Department of Zoology, Riazan Pedagogical Institute, 46 Ulitsa Svobody, 390000 Riazan, Russia (bees). Frantsevich, L. I.: Institute of Zoology, Ukranian Academy of Sciences, 15 Lenina Ulitsa, 252650 Kiev, Ukraine (spatial orientation of many different insects).

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Kartsev, V. M.: Department of Entomology, Biological Faculty, Moscow University, Leninskiye Gory, 119899 Moscow, Russia (Hymenoptera, learning). Karas, A. Ja.: Department of Higher Nervous Activity, Biological Faculty, Moscow State University, Leninskiye Gory, 119899 Moscow, Russia (ants, learning). Kaul, R. M.: Research Institute of Biology and Biophysics, Tomsky State University, 36 Lenina Prospekt, 634010 Tomsk, Russia (ants). Kipiatkov, V. Ye.: Department of Entomology, Biological Faculty, St. Petersburg State University, 7/9 Universitetskaya Naberezhnaya, 199164 St. Petersburg, Russia (ants). Komissar, A. D.: Department of Insect Physiology, Institute of Zoology, Ukrainian Academy of Sciences, 15 Lenina Ulitsa, 252030 Kiev, Ukraine (bees). Krivtsov, N. I.: Research Institute for Bee-keeping, 22 Pochtovaya Ulitsa, Rybnoye, Riazan Region, 391110 Russia (bees). Mazokhin-Porshnyakov, G. A.: Department of Entomology, Biological Faculty, Moscow University, Leninskiye Gory, 119899 Moscow, Russia (Hymenoptera, dragonflies). Nepomniashchikh, V.: Institute of Freshwater Biology, Russian Academy of Sciences, Borok, Nekouz District, Yaroslavl Region 152742, Russia (Trichoptera, caddis worms). Popov, A. V.: Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, 44 M. Toreza Prospect, 194223 St. Petersburg, Russia (Orthoptera, neurobiology of hearing). Reznikova, Zh. I.: Laboratory of Insect Ecology, Institute of Biology, Sibirian Branch of Russian Academy of Sciences, 11 Fmnze Ulitsa, 630091 Novosibirsk, Russia (ants). Riazanova, G. I.: Department of Entomology, Biological Faculty, Moscow University, Leninskiye Gory, 119899 Moscow, Russia (dragonfly larvae). Sulkhanov, A. V.: Institute of Animal Evolutionary Morphology and Ecology, Russian Academy of Sciences, 33 Leninsky Prospect, 117071 Moscow, Russia (ants). Tschemyshev V. B.: Department of Entomology, Biological Faculty, Moscow State University, Leninskiye Gory, 119899 Moscow, Russia (beetles, biological rhythms). Udalova, G. P.: Biological Faculty, St. Petersburg State University, 7/9 Universitetskaya Naberezhnaya, 199164 St. Petersburg, Russia (ants). Zakharov, A. A.: Institute of Animal Evolutionary Morphology and Ecology, Russian Academy of Sciences, 33 Leninsky Prospect, 117971 Moscow, Russia (ants). Zhantiyev, R. D.: Department of Entomology, Biological Faculty, Moscow State University, Leninskiye Gory, 119899 Moscow, Russia (Orthoptera, bioacoustics).


Appendix B Bibliography of Invertebrate Articles Appearing in Neuroscience and Behavioral Physiology

Appendix B provides the citations of every invertebrate article appearing in Neuroscience and Behavioral Physiology from Volume 1, Number 1 (1967) through Volume 25, Number 6 (1995), for a total of 133 articles. From volume 1 to volume 4, the journal was known as Neuroscience Translations. There are years, however, when the journal was not published. Articles marked with an asterisk (*) were written by contributors of this book. Volume 24, number 1, appearing in 1994, is a special issue on the neurobiology of the snail. It is available as a separate item. Additional information on Russian material follows. To facilitate acquiring Russian material, e-mail numbers for the editors of this book are provided, as is information on a Russian e-mail journal. NEUROSCIENCE TRANSLATIONS Volume 1: 1967/1968 *Cherkashin, A. N., Sheiman, I. M, and Bogorovskaya, G. I. Conditioned reflexes in planarians and regeneration experiments (pp. 12-14). Lebedev, A. N. Spontaneous spike potentials of single neurons in situ in ganglia of the leech (pp. 285-290). Rusinov, V. S., and Ezrokhi, V. D. Local and propagating excitation in different parts of stretch receptor neurons in crustaceans (pp. 320-326). Rusinov, V. S., and Ezrokhi, V. D. Possibility of ephaptic interaction of neurons through an electric field generated by them (pp. 469-476). NEUROSCIENCE TRANSLATIONS Volume 2: 1968/1969 Sokolov, E. N., and Dulenko, V. P. Neuronal responses of Helix pomatia to tactile stimu-

