Progress in Brain Research Volume 26A Correlative Neurosciences: Fundamental Mechanisms

PROGRESS I N BRAIN RESEARCH VOLUME 21A CORRELATIVE NEUROSCIENCES PART A: F U N D A M E N T A L MECHANISMS

PROGRESS IN BRAIN RESEARCH

ADVISORY BOARD W. Bargmann

M. T. Chang E. De Robertis

J. C. Eccles J. D. French

H. H y d h J. Ari8ns Kappers S. A. Sarkisov J. P,Schad6

F. 0. Schmitt

Kiel Shanghai Buenos Aires Canberra Los Angeles

Giiteborg Amsterdam Moscow Amsterdam Brookline (Mass.)

T. Tokizane

Tokyo

H. Waeisch

New York

J. Z. Young

London

PROGRESS I N BRAIN RESEARCH VOLUME 21A

CORRELATIVE NEUROSCIENCES PART A :

F ~ N ~ A M E N T AMECHANISMS L EDITED BY T. TOKIZANE Institute of Brain Research, University of Tokyo, Tokyo (Japan) AND

J. P. SCHADI? Netherlands Central Institute for Brain Research, Anisterdam (The NetherlanrJs)

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List of Contributors

T. ABE,Department of Neuroanatomy, Institute of Higher Nervous Activity, Osaka University Medical School, Osaka (Japan).

H. AKIMOTO, Department of Neuropsychiatry, Faculty of Medicine, University of Tokyo, Tokyo (Japan). T. BAN, Department of Anatomy, Osaka University Medical School, Osaka (Japan). T. FURUKAWA, Department of Physiology, Osaka University Medical School, Osaka (Japan).

K. HAMA,Department of Anatomy, School of Medicine, Hiroshima University, Hiroshima (Japan). T. HUKUHARA, Department of Pharmacology, Faculty of Medicine, University of Tokyo, Tokyo (Japan).

M. ITO,Department of Physiology, Osaka University Medical School, Osaka (Japan). M. KATO,Department of Neuropsychiatry, Faculty of Medicine, University of Tokyo, Tokyo (Japan). Y. KATSUKI,Department of Physiology, Tokyo Medical and Dental University, Tokyo (Japan). E. KAWANA, Department of Neuroanatomy, Institute of Brain Research, Faculty of Medicine, University of Tokyo, Tokyo (Japan). H. KUMAGAI, Department of Pharmacology, Faculty of Medicine, University of Tokyo, Tokyo (Japan). M. KUROKAWA, Institute of Brain Research, Faculty of Medicine, University of Tokyo, Tokyo (Japan). T. KUSAMA, Department of Neuroanatomy, Institute of Brain Research, Faculty of Medicine, University of Tokyo, Tokyo (Japan).

H. MANNEN, Anatomical-PhysiologicalSection, Institute of the Deaf, Tokyo Medical and Dental University, Tokyo (Japan). K. MIYAMOTO, Department of Physiology, Osaka University Medical School, Osaka (Japan).

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LIST OF C O N T R I B U T O R S

K. MOTOKAWA, Department of Physiology and Institute of Brain Diseases, Tohoku University School of Medicine, Sendai (Japan). H. NAKAHAMA, Department of Physiology, Keio University School of Medicine. Tokyo (Japan).

H. NARUSE, Institute of Brain Research, Faculty of Medicine, University of Tokyo, Tokyo (Japan).

S. NISHIOKA, Department of Physiology, Keio University School of Medicine, Tokyo (Japan). K. OTANI,Department of Anatomy, School of Medicine, Chiba University, Chiba (Japan). T. OTSUKA,Department of Physiology, Keio University School of Medicine, Tokyo (Japan).

Y. SAITO,Department of Neuropsychiatry, Faculty of Medicine, University of Tokyo Tokyo (Japan). F. SAKAI,Department of Pharmacology, Faculty of Medicine, University of Tokyo, Tokyo (Japan). A. SAKUMA, Department of Pharmacology, Institute of Cardiovascular Diseases, Tokyo Medical and Dental University, Tokyo (Japan). N. SHIMIZU,Department of Neuroanatomy, Institute of Higher Nervous Activity, Osaka University Medical School, Osaka (Japan). M. SHIMOKOCHI, Department of Physiology, Osaka University Medical School, Osaka (Japan). H. SUZUKI,Department of Physiology and Institute of Brain Diseases, Tohoku University School of Medicine, Sendai (Japan).

Y. TSUKADA, Department of Physiology, Keio University School of Medicine, Tokyo (Japan). N. YOSHII,Department of Physiology, Osaka University Medical School, Osaka (Japan).

Other volumes in this series:

Volume 1 : Brain ~echanisms Specific und aspecific Mechanisms of Sensory Motor ~ntegrut~on Edited by G. Moruzzi, A. Fessard and H. H. Jasper

Volume 2: Nerve, Bruin and Memory Models Edited by Norbert Wiener? and J. P. Schadt

Volume 3: The Rhinencephalon and Related Structures Edited by W.Bargmann and J. P. Schadi:

Volume 4: Growth and Maturation of the Brain Edited by D. P. Purpura and J. P. Schadk

Volume 5 : Lectures on the Diencephalon Edited by W. Bargmann and J. P. Schade

Volume 6: Topics in Basic Neurology Edited by W . Bargmann and J. P. Schadt Volume 7: Slow Electrical Processes in the Brain by N . A. Aladjalova

Volume 8: Blogenic Amhes Edited by Harold E. Himwich and Williamina A, Himwich

Volume 9: The Developing Brain Edited by Williamina A. Himwich and Harold E. Himwich

Volume 10: The Structure and Function ofthe Epiphysis Cerebri Edited by 1. Ariens Kappers and J. P. Schadi:

Volume 11 : Organization of the Spinal Cord Edited hy J . C. Eccles and J. P. Schade

Volume 12: Physiology of Spinal Neurons Edited by J. C. Eccles and J. P. Schadi:

Volume 13: Mechanisms of Neural Regeneration Edited b y M. Singer and J. P. Schadt

VlII

Volume 14: Degeneration Patterns in the Nervous System Edited by M. Singer and J. P. Schad6

Volume 15 : Biology of Neuroglia Edited by E. D. P. De Robertis and R. Carrea

Volume 16 : Horizons in Neuropsychopharmacology Edited by Williamina A. Himwich and J. P. Schad6

Volume 17: Cybernetics of the Nervous System Edited by Norbert Wiener1 and J. P. Schadk

Volume 18 : Sleep Mechanisms Edited by K. Akert, Ch. Bally and J. P. Schadk

Volume 19: Experimental Epilepsy by A. Kreindler

Volume 20: Pharmacology and Physiology of the Reticular Formation Edited by A. V. Valdman

Volume 21B : Correlative Neurosciences Part B: Clinical Studies Edited by T. Tokizanc and J. P. Schad6

Volume 22: Brain Reflexes Edited by E. A. Asratyan

Volume 23 : Sensory Mechanisms Edited by Y . Zotterman Volume 24: Carbon Monoxide Poisoning Edited by H.Bow and I. McA. Ledingham Volume 25: The cerebellum Edited by C. A. Fox and R. S. Snider Volume 26 : Developmental Neurology Edited by C . G. Bernhard

Volume 21 : Structure and Function of the Limbic System Edited by W. Ross Adey and T.Tokizane

1x

Preface

Medical and biological sciences in Japan have a long history. As far back as 562 AD medical books were introduced from China, initiating a long period of fruitful medical education and practice. An important era of scientific interest in the structure and function of the nervous system began in 19 11 with the publication by Prof. Shiro Tashiro on the carbon dioxide production of nerve fibers. Prof. Genichi Kato announced in 1920 his famous theory of non-decremental nerve conduction and presented all the evidence at the International Physiological Conference in 1926. His research was a major breakthrough in the physiology of single nerve fibers. He had a profound influence on the development of physiology in Japan and directing interest toward neurophysiology. From that time on the majority of Japanese scientists have been engaged in research in the brain sciences. The present volume is the first of a set of two, containing reviews and surveys of brain research in the majot Japanese laboratories and institutes. It particularly reflects the progress of Japanese research in the basic and clinical neurological sciences. Part A covers important fields such as: neural regulations of autonomic functions, basic mechanisms of vision and hearing, histochemistry and submicroscopy of synapses and dendrites, enzymatic and metabolic parameters of behavior and convulsive states. Part B will deal with clinical neurological studies and the relationship of neuroanatomy, neurophysiology and neurochemistry to the clinical sciences. It is a rare occasion that one acquires an overall view of the research activities of a large country in such an important field of the medical sciences. We trust this volume will provide a means of evaluating the level of brain research in Japan. The Editors.

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Con tents

List of contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX The septo-preoptico-hypothalamicsystem and its autonomic function T. Ban (Osaka, Japan). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Synaptic interaction at the Mauthner cell of goldfish T. Furukawa (Osaka, Japan) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 Neural mechanism of hearing in cats and monkeys Y.Katsuki (Tokyo, Japan). . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 Relationship between activity of respiratory center and EEG H. Kumagai, F. Sakai, A. Sakuma and T. Hukuhara (Tokyo, Japan) . . . . . . . . . . 98 Metabolic studies on ep mouse, a special strain with convulsive predisposition M. Kurokawa, H. Naruse and M. Kato (Tokyo, Japan) . . . . . . . . . . . . . . . . 112 Contribution to the morphological study of dendritic arborization in the brain stem H. Mannen (Tokyo, Japan) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 Central mechanism of vision 163 K. Motokawa and H. Suzuki (Sendai, Japan). . . . . . . . . . . . . . . . . . . . . Excitation and inhibition in ventrobasal thalamic neurons before and after cutaneous input deprivation H. Nakahama, S. Nishioka and T. Otsuka (Tokyo, Japan) . . . . . . . . . . . . . . . 180 Histochemical studies of the brain with reference to glucose metabolism N. Shimizu and T. Abe (Osaka, Japan) . . . . . . . . . . . . . . . . . . . . . . . 197 Studies on the neural basis of behavior by continuous frequency analysis of EEG N. Yoshii, M. Shimokochi, K. Miyamoto and M. Ito (Osaka, Japan) . . . . . . . . . . 217 Studies on fine structure and function of synapses K. Hama (Hiroshima, Japan) . . . . . . . . . . . . . . . . . . . . . . . . . . . 251 Amino acid metabolism and its relation to brain functions Y.Tsukada (Tokyo, Japan) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268 Projections of the motor, somatic sensory, auditory and visual cortices in cats T. Kusama, K. Otani and E. Kawana (Chiba, Japan) . . . . . . . . . . . . . . . . . 292 Synchronizing and desynchronizing iduences and their interactions on cortical and thalamic neurons H. Akimoto and Y.Saito (Tokyo, Japan) . . . . . . . . . . . . . . . . . . . . . . 323 Author index. 352 Subject index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358

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The Septo-Preoptico-Hypothalamic System and its Autonomic Function TADAYASU BAN Deparrtiient of Anaroniy, Osaka University Medical School, Osaka (Japan)