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lation (pp. 593-599). Tors'ka, I. V., Bilokrinits'kyi, V. S., Burchins'ka, L. F., and Genis, Ye. D. Properties of neurons of the central nervous system of the freshwater gastropod mollusc Planorbis corneus (pp. 743-755). Sokolov, E. N., and Dulenko, V. P. Relations between adjacent units of the sub-esophageal ganglion of Helix pomatia (pp. 756-764). Burchinskaya, L. F. Neurohistological and histochemical properties of neurons of the buccal ganglia of Planorhis corneus (pp. 765-767). Karnaukhov, V. N. Spectrometric investigations of the energy system of living neurons (pgs. 867-884). NEUROSCIENCE TRANSLATIONS Volume 3:1969/1970 Airapetyan, S. N. Effect of temperature on membrane potential of giant neurons in snails (pp. 5-9). Tereshkov, O. D., & Fomina, M. S. Electrophysiological investigation of the system of paired giant cells in the ventral chain of Aulastoma gula (pgs. 10-16). Tereshkov, O. D, Fomina, M. S., and Gurin, S. S. Electrophysiological properties of paired giant cells of the leech Aulastoma gulo (pp. 11-SO). Yarmizina, A. L., Sokolov, E. N., and Arakelov, G. G Identification of neurons of the left parietal ganglion in Limnaea stagnalis (pp. 119-127). NEUROSCIENCE TRANSLATIONS Volume 4:1970/1971 Airapetyan, S. N. Metabolically dependent fraction of membrane potential and electrode properties of the membrane of giant neurons in mollusks (pp. 53-57). Vereshchagin, S. M., Lapitskii, V. P., and Tyshchenko, V. P. Functional characteristics of neurons in the central nervous system of Gryllus domesticus (pp. 89-98). Frantsevich, L. I. Properties of directionally sensitive neurons in scarabaeid beetles (pp. 99104). Stepushkina, T. A., Kan, G. S., and Kosolapov, V. N. Efferent activity during reflex opening of the crayfish dactylopodite (pp. 101-106). Malyuk, V. I., and Maiskii, V. A. Incorporation of radioactive methionine into giant neurons of the mollusk Planorhis corneus (pp. 107-114). Ger, B. A., Dardymov, I. V., Lavrent'eva, V. V., and Mikhel'son, M. Ya. Pharmacological characteristics of some muscles of sipunculids and annelids (pp. 111-120). NEUROSCIENCE AND BEHAVIORAL PHYSIOLOGY Volume 5:1972 Tsvileneva, V. A. Sensory neurons in the central nervous system of the crayfish (pp. 86-90). Fomina, M. S., and Tereshkov, O. D. Electrical transmission between symmetrical neurons in leech ganglia (pp. 91-96). Manokhina, M. S., and Kuz'mina, L. V. Biogenic monoamines in the central nervous system of mollusk Tritonia sp. (pp. 212-218).