THREE Z O N E S I N THE H Y P O T H A L A M U S

In 1935, Hasegawa reported that the body temperature rose after needle (0.1-0.2 mm in diameter) puncture in Griinthal’s (1929) b cell-group of the hypothalamus in guineapigs. On the other hand, Megawa reported in 1940 that needle puncture of Griinthal’s a and c cell-groups of the hypothalamus and the lateral part of the midbrain tegmentum in guinea-pigs showed a fall in body temperature. The b cell-group also showed increases in blood sugar (Shimizu, 1941) and in number of leucocytes (Satani, 1943) with increased mononuclear leucocytes after needle puncture in rabbits, although in the a and c cell-groups blood sugar (Shimizu, 1941) and leucocytes (Satani, 1943) decreased. In these cases, the coagulation time of the blood was shortened and the sedimentation rate was raised by the puncture of the b cell-group, although the coagulation time was prolonged and the sedimentation rate was lowered by the puncture of the a and c cell-groups (Iwakura, 1944; Kurotsu et al., 1943). Electrical stimulation of the cell-groups mentioned above showed almost the same results as shown by needle puncture. These results prompted Kurotsu and his associates (1947) to propose the hypothesis that the wall of the third ventricle in the hypothalamus was physiologically divided into three zones medio-laterally, namely, a-parasympathetic, b-sympathetic and c-parasympathetic zones respectively. The a-parasympathetic zone corresponds to the hypothalamic periventricular stratum (Simidu, 1942) and the medial mamillary nucleus, and the c-parasympathetic zone to the lateral hypothalamic area (the lateral hypothalamic nucleus). The b-sympathetic zone corresponds to the medial hypothalamic area including the anterior, supraoptic, paraventricular, dorsomedial, ventromedial, posterior and the lateral mamillary nuclei, but the stimulation of the anteromedial part of the paraventricular nucleus near the periventricular stratum decreased the blood sugar level (Shimizu, 1941). The b and c zones are separated from each other by the fornix (Fig. l). STIMULATION A N D DESTRUCTION EXPERIMENTS

O F THE H Y P O T H A L A M U S

( I ) Circulatory system Generally speaking, the blood pressure (Ban et al., 1949, 1951a, 1953; Kurotsu et al., Rrfirenccs p . 39-43

T. B A N

Fig. 1. Frontal sections of the septa1region (SEP) and the preoptic and hypothalamic areas are shown from left to right. ACA, anterior limb of the anterior commissure; AH, anterior hypothalamic nucleus; ARC, arcuate nucleus; CA, anterior commissure; CAU, caudate nucleus; CC, corpus callosum; CI, internal capsule; CHOP, optic chiasm; COMH, commissura fornicis; CORA, Ammon's horn; DM, dorsomedial hypothalamic nucleus; DSM, supramamillary decussation ; F, fornix; FM, fasciculus retroflexus; HYP, hypophysis; LH, lateral hypothalamic nucleus; ML, lateral mamillary nucleus; MM, medial mamillary nucleus; MT, mamillothalamic tract; PC, cerebral peduncle; PCA, posterior limb of the anterior commissure; PH, posterior hypothalamic nucleus; PMD, dorsal premamillary nucleus; PMV, ventral premamillary nucleus; POL, lateral preoptic area; POM, medial preoptic area; PV, paraventricular hypothalamic nucleus; SCH, suprachiasmatic nucleus; SM, supramamillary nucleus; SOP, supraoptic nucleus; SPVH, hypothalamic periventricular stratum; SPVP, preoptic periventricular stratum; STM, stria medullaris; STT, stria terminalis; SUB, subthalamic nucleus; TOL, lateral olfactory tract; TOP, optic tract; VL, lateral ventricle; VM, ventromedial hypothalamic nucleus; VIII, third ventricle.

19%) was increased by electrical stimulation of the nuclei in the medial hypothalamic area, but it was decreased after a longer latent period by stimulation with low frequency and voltage. This decrease could not be prevented by administration of atropine, and it was slightly accelerated by administration of Imidalin. The blood pressure was decreased (Kurotsu et al., 1954c) by electrical stimulation with low frequency and, after bilateral adrenalectomies, was increased by the same stimulation. However, even in normal rabbits, the same stimulation produced an increase in blood sugar, inhibition of gastric motility and a decrease in renal volume. In hypophysectomized, thyroidectomized or adrenalectomized rabbits, the latent period was about 1.0 sec, which was similar to that in normal rabbits (Ban et al., 1953). The pressor response obtained by the stimulation was pronounced in bilaterally adrenalectomized rabbits. In hypophysectomized rabbits, pressor response was rapid and the secondary rise of blood pressure became apparent as the stimulation was repeated. This secondary rise was not modified by extirpation of the thyroid gland, but disappeared after extirpation of the suprarenal glands. Even when all three glands were extirpated, the blood pressure still increased after medial hypothalamic stimulation (Ban et al., 1953). On the other hand, blood pressure was decreased by electrical stimulation of the lateral hypothalamic area as well as the periventricular stratum in normal rabbits (Ban e l al., 1949, 1951a, 1953; Kurotsu er al., 1954~). On strong stimulation, the blood pres-