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Popov, A. V., and Svetlogorskaya, I. D. Receptor interaction and ultrastructural organization of the auditory nerve in Logusta migratoria (pp. 282-288). Sorokina, Z. A. Hydrogen ion role in active transport of potassium and sodium through neuron membranes in the snail Helix pomatia (pp. 291-296). Shtark, M. B., Korochkin, L. I., Maksimovskii, L. F., Khizhnyak, E. V., and Kudryavtseva, N. I. Correlation between RNA synthesis and electrogenesis in the sensory neuron of the crayfish stretch receptor (pp. 319-324.) NEUROSCIENCE AND BEHAVIORAL PHYSIOLOGY Volume 6: 1973 Glezer, I. 1., and Erokhin-Peretolchina, N. M. Patterns of postnatal development of the ultrastructures of invertebrate and vertebrate nerve tissue (pp. 51-61). Gerasimov, V. D. Intercellular potentials of CNS neurons in the pteropod mollusk Clione limacina (pp. 62-68). Adzhimolaev, T. A., Murav'ev, R. A., and Rogovin, V. V. Electron cytochemistry of acid phosphatase in giant neurons of the mollusk Tritonia diomedia (pp. 131-138). Arakelov, G. G. Relation between synaptic and pacemaker potentials of giant neurons in the snail Helix pomatia (pp. 260-270). Mandel'shtam, Yu. E. Structual and functional properties of synapses in insects (pp. 271282). Didenko, A. V., Bazanova, I. S., Evdokimov, S. A., and Merkilova, O. S. Correlation between unit activity of the leech retzius cells and fluorescence of the RNA acridine derivative in the same neurons (pp. 283-292). Sveshnikov, V. G. Head receptors controlling wing muscle activity in the dragonfly Aeschna grandis (pp. 293-300). NEUROSCIENCE AND BEHAVIORAL PHYSIOLOGY Volume 7: 1976 (No 1974 or 1975 issue released) Popov, A. V., Shuvalov, V. F., and Markovich, A. M. The spectrum of the calling signals, phonotaxis, and the auditory system in the cricket Gryllus bimaculatus (pp. 56-62). Gorelkin, V. S. Functional characteristics of stretch receptors of flight apparatus of cockroach Periplaneta americana (pp. 63-68). NEUROSCIENCE AND BEHAVIORAL PHYSIOLOGY Volume 8: 1977 No papers cited employing invertebrates NEUROSCIENCE AND BEHAVIORAL PHYSIOLOGY Volume 9:1978 No papers cited employing invertebrates

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NEUROSCIENCE AND BEHAVIORAL PHYSIOLOGY Not published in 1979 NEUROSCIENCE AND BEHAVIORAL PHYSIOLOGY Volume 10:1980 Tsitolovskii, L. E., and Tsaturyan, O. I. Selective depression of neuronal excitability during habituation (pp. 333-339). *Balaban, P. M. Sensitization and habituation in command neurons of the defensive reflex in Helix lucorum (pp. 340-345). Litvinov, E. G., and Logunov, D. B. Changes in excitability of a command neuron in the initial period of conditioning in Helix pomatia (pp. 539-547). NEUROSCIENCE AND BEHAVIORAL PHYSIOLOGY Volume 11: 1981 Logunov, D. B. Conditioned reflexes to time in Helix lucorum(pp. 234-240). Maksimova, O. A. Formation of a motor food-getting conditioned reflex with two-way connection in Helix lucorum (pp. 241-246). Doroshenko, P. A., Kostyuk, P. G., and Tsyndrenko, A. Ya. Separation of potassium and calcium channels in the nerve cell soma membranes (pp. 305-312). *Maksimova, O. A., and Balaban, P. M. Relations between command neurons of feeding and avoidance behavior in Helix lucorum (pp. 558-562). NEUROSCIENCE AND BEHAVIORAL PHYSIOLOGY Volume 12: 1982 Arakelov, G. G., and Sakharova, T. A. Structural-functional analysis of identified neurons in the snail Helix pomatia (pp. 75-81). NEUROSCIENCE AND BEHAVIORAL PHYSIOLOGY Volume 13: 1983 Chesnokova, E. G., and Ponomarenko, V. V. Effect of mutant genes with a known biochemical effect on rate of formation of motor conditioned reflexes in honey bees (pp. 72-75). Arakelov, G. G., and Sakharova, T. A. Functional role of processes of an identified neuron in Helix pomatia (pp. 77-83). Maksimova, O. A. Defensive conditioning in Helix lucorum and associated changes in command neuron activity (pp. 209-215). Arakelov, G. G. Integrative processes in an identified snail neuron with two trigger zones (pp. 222-227). *Zakharov, I. S., and Balaban, P. M. Changes in defensive reflexes of Helix lucorum in ontogeny (pp. 248-251). Sotnikov, O. S., and Kostenko, M. A. Reactive changes of live nerve endings in a culture of isolated glia-deprived neurons (pp. 257-268).