SEPTO-PREOPTICO-HYPOTHALAMIC S Y S T E M

3

sure sometimes increased and then decreased. When the basic level was markedly lowered by extirpation of the adrenal glands, stimulation of the lateral hypothalamic area did not produce a fall but a small rise of the pressure. However, when the basic level was elevated again by intravenous injection of physiological saline solution, the same stimulation decreased the blood pressure (Ban et at., 1953). These results suggest that the effect of nervous stimuli is subject to the internal environment of animals. The electrocardiographic changes (Morimoto, 1951 ; Yuasa et a]., 1957) during medial hypothalamic stimulation under ether or chloralose anesthesia in rabbits were as follow. The RR intervals were shortened after a latent period of 0.5-1.0 sec. The RQ and QT intervals were also shortened and the P wave increased by the stimulation (Fig. 2). Lateral hypothalamic stimulation markedly prolonged the RR intervals after a latent period of 0.4-0.8 sec. The PQ and QT intervals were also prolonged and the P wave was decreased. At the same time, sinus bradycardia, sinoauricular block or auriculoventricular block was observed. Sometimes auriculoventricular or ventricular automatism was recognized (Fig. 2). These reactions induced by the stimulation of the lateral hypothalamic nucleus were suppressed by bilateral vagotomies, but sometimes slight temporary prolongation of RR intervals could be observed 4-10 sec after the beginning of the stimulation in bilaterally vagotomized rabbits, which might be caused humorally. Effects of stimulation of the periventricular stratum on the electrocardiogram were almost the same to those mentioned above. According to Iwakura (1944), an increase in fibrinogen and thrombin was demonstrated with a decrease in the coagulation time of blood after medial hypothalamic stimulation. At the same time, the sedimentation rate was accelerated (Iwakura, 1944) and the total amount of protein, albumin and globulin, especially y-globulin, in serum increased (Morimoto, 1950). An increase in aspartic acid in serum was also demonstrated (Tazuke, 1951). On the other hand, after lateral hypothalamic stimulation, the coagulation time was prolonged and the sedimentation rate was retarded (Iwakura, 1944), and the total amount of protein, albumin and globulin in serum was gradually reduced (Morimoto, 1950). Kotake asserted in 1930 that the method for estimating the serum-iodometric titration value was the most suitable for ascertaining the state of intermediate metabolism of protein. Tazuke (Kurotsu et a]., 3954d), using this method, reported that thevalue was rapidly increased by 40-90 % after medial hypothalamic stimulation, and stated that this increase was due to an increase in the ether-insoluble material and not to an ether-soluble one such as a-ketonic acid. From the results of these experiments, it is concluded that the medial hypothalamic area can accelerate protein metabolism and the lateral hypothalamic area as well as the periventricular stratum suppress it. The total nonprotein nitrogen in blood also increased up to 30% after medial hypothalamic stimulation (Kurotsu et at., 1954d). The total nonprotein nitrogen and albumin in blood are closely related to renal function, which will be discussed later. At any rate, albuminuria was observed until 3 days after medial hypothalamic stimulation in rabbits, even in anesthetized rabbits (Ban et at., 1951a). An increase in blood sugar after medial hypothalamic stimulation has been mentioned above (Shimizu, 1941), but even when the hypophysis, thyroid and adrenal glands had been ReJermcrr p. 39-43

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T. B A N

all extirpated, an increase in blood sugar occurred on stimulation (Kurotsu et al., 1953~).This fact is very interesting for studying liver metabolism. The changes in the total cholesterol and lipid phosphorus in blood and total lipid in serum induced by the stimulation of the ventromedial hypothalamic nucleus were

2

3

Fig. 2. 1 shows shortening of PR, PQ and QT and increase of P induced by the stimulation of the nucleus hypothalamicus posterior under ether anesthesia. 2 shows shortening of RR and PQ and increase of P induced by the stimulation of the nucleus hypothalamicus ventromedialis under chloralose anesthesia. 3 shows the ventricular automatism induced by the stimulation of the nucleus hypothalamicuslateralis under chloralose anesthesia.

SEPTO-PREOPTICO-HY POTHALAMIC SYSTEM

5

measured by means of Bloor’s and Fiske-Subbarow’s methods and the phenol turbidity method of Kunkel and the results were as follow (Inoueetal., 1954).Totalcholester01 decreased in all 15 rabbits, lipid phosphorus decreased in 8, increased in 5 and remained unchanged in 2. Total lipid decreased slightly in 7 rabbits and remained unchanged in 5. After lateral hypothalamic stimulation, total cholesterol remained almost unchanged, but slightly increased (8 mg/dl) in 3 out of 10 rabbits. Lipid phosphorus increased in 1 1 out of 15 rabbits, remained unchanged in 3 and decreased in 1. Total lipid in serum remained unchanged in 7 out of 12 rabbits, while it increased in 5 (Inoue et al., 1954). As to the histamine content in total blood (Kurotsu et al., 1955a; Tane et al., 1958) measured by Code’s method, medial hypothalamic stimulation was inclined to lower the blood histamine, but an increase was observed in rabbits that died after the stimulation. In bilaterally adrenalectomized rabbits, the same stimulation caused an increase in blood histamine as shown in thyroidectomized rabbits, but the histamine content tended to decrease on stimulation when adrenocortical extract (Interenin) was satisfactorily administered to the adrenalectomized rabbits. In hypophysectomized rabbits, a decrease in blood histamine was observed on the same stimulation. On the other hand, lateral hypothalamic stimulation produced an increase in blood histamine in all normal rabbits, whereas the same stimulation showed a decrease of blood histamine content in adrenalectomized or thyroidectomized rabbits. In hypophysectomized rabbits, the same stimulation showed an increase in the same manner as in normal rabbits. Regarding the changes (Ban et al., 1951b) of K+ and Ca2+in total blood induced by the hypothalamic stimulation measured by Kramer-Tisdall’s method, K+ increased while Ca++ decreased slightly on ventromedial hypothalamic stimulation. On lateral hypothalamic stimulation, K+ decreased while Ca2+ was apt to increase. According to Okamoto and Oda (1952) mobilization of lymph from the lymph gland was accelerated by medial hypothalamic stimulation : the lymphocyte count in the efferent lymphatic vessels was increased and the related lymph gland was reduced in size by the stimulation. On the other hand, they (Okamoto and Oda, 1952) reported that production of lymph in the lymph gland was accelerated by lateral hypothalamic stimulation, because the lymphocyte count in the efferent lymphatic vessels remained almost unchanged and the related lymph gland was enlarged. (11) Cerebrospina1,fluidand choroid plexus (Kurotsu et al., 19536)

The cerebrospinal fluid pressure was markedly elevated up to 200 mm HzO in a glass tube (1.5 mm in diameter) immediately after the ventromedial hypothalamic nucleus was stimulated. In the course of repetition of the stimulation, a marked antagonistic action occurred between the sympathetic and parasympathetic systems. The stimulation resulted in positive globulin reaction and proportionate increases i n cell count, total protein and sugar contents. Portal permeability from blood into cerebrospinal fluid was increased by the stimulation. At the same time, the vitamin C content was noticeably lowered and the epithelial layer cells of the choroid plexus