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*Bravarenko, N. I., Balaban, P. M., and Sokolov, E. N. Organization of sensory input of command neurons (pp. 269-274). Arakelov, G. G., Shekhter, E. D., and Sokolov, E. N. Plasticity of the receptive field of a molluscan polyfunctional neuron (pp. 302-306). NEUROSCIENCE AND BEHAVIORAL PHYSIOLOGY Volume 14:1984 Zaitseva, O. V. Innervation of the integument of pulmonata (pp. 23-29) Tsitolovskii, L. E., and Kraevskii, A. A. Possible dependence on learning of nontemplate RNA synthesis in neurons (pp. 121-127). Vladimirova, O. O., and Fomichev, N. I. Ultrastructural organization of the gastric ganglion in the crayfish (pp. 290-296) Shuvalov, V. F. Behavioral reaction of the cricket Gryllus bimaculatus to change in sonic stimulation (pp. 524-527). NEUROSCIENCE AND BEHAVIORAL PHYSIOLOGY Volume 15:1985 Mashanskii, V. F., Bazanova, I. S., Kazanskii, V. V., and Merkulova, O. S. Functional rearrangements of the ultrastructure of the giant (retzius') neuron of the medicinal leech and possible role of Ca~ ions in these processes (pp. 337-342) NEUROSCIENCE AND BEHAVIORAL PHYSIOLOGY Volume 16:1986 Sokolov, E. N., and Ter-Margaryan, A. G. Longterm habituation in neurons LPa3 and PPa3 of the snail (pp. 246-248) Grechenko, T. N. Conditioned suppression of action potential generation in an isolated snail neuron (pp. 274-276). D'yakonova, T. L. Two types of neurons differing in plastic properties: Study of ionic mechanisms (pp. 277-284). Savostin, V. A., and Arkhipenko, S. V. Variability of morphological parameters of individual neurons and their aggregations in visceral ganglion of the mollusk Lymnaea stagnalis (pp. 436-441). NEUROSCIENCE AND BEHAVIORAL PHYSIOLOGY Volume 17:1987 *Ierusalimskii, V. N., and Balaban, P. M. Magnitude of potential induced in a mollusk nerve cell in a low-frequency electric field (pp. 125-130). Pivovarov, A. S., and SaganeHdze, G. N. Modulation of Ca ions of short-term plasticity of the cholinoreceptive membrane in molluscan neurons (pp. 288-296). Lopatina, N. G. and Dolotovskaya, L. Z. Effect of tryptophan and its metabolites on conditioned reflex activity of the honey bee (pp. 332-339). Kudryashova, I. V., & Logunov, D. B. Action of vasopressin analog on neural excitability

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in Helix lucorum (pp. 407-414) Chistyakova, M. V. influence of serotonin and noradrenaline on response amplitude in defensive-behavior command neurons in the garden snail (pp. 506-510). NEUROSCIENCE AND BEHAVIORAL PHYSIOLOGY Volume 18:1988 Sokolov, E. N. Endoneural mechanisms of reinforcement (pp. 1-4). Mashanskii, V. F., Bazanova, I. S., and Maiorov, V. N. Protective action of serotonin against acetylcholine-induced changes in ultrastructure of the retzius neuron (pp. 122-126). Pivovarov, A. S., and SaganeHdze, G. N. Differences in habituation of nicotinic and muscarinic acetylcholine receptors of snail neuron RPa4 (pp. 139-146). *Stepanov, I. 1., Lokhov, M. I., Satarov, A. S., Kuntsevich, S. V., and Vartanyan, G. A. Specific and nonspecific components of the neurohumoral link of food refusal conditioned response in the snail (pp. 207-216). *Stepanov, I. L, Lokhov, M. 1., Satarov, A. S., Kuntsevich, S. V., and Vartanyan, G. A. Humoral link in the mechanism of formation of the food refusal conditioned response in the snail (pp. 257-264.) Kudryashova, I. V. Habituation of command neurons against the background of introducing an analogue of vasopressin in snails (pp. 280-281) Verbnyi, Ya. I. Formation of cellular analog of instrumental reflex on identified Lymnaea stagnalis neurons in response to automatic intracellular electrical stimulation (pp. 440-442). *Balaban, P. M., and Maksimova, O. A. Differences in responses of identified neurons to chemostimuli in satiated and hungry grape snails (pp. 469-474). NEUROSCIENCE AND BEHAVIORAL PHYSIOLOGY Volume 19:1989 Berezhnaya, L. A. Avesicular intercellular junctions in the neuropil of ganglia of the subpharyngeal complex and in nerves of the snail (pp. 208-211). Zapara, G. A., Ratushnyak, A. S., and Shtark, M. B. Local changes in transmembrane ionic currents during plastic reorganization of electrogensis of isolated neurons of the pond snail (pp. 224-229). Verbnyi, Ya. I., and Mogilevskii, A. Ya. Cellular analog of "instrumental behavior" in individual neurons of a mollusk with intracellular automatic electrostimulation (pp. 235-241).

NEUROSCIENCE AND BEHAVIORAL PHYSIOLOGY Volume 20:1990 Babkina, N. V., and Tsitolovskii, L. E. Model of classical conditioning on the isolated mollusk CNS (pp. 1-4). *Dashevskii, B. A., Karas, A. Ya., and Udalova, G. P. Behavioral plasticity of Myrmica rubra ants during learning in a multi-alternative symmetrical labyrinth (pp. 18-26). Kudryashova, I. V. Comparative analysis of the action of a vasopressin analogue on

APPENDIX B

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functionally different neurons in the edible snail (pp. 26-31). *Berezhnaya, L. A., and Balaban, P. M. A monosynaptic connection between identified snail neurons (pp. 174-177). Inozemtsev, A. N. Changes in behavioral responses of nereids to vibration after unconditioned stimulation (pp. 185-187). Berezhnaya, L. A., and Leontovich, T. A. Some principles of the organization of the preterminal and terminal ramifications of the afferent conductors in the neuropil of the dorsal ganglia of the edible snail (pp. 224-229). *Storozhuk, M. V., and Balaban, P. M. Role of cyclic adenosine monophosphate in simple forms of plasticity in the edible snail (pp. 267-271). Arakelov, G. G., Marakueva, I. V., and Palikhova, T. A. Monosynaptic connection: Identifiable synapses in the CNS of the edible snail (pp. 331-338). Chistyakova, M. V. Role of dopamine and serotonin in modulation of snail defensive behavior (pp. 446-452). Grechenko, T. N. Conditioned inhibition of action potential generation in isolated Helix pomatia neurons (pp. 452-459). Grechenko, T. N. Features of formation of conditioned responses in isolated LPa3 neuron of the edible snail (pp. 546-548). NEUROSCIENCE AND BEHAVIORAL PHYSIOLOGY Volume 21:1991 Grechenko, T. N. Features of associative learning of snail isolated neurons of the edible snail (pp. 41-43). *Balaban, P. M., and Chase, R. Interrelationships of the emotionally positive and negative regions of the brain of the edible snail (pp. 172-180). Gapon, S. A., and Rosza, K. S. Habituation of completely isolated neurons of the edible snail to electrical stimulation (pp. 249-254). Pivovarov, A. S., Drozdova, E. I., and Kotlyar, B. I. Calmodulin blockers decrease shortterm plasticity of the cholinorecptors of neurons of the edible snail (pp. 289-295). Beregovoi, N. A., Gainutdinov, Kh. L., Safronova, O. G., and Savonenko, A. V. Change in behavior with the development of long-term sensitization of a defensive reflex in the edible snail (pp. 321-323). Kabotyanskii, E. A., and Sakharov, D. A. Neuronal correlates of the serotonin-dependent behavior of the pteropod mollusc Clione limacina (pp. 422-435). Kudryashova, I. V., and Kruglikov, R.I. Influence of an analog of vasopressin on the reaction of command neurons of defensive behavior of the edible snail during the stimulation of nerves (pp. 513-519). NEUROSCIENCE AND BEHAVIORAL PHYSIOLOGY Volume 22:1992 Kozyrev, S. A., Nikitin, V. P., and Sherstnev, V. V. Selective participation of brain-specific nonhistone Np-3.5 proteins of chromatin in the processes of the reproduction of a defensive habit in response to food in edible snails (pp. 120-127). *Maksimova, O. A., and Balaban, P. M. Reinforcing effect of stimulation of the mesocerebral region of the brain of the edible snail (pp. 137-141).