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T. B A N

seemed to indicate enhancement of their secretion cytologically. After repetition of the ventromedial hypothalamic stimulation, hydrocephalus internus could often be observed. On the other hand, a decrease in cerebrospinal fluid pressure was observed down to -100 mm HzO when the lateral hypothalamic nucleus was stimulated. No changes occurred in permeability from blood to cerebrospinal fluid, in vitamin C or sugar contents. Epithelial layer cells of the choroid plexus showed features which made it seem that secretory function was at rest cytologically. (III) Eye and intraorbital glands

When the medial hypothalamic area was stimulated in rabbits, exophthalmos and mydriasis were observed (Ban et al., 1951a, b). At the same time, the intraocular pressure rose markedly (Nagai et al., 1951), even when the common carotid artery was ligated. This rise in pressure was believed to be due first to the contraction of Miiller’s muscles (Nagai, 1951) and then to an increase in blood pressure. The total protein content in the aqueous humor (Nagai and Ito, 1951) and the permeability from blood to aqueous humor (Nagai and Morimoto, 1952) were also increased by the same stimulation. According to histochemical tests, glycogen in the retina decreased during the stimulation and then increased after the stimulation (Matsumoto and Ishino, 1957). The lacrimal gland and Harder’s gland showed features of intracellular production of secretion on stimulation (Kurotsu et al., 1956b). On the other hand, when the lateral hypothalamic nucleus was stimulated, enophthalmos and miosis were observed (Ban et al., 1951a, b). At the same time, the intraocular pressure fell slightly after the drop in the blood pressure, even when the common carotid artery was ligated. Thus the fall in the intraocular pressure was presumed to be due partly to a decrease in blood pressure and partly to extension of Miiller’s muscles as well as pupillary constriction (Nagai et al., 1951). Glycogen in the retina seemed to be increased, according to histochemical examination (Matsumoto and Ishino, 1957). After lateral hypothalamic stimulation, the lacrimal and Harder’s glands showed features of secretion cytologically (Kurotsu et al., 1956b). ( I V ) Digestive system

In 1943, Fujita (1943; Fujita and Amano, 1943) in our laboratory reported that lateral hypothalamic stimulation in rabbits produced stomach bleeding which was prevented by bilateral vagotomies or administration of atropin before the stimulation. In coeliac gangliectomized rabbits, marked stomach bleeding or ulcer (Fig. 3) occurred after the same stimulation. These phenomena were presumed to be produced by rupture of the blood capillaries due to the high pressure of arterial blood caused by venal constriction induced by muscular contraction of the gastric body. Lateral hypothalamic stimulation increased the intragastrointestinal pressure and motility, and produced hemorrhage in the gastric mucosa (Kurotsu et al., 1951c, 1952~).The impulse from the lateral hypothalamic nucleus to the stomach and small intestine was transmitted chiefly through the vagi, but the rectum had no relation with the vagi and

SEPTO-PREOPTICO-HYPOTHALAMIC S Y S T E M

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coeliac ganglia, because their extirpation did not modify the responses of the rectum to lateral hypothalamicstimulation (Kurotsu el a/., 1951c, 1952~).The same stimulation increased intraesophageal pressure (Kurotsu et a/., 1953a), but it decreased the motilities of the cardia and pylorus (Takeda and Ito, 1951). According to Fujita (1943; Fujita and Amano, 1943), the stimulation ofthe medial hypothalamic area in rabbits produced small dotted bleeding in the stomach in 50%

Fig. 3. Stomach ulcer induced by lateral hypothalamic stimulation in the coeliac gangliectomized rabbit (Kurotsu e/ a/., 1951~).

which was prevented by extirpation of the coeliac ganglia but not influenced by bilateral vagotomies or administration of atropine. Medial hypothalamic stimulation decreased the intragastrointestinal pressure and obliterated their motilities completely through both coeliac ganglia (Kurotsu et a/., 195lc, 1952~).Intraesophageal pressure also showed a slight fall (Kurotsu et af., 1953a) but the cardiac and pyloric motilities were increased by the same stimulation (Takeda and Ito, 1951). We sometimes observed minor bleeding or ulcers in the cardia or pylorusafter medial hypothalamic stimulation. The complete obliteration of the rectal motility induced by the same stimulation had no relation with the coeliac ganglia. The sexual cycle in female rabbits markedly affected all responses to the hypothalamic stimulation especially in the gastrointestinal system as well as genital organs (Kurotsu et a/., 1952b). The alveolar cells of the parotid and submandibular glands in rabbits (Kurotsu et a/., 1951b), and the chief and parietal cells of the fundus gland (Amano, 1947) and the surface epithelium cells in cats (Kurotsu eta/., 1954a), as well as the duodenal gland cells (Kurotsu et al., 1958a) and the acinus cells of the pancreas (Kurotsu, 1954) in Rr/i,renrrs p 39-43

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T. B A N

rabbits, after lateral hypothalamic stimulation, all had features observable cytologically in which they seemed to discharge their intracellular contents to the ducts, whereas after medial hypothalamic stimulation, they showed features in which they seemed to produce secretory substances in the cells. The epithelium cells of the submandibular duct discharged supranuclear vacuoles to the duct and large vacuoles along the basic membrane to the intercellular space outside the duct after medial hypothalamic stimulation. The former was taken to be the sympathetic salivary fluid and the latter to be an endocrine substance of the salivary gland. On the other hand, the surface epithelium cells of the stomach also showed features in which they discharged the contents to the lamina propria after lateral hypothalamic stimulation. This is likely to be an endocrine function of the gastric mucous membrane. ( V ) Genital organs and ejection of milk