224


Bravarenko, N. I. Influence of small cardioactive peptide (b) on the efficiency of synaptic transmission and the excitability of command neurons of the defensive behavior of the edible snail (pp. 148-152). Zakharov, I. S., and lemsalimskii, V. N. Role of the cerebral ganglia in the organization of alimentary behavior of the pteropod mollusc Clione limacina (pp. 179-186). Nikitin, V. P., and Kozyrev, S. A. Dynamics of defense and alimentary reactions in the development of sensitization in edible snails (pp. 259-267). Vislobokov, A. I., Mantsev, V. V., Kopylov, A. G., and Gurevich, V. S. Influence of taurine on the electrically regulated ionic channels of the somatic membrane of neurons of the pond snail (pp. 315-319). Babkina, N. V., and Tsitolovskii, L. E. Active electrogenesis of command neurons of defensive behavior of the snail during conditioning (pp. 380-385). D'yakonova, T. L., and Arakelov, G. G. The monosynaptic connections: Modulating influence of opiod peptides on the plasticity of presynaptic neurons and identified synapses (pp. 386-392). Pivovarov, A. S., Drozdova, E. I., and Kotlyar, B. I. Arachidonic acid and its acyclic derivatives regulate short-term plasiticity of the cholinoreceptors of neurons in the edible snail (pp. 393-400). NEUROSCIENCE AND BEHAVIORAL PHYSIOLOGY Volume 23:1993 Norekyan, T. P., and Satterlie, R. Neuronal analysis of hunting behavior of the pteropod mollusc Clione limacina (pp. 11-23). Pivovarov, A. S., Drozdova, E. I., Zabolotskii, D. A., and Myagkova, G. I. Eicosapolynoic acids, inhibitors of Hpoxygenases, weaken the short-term plasticity of cholinoreceptors of neurons of the edible snail (pp. 176-181). Sergeeva, S. S. An investigation of the effect of amtizol on the plastic properties of the membrane of the retzius neuron on the leech (pp. 476-479). NEUROSCIENCE AND BEHAVIORAL PHYSIOLOGY Volume 24:1994 Note: Volume 24, Number 1, is a special issue devoted to the neurobiology of the snail. It is available as a separate item. Sakharav, D. A. The long path of the snail (pp. 1-4). Sokolov, E. N. The architecture of the reflex arc (pp. 5-11). *Lerusalimsky, V. N., Zakharov, I. S., Palikhova, T. A., and Balaban, P. M. (Nervous system and neural maps in gastropod Helix lucorum L. (pp. 13-22). Altmp, U., and Speckmann, E. J. Identified neuronal individuals in the buccal ganglia of Helix pomatia (pp. 23-32). Lerusalimsky, V. N., and Zakharov, I. S. Mapping of neurons participating in the innervation of the body wall of the snail (pp. 33-39). Koval, L. M., and Kononenko, N. I. Newly identified nerve cells of the snail, Helix pomatia, associated with the generation of pacemaker activity (pp. 41-46). Zaitseva, O. V. Structural organization of the sensory systems of the snail (pp. 47-57).

APPENDIX B

225

Chernorizov, A. M., Shekhter, E. D., Arakelov, G.G., and Zimachev, M. M. The vision of the snail: The spectral sensitivity of the dark-adapted eye (pp. 59-62). Zakharov, I. S. Avoidance behavior of the snail (pp. 63-69). Palikhova, T. A., Marakurva, I. V., and Arakelov, G. G. Mono- and poly synaptic connections between identified neurons in the system of the passive avoidance reflex of the snail (pp. 71-76). Kemenes, G. Processing of mechano- and chemosensory information in the lip nerve and cerebral ganglia of the snail Helix pomatia L. (pp. 11-SI). Bychkov, R. E., Safonova, T. A., and Zhuravlev, V. L. Viscerocardiac reflexes of the snail (pp. 89-96). *Balaban, P. M., Maksimova, O. A., & Bravarenko, H. I. Behavioral plasticity in a snail and its neural mechanisms (pp. 97-104). Grinkevich, L. N. Protein metabolism in the formation of the conditioned avoidance reflex of molluscs (pp. 105-110). Gainutdinov, Kh. L. Dynamics of defense and feeding conditioned reactions in the snail during long-term sensitization (pp. 111-114). Shevelkin, A. V. Facilitation of defense reactions during the consumption of food in snails: The participation of glucose and gastrin/cholecystokinin-like peptide (pp. 115-124). Nikitin, V. P., Samoilov, M. O., and Kozyrev, S. A. Mechanisms of the development of sensitization in the snail: The participation of calcium and calmodulin (pp. 125131). Nikitin, V. P., Kozyrev, S. A., and Samoilov, M. O. Conditioning and sensitization in the snail: Neurophysiological and metabolic characteristics (pp. 133-140). Pivovarov, A. S. Cholinoreceptor neurons of the snail: Identification, plasticity, and its regulation by opioids and second messengers (pp. 141-151). Osipenko, O. N. Influence of oxytocin on identified neurons of the brain of the snail Helix pomatia L. (pp. 153-157). (End of special issue) Kononenko, N. I. Excitatory and inhibitory monosynaptic peptidergic transmissions in the CNS of the snail Helix pomatia (pp. 203-208). Nikitin, V. P. Molecular-cellular mechanisms of learning of the common snail (pp. 321328). Mogilevskii, A. Ya., and Verbnyi, Ya. I. Influences of different intracellular electrostimulation regimes on the dynamics of the adaptational processes of neurons (pp. 386393). Burov, Yu. V., Drozdova, E. I., Pivovarov, A. S., and Robakidze, T. M. Amiridin and tacrine modulation of the activity and plasticity of the cholinoreceptors of neurons of the common snail: Phenomenology and mechanisms (pp. 507-512). NEUROSCIENCE AND BEHAVIORAL PHYSIOLOGY Volume 25:1995 Gainutdinov, K. L., and Beregovi, N. A. Long-term sensitization in the common snail: Electrophysiological correlates in the defensive behavior command neurons (207214).