The electrical stimulation of the medial hypothalamic area, medial preoptic area or the midbrain central gray substance produced ovulation in mature rabbits (Kurotsu et a/., 1950). In rabbits whose ovarial nerve or internal carotid nerves, including the superior cervical ganglia, were extirpated, or whose ovary was autotransplanted in the anterior chamber of the eye, follicular hematomata were also produced by the stimulation. In pregnant or pseudopregnant rabbits as well as hypophysectomized rabbits (Kurotsu et al., 1952a), ovulation could not be observed after the same stimulation. From these results we conclude that the gonadotropic stimulus in the hypothalamus was transmitted to the anterior lobe of the pituitary gland through the pituitary stalk. On the other hand, the lateral hypothalamic stimulation inhibited ovulation induced by medial hypothalamic stimulation, but it could not prevent ovulation produced by the injection of urine of pregnant women (Kurotsu eta/., 1950). The motility and tone of the uterus were increased by medial hypothalamic stimulation, but these reactions varied according to the sexual cycle (Kurotsu ef a/., 1952b). Three days after castration, spontaneous motility and reactions of the uterus to the hypothalamic stimulation disappeared, but they reappeared on administration of the follicular hormone. Spontaneous motility of the uterus and its reactions to sympathetic stimulation became evident in accord with disappearance of the corpora luteal function in pregnant or pseudopregnant rabbits. The tone of the uterus was increased, while the frequency and amplitude of the uterine motility were decreased by the lateral hypothalamic stimulation in normal mature rabbits (Kurotsu et a/., 1952b). Regarding the influence of the hypothalamus upon pregnancy in the rabbit (Tsutsui et al., 1957), ventromedial hypothalamic stimulation at the last stage of pregnancy often caused delivery, but lateral hypothalamic stimulation had no effect on the delivery or the puerperium. The gestation was prolonged by bilateral destruction of the medial hypothalamic areas during pregnancy. After bilateral destruction of the lateral hypothalamic areas at various stages of pregnancy, different changes were found as follow. Destruction on the seventh day of pregnancy caused abortion without placentation. Destruction on the 14th day of pregnancy produced necrotized uterine contents which

SEPTO-PREOPTICO-HYPOTHALAMIC S Y S T E M

9

were absorbed or discharged later and promoted atrophy of the corpus luteum gravidarum. Destruction on the 25th day of pregnancy caused premature labor. However, even with this destruction of the lateral hypothalamic nuclei pregnancy safely could be maintained by administration of more than 40 mg of progesterone, but not by administration of follicular hormone. Medial hypothalamic stimulation in rabbits on the 3rd postpartum day increased the ejection of milk (Shimizu et al., 1956; Ban et al., 1958), to the maximum value of 38 mm3 in a glass cannula of 0.8 mm in diameter inserted in a teat duct, which was almost equal to the value induced by 100 mU of oxytocin. The same stimuldtion could not produce any ejection of milk in hypophysectomized rabbits, but it showed a vigorous ejection in thyroidectomized rabbits. It is probable that the medial hypothalamic stimulation induces milk ejection by the posterior pituitary hormone via the hypothalamohypophysial tract. Stimulation of the lateral hypothalamic nucleus or the periventricular stratum did not increase milk ejection. Bilateral destruction of the ventromedial hypothalamic nuclei of rabbits at postpartum caused reduction of the mammary gland cells as early as the 4th day after the destruction and often the sucklings died. Even though they could continue to live, their growth was not satisfactory. In these cases, milk secretion could be maintained by administration of more than 5 R.U. of the anterior pituitary hormone (Hypophorin) after the bilateral destruction of the medial hypothalamic areas. On the other hand, bilateral destruction of the lateral hypothalamic nuclei maintained milk secretion well and all sucklings showed satisfactory growth. Histological changes in the testis and prostate in mature rabbits induced by ventromedial hypothalamic stimulation were as follow (Nakamura et al., 1962). In the seminiferous tubules, marked dilatation of the lumen, discharge of spermium and reduction of fat granules were observed, while in the interstitial cells, diminution of the cell body, disappearance of vacuoles and reduction of fat granules were observed. At the same time, the prostate showed marked secretory activity similar to that in apocrine glands. Accordingly, Leydig’s interstitial cell as well as the prostate were presumed to secrete on medial hypothalamic stimulation. On the other hand, lateral hypothalamic stimulation induced contraction of the lumen, acceleration of spermatogenesis and increase of fat granules in the seminiferous tubules, while in the interstitial cells, swelling of the cell body and increase of vacuoles and fat granules were observed after the stimulation. In the prostate also fat granules were increased. ( V I ) Neurosecretion

In 1940, Kurotsu and Kondo reported the seasonal changes of neurosecretion, an increase in summer and a decrease in winter in the hypothalamus of the toad. In rabbits, some neurosecretory granules were seen which were transmitted partly to the intracellular spaces of the pars tuberalis and the frontal part of the pars distalis via primary capillaries or the perivascular spaces or the hypophysial portal system, and partly to the intercellular spaces in the caudal part of the pars distalis via the posteRcVc.rmcrs p . 39-43

10

T. B A N

rior and intermediate lobes from the hypothalamus (Okada et at., 1955) (Fig. 4). These observations may be related to the hypothalamic control of the anterior lobe. We also observed morphological changes which made it seem likely that the neurosecretory material was released into the hypothalamic and hypophysial blood vessels, and partly into the third ventricle, by the ventromedial hypothalamic stimulation, whereas after lateral hypothalamic stimulation its outflow was suppressed and it was

- - - .c

,

-- PI

PD Fig. 4. Hypothalamohypophysialneurosecretory pathways in the rabbit hypophysis(sagittal section). HS, hypophysial stalk; NR, posterior lobe; PD, anterior lobe; PI, intermediate lobe; PT, pars

tuberalis; a, b and c, descending course of neurosecretory granules to the anterior lobe.

retained in the axons (Shimazu et at., 1954). By irradiating rat heads with X-rays, neurosecretory granules in the hypothalamus and hypophysis were increased in one or two days (Tanimura, 1957). During pregnancy, parturition and post-partum periods in rabbits, neurosecretory material showed some changes as follow (Tanimura et at., 1960). Early in the pregnancy the supraoptic and paraventricular nuclei contained many vacuoles and comparatively few granules. At mid-pregnancy, granules increased markedly in the nuclei, infundibular area and neurohypophysis. Granules and droplets also invaded the intercellular spaces of the pars intermedia. Immediately before parturition neurosecretory granules decreased rapidly, and Herring-bodies of the neurohypophysis became vacuolated and irregularly shaped. This decrease in neurosecretory material continued to the 7th day post-partum. In rabbits which were allowed to suckle their young, neurosecretory granules in the hypothalamohypophysial system tended to increase from the 7th day.