226


Lopatina, N. G., Chesnokova, E. G., Sezontenko, V. N., and Ponomarenko, V. V. Role of kynurenines in the regulation of the honeybee's memory process (225-227). Pivovarov, A. S. Plasticity of cholinoreceptors of neurons of the common snail after effects on inositol-l,4,5-triphosphate-and Ca2+-dependent mobilization of stored Ca2+ and the level of phosphatidic acid (474-482). Pivovarov, A. S., and Egido-Villareal, W. The influence of an inhibitor of lipoxygenases on the modulation of the plasticity of cholinoreceptors by 15-Hete (483-487). OTHER TRANSLATED MATERIAL RELATED TO NEUROSCIENCE Koltsova, V. A., Oleinik, Y. N., Gilgen, A. R., and Gilgen, C. K. 1996. Post-Soviet Perspectives on Russian Psychology. Westport, Conn.: Greenwood Press. Krushinsky, L. V. 1990. Experimental Studies of Elementary Reasoning: Evolutionary, Physiological and Genetic Aspects of Behavior. Bethesda, Md.: National Library of Medicine. Louttit, R. T., and Hanik, M. J. (1967). A Bibliography of Translation in the Neural Sciences 1950-1966. Public Health Service Publication No. 1635. (Contains a subject index that lists more than 1000 items.) Mazohkin-Porshnyakov, G. A. 1968. Insect Vision. New York: Plenum. E-MAIL JOURNAL OF TRANSLATED RUSSIAN WORKS Man, Neuron, Model: E-Mail Communications in psychophysiology publishes translated articles that are available through e-mail. Information is available from E. N. Sokolov ([email protected]) and P. M. Balaban ([email protected]). E-MAIL ADDRESSES OF THE EDITORS The e-mail addresses of the editors are listed below. Feel free to contact us. C. I. Abramson:[email protected] Yu. M. Burmistrov:[email protected] Zh. P. Shuranova:[email protected]

Index

Ant behavior: conditioned reflexes, 148149; forest defense, 145-146; investigations of learning and memory, 146-151; learning as a function of motivation, 157-164; maze learning, 149-151; performance in a multi-choice maze under different motivation, 157-164; role of motivation in learning, 151-153; significance of motivation for estimating the plasticity of ant behavior, 165-167 Apparatus for the measurement of behavior: in ants, 155; in bees, 186; in beetles, 60; in crayfish, 123; in mollusks, 80; in planarians, 45, 48-49 Beritashvili, I. S., 20-21, 33 Bibliography of invertebrate articles appearing in Neuroscience and Behavioral Physiology from 1967-1995, 217-226 Crayfish behavior: advantages of Procambarus cubensis as an experimental subject, 117-119; autonomic reactions to sudden changes in the environment, 127; behavior of the adult, 119-122; "emotional" reactions, 133-134; locomotor activity as an indicator of behavior, 123-126; parental behavior, 122123; reactions to electromagnetic fields,

128-129; responses to sudden environmental changes, 126-134 Crustacean behavior: advantages for the comparative study of individual behavior, 115; Russian investigations, 111115; structure of the central nervous system, 115-117 Differences between Russian and Western studies of invertebrate learning and behavior, 3-4 Fabri, K. E., 22 Grain beetles: effects of hydra head activator on memory, 67-68; effects of Luliberin on development in adults and larvae, 68-71; memory and metamorphosis, 60-65; memory trace formation in larvae, 61-64; regulators of memory and morphogenesis, 65-71 Helix behavior: age dependent changes in learning, 104-106; associative behavior, 88; defensive behavior, 79-82; differential conditioning, 91; environmental conditioning, 86-88; extinction and spontaneous recovery, 91; feeding behavior, 82-84; food-aversion condition-