SE PTO- PR EO PTI C O - H Y POTH A L A M I C SYSTEM

11

( VII) Urinary system

Ventromedial hypothalamic stimulation in normal rabbits anesthetized with urethane showed a marked diminution in renal volume recorded by an oncometer, followed by a decreasing number of urine drops, and then marked dilatation of the kidney followed almost simultaneously by an increase in urine drops. The same stimulation in bilaterally splanchnicotomized, hypophysectomized or bilaterally adrenalectomized rabbits showed a marked decrease in renal volume, but it recovered without exceeding the initial renal volume (Hirahara et al., 1953). On the other hand, lateral hypothalamic stimulatjon in normal rabbits showed an increase in renal volume followed by an increasing number of urine drops and then reduction of the renal volume with diminution of urine drops. In biIaterally splanchnicotomized, hypophysectomized or bilaterally adrenalectomized rabbits, the renal volume was increased by the stimulation and recovered to the initial volume after the stimulation without any rebound response. The number of urine drops in the former 2 groups was almost normal, but in the adrenalectomized rabbits, no urine drop was observed in the course of our experiments (Hirahara ef al., 1953). The histological changes in the kidney after hypothalamic stimulation were as follow. During the ventromedial hypothalamic stimulation, the majority of the renal corpuscles and the intracapsular spaces became smaller, and the permeability of the blood vessels decreased simultaneously. Consequently the filtration activity was diminished. At the same time, the proximal convolution cells showed changes in their fine structures, in which the cells were presumed to absorb the filtrate from the lumina during the stimulation. During lateral hypothalamic stimulation, the renal corpuscles became much larger, and the intracapsular spaces dilated strikingly up to 18 /.I in diameter. The glomerular capillaries also dilated from 9 to I 1 p in diameter. These features were taken to indicate promoted glomerular filtration, while the proximal convolution cells showed changes in their finer structures, in which thecells were presumed to discharge the absorbed substance into the blood vessels (Kurotsu et al., 1954b). These results show that the changes in the renal volume took place in parallel with the changes in dimensions of the renal corpuscles and the inner diameter of the uriniferous tubules. In bilaterally adrenalectomized rabbits (Kurotsu et al., 1955b), the renal corpuscles seemed to decrease in size slightly during ventromedial hypothalamic stimulation, and then they gradually enlarged after the stimulation; whereas during the lateral hypothalamic stimulation they enlarged with dilated intracapsular spaces, and after the stimulation they gradually returned to their initial size. The proximal convolution cells always showed features which suggested that they absorbed the filtrate and then discharged it to the blood stream. It was also probable in these adrenalectomized rabbits that the changes in the renal volume were mainly due to changes in size of the renal corpuscles and the other blood vessels. The anuria following bilateral adrenalectomy, which continued even at the stage of the hypothalamic stimulation, was thought to be mainly due to the intensive fall of the general blood pressure and the absorption of the proximal convolution cells. Rrfprenres p . 39-43

12

T. B A N

According to Yokoyama (Yokoyama et al., 1960) who studied urinary bladder responses to the electrical stimulation of the hypothalamus in male mature rabbits anesthetized with small doses of urethane (0.5-0.7 g per kg in body weight), the stimulation of the medial hypothalamic area or the mamillary peduncle produced relaxation response only or relaxation response after an initial contraction, whereas stimulation of the lateral hypothalamic area, mamillotegmental tract or the periventricular stratum produced a prompt, vigorous and sustained contraction as well as miosis and somatic urinary movement. Stimulation of the boundary of the three zones showed almost biphasic responses. (VIII) Respiratory system

In 1951,Ban et al. (1951a) reported hemorrhage of the lung induced by ventromedial hypothalamic stimulation in rabbits (Fig. 5 ) . Accordingly the effects of hypothalamic stimulation on the lung were studied histologically in rabbits (Kurotsu et al., 1956a).

Fig. 5. Hemorrhage of the lung induced by the stimulation of the ventromedial hypothalamic nucleus in the rabbit.

After ventromedial hypothalamic stimulation, the alveolar lumina enlarged, walls thinned and capillaries contracted. In 96 % of all cases, many scattered hemorrhages occurred at the beginning of the stimulation. This hemorrhage was due to rupture of the capillaries by an increase of blood pressure. Immediately after the stimulation, bronchial and bronchiolar dilatations were observed. Goblet cells of the bronchi and bronchioles were also distended, mitochondria increased in number, and then vacuoles began to appear. Forty min after the stimulation, vacuoles began to be discharged. On the other hand, after lateral hypothalamic stimulation, narrowing of the alveolar