228 ing, 89-90; mathematical models of conditioning, 93-97; motivation, reward and learning, 100-104; nonassociative behavior, 85-86; parameters of conditioning, 90-93; self-stimulation, 100104; sexual behavior, 85; transfer of learning through humoral components, 97-100 History of behavioral investigations in Russia, 8-22 Honey bee behavior: ability to solve logical tasks, 195-196; abstraction, 196199; aversive conditioning, 182; compared to wasp behavior, 189-190, 192, 194, 200-208; compound conditioning, 191; conditioned inhibition, 183; conditioning to various types of stimuli, 185-186, 188-189; delayed conditioning, 183; discrimination of 2 and 3 color combinations, of left and right, 181-182, 194-195; role of mushroom bodies in conditioning, 185; solving multidecision tasks, 201-205; stage dismemberment, 188; transfer of training, 199-201 Kogan, A. B., 24-25 Kreps, E. M., 7, 24, 27-28 Krushinsky, L. V., 18-20,29 Ladygina-Kots, N. N., 14-16

INDEX

reaction of the Pavlovian school, 13-14; relationship to Pavlov's physiological approach to behavior analysis, 12-14 Orbeli, L. A., 16-18,23 Pavlov, I. P., 10, 13, 24, 77, 177-178 Pavlovian sessions, 7-9, 21 Planarian behavior: cellular destruction during regeneration, 50; classical conditioning, 45-48; computer morphometry of regeneration, 48-49; functional changes during regeneration, 50-51; morphometric observation of regeneration, 48-52; the nervous system and conditioning, 44-45; regeneration factors, 51-53; storage of conditioned reflexes after regeneration, 53-60 Plavilstchikov, N. N., 26 Promptov, A. N., 18 Russian behavioral studies: of ascidians, 27-29; of crustaceans, 29-30, 111-115; of insects, 30-32, 145-153, 177-184; of mollusks, 77-79; of planarians, 45 Russian investigators engaged in the study of invertebrates, 213-215 Social life and status of science and scientists in Russia, 5-8

Malyshev, S. I., 30-31

Vagner, V. A., 9-10, 13, 30, 32-33 Vaskhnil session, 7 Voronin, L. G., 24-25, 34-35

Objective Biopsychology, 9-10, 14;

Wasp behavior. See Honey bee behavior

Addresses of the Editors and Contributors

Charles I. Abramson, Department of Psychology, Oklahoma State University, 215 North Murray, StiUwater, OK 74075, USA Pavel M. Balaban, Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, 117865 Moscow, Russia Yuri M. Burmistrov, Institute of Information Transmission Problems, Russian Academy of Sciences, 101447 Moscow, Russia Anna Ja. Karas, Biological Faculty, Moscow State University, 119899 Moscow, Russia Vladimir M. Kartsev, Biological Faculty, Moscow State University, 119899 Moscow, Russia Inna M. Sheiman, Institute of Cell Biophysics, Russian Academy of Sciences, 142292 Pushchino-na-Oke, Russia Zhanna P. Shuranova, Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, 117865 Moscow, Russia Igor I. Stepanov, Institute of Experimental Medicine, Russian Academy of Medical Sciences, 197376 St. Petersburg, Russia Kharlampi P. Tiras, Institute of Cell Biophysics, Russian Academy of Sciences, 142292 Pushchino-na-Oke, Russia Galina P. Udalova, Biological Faculty, St. Petersburg State University, 199034 St. Petersburg, Russia


About the Editors CHARLES I. ABRAMSON is Assistant Professor of Psychology at Oklahoma State University. He is the author of Invertebrate Learning: A Laboratory Manual (1990) and A Primer of Invertebrate Learning: The Behavioral Perspective (1994). ZHANNA P. SHURANOVA is on the faculty at the Institute of Higher Nervous Activity and Neurophysiology of the Russian Academy of Sciences in Moscow. She is the author of Neurophysiology of the Crayfish (1988) and Progress in Crustacean Neurobiology (1991). YURI M. BURMISTROV is on the faculty at the Institute of Information Transmission Problems of the Russian Academy of Sciences in Moscow. With his collaborator, Zhanna Shuranova, he is the author of Neurophysiology of the Crayfish (1988) and Progress in Crustacean Neurobiology (1991).

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