SEPTO- PR EOPTI C O - H Y P O T H A LAM I C SYSTEM

13

lumina, thickening and loosening of the walls and dilatation of the capillaries were observed. Sometimes, leucocytes and emigrated cells were found to be more numerous in the alveolar sacs. Pulmonary hemorrhage occurred in 31 % in gross solitary form. This hemorrhage was believed to occur through an increase in permeability of blood vessels. Pulmonary edema accompanied by congestion was seen in the bleeding area. Atelectasis was observed in 40 %. Two out of 8 cases showed pneumonia-like features. The bronchi constricted into asteroid shape, and the bronchial lumina were covered with mucous secretion. Goblet cells were constricted and mucous secretion was observed in both apocrine and ecrine types. Shinoda studied the types of respiratory reactions induced by the hypothalamic stimulation (Shinoda et al., 1958). Electrical stimulation of the various nuclei of the medial hypothalamic area caused respiratory acceleration and also marked levelshifting towards inspiration. At the same time, enlargement of the alveoli was perceived histologically (Kurotsu et al., 1956a). Strong stimulation caused various types of panting with periodical gasping. Stimulation, if repeated, caused marked continuous acceleration in respiratory activity. The effect was greatest after stimulation of the ventromedial hypothalamic nucleus. On the other hand, electrical stimulation of the lateral hypothalamic nucleus and the periventricular stratum caused level-shifting towards expiration. On the whole, weak stimulation gave slight and gradual decrease of respiratory activity (generally, decrease in frequency and amplitude), slightly stronger stimulation caused paroxysmal hyperpnea with preponderance of expiration, and a strong one produced panting attended by marked inhibition of inspiration, also maintaining the shift towards expiration. This panting was smaller in amplitude and shallow, extremely rapid and convulsive in respiration, with gasping hardly intermingled. During these types of respiration, deflation of the alveoli of lungs was also perceived histologically (Kurotsu et al., I956a). Stimulation, if repeated, induced continuous decrease in respiratory activity. This decrease, different from the one seen in the non-narcosis, non-stimulation and untreated, soon (1 5-20 min later) reached the same degree as in natural sleep, but had no such peculiarity of respiratory waves as was seen in natural sleep. On stimulation of the periventricular stratum, the only peculiarity was that respiration was often made to stop by strong stimulation (Shinoda et at., 1958). ( I X ) Gaseous metabolism

The apparatus used was Knipping Gas Metabolimeter produced by Ei-Ken Co. combined with Saeki’s Respiration Chamber for animals. Mature male rabbits weighing about 2.5 kg were used. Experimental results were as follow (Ban el al., 1955). Gaseous metabolism was increased markedly by ventromedial hypothalamic stimulation, whereas lateral hypothalamic stimulation showed a decrease in gaseous metabolism or a slight increase directly after the stimulation followed by a decrease soon after. Choralose or urethane anesthesia produced a slight increase in gaseous metabolism, while lsoamytal(5,5-isoamylethylbarbit~ric acid) anesthesia markedly inhibited the increase in gaseous metabolism induced by ventromedial hypothalamic stimuReferences p . 3 W 3

14

T. B A N

lation. TEAB (tetraethylammoniumbromide) administered to rabbits showed almost the same change in gaseous metabolism as seen in non-anesthetized rabbits. Gaseous metabolism was increased by ventromedial hypothalamic stimulation even in thyroidectomized, unilaterally adrenalectomized or hypophysectomized rabbits, but the increase was less than that in non-operated rabbits. The increase in gaseous metabolism produced by the same stimulation in bilaterally adrenalectomized rabbits was much less than that of healthy rabbits. Bilateral adrenalectomy caused a marked decrease in gaseous metabolism : therefore it was difficult to determine whether the decrease was due to bilateral adrenalectomy only, or partly due to the lateral hypothalamic stimulation.

( X ) Endocrine glands and some other glands According to histological and cytological studies on some gland cells induced by the hypothalamic stimulation, ventromedial hypothalamic stimulation in rabbits caused swelling of the cell body by vacuolization, whereas lateral hypothalamic stimulation induced shrinkage of the cell body, and the intercellular space became much dilated in the medulla of the suprarenal gland (Kurotsu, 1954; Ishida, 1944). The thyroid follicular cells also showed an increase in vacuole and became taller, and the size of the follicle became smaller due to discharge of its contents on ventromedial hypothalamic stimulation, whereas after lateral hypothalamic stimulation, the cells became lower again due to gradual discharge of the vacuole contents to the follicular lumen, and the lumen became larger (Kurotsu, 1954; Fujita, 1947). The cytological changes of the anterior lobe of the hypophysis in rabbits studied by Heidenhain, Mallory and Gomori methods were as follow (Okada, 1954). Lateral hypothalamic stimulation caused accumulation of the secretion in the intercellular spaces. Dilatation of the capillaries, dark cytoplasm and obliteration of the fine structure of the cells were observed. The cell bodies shrunk and the intercellular space dilated markedly, while the ventromedial hypothalamic stimulation showed constriction of the capillaries and swelling of the cell body due to an increase in vacuoles and mitochondria. Concomitantly, the intercellular spaces were reduced markedly. In the pancreatic islets (Kurotsu et al., 1957), an increase in the number of cells was induced, and the cell body became larger by vacuole formation after ventromedial hypothalamic stimulation, whereas lateral hypothalamic stimulation caused a decrease in the number of b cells, and the cell body became smaller by discharging its content. Even in bilaterally adrenalectomized rabbits, ventromedial hypothalamic stimulation induced an increase in the number of @ cells whereas lateral hypothalamic stimulation caused a decrease. The cytological changes in the gland of the mucous membrane of the maxillary sinus induced by hypothalamic stimulation were as follow (Kato, 1958). After ventromedial hypothalamic stimulation, the serous gland cells as well as the mucous gland cells showed secretory production, even in bilaterally adrenalectomized rabbits, whereas lateral hypothalamic stimulation produced a secretory discharge, even in bilaterally adrenalectomized rabbits. Argentaffine cells (Kubo, 1960) in the epithelium of the digestive tube decreased in number on ventro-

-

-

S E P T 0 P R E 0 P T 1C 0 H Y P O T H A L A M I C

S Y ST E M

15

medial hypothalamic stimulation. This result was presumed to be due to a decrease in argentaffinity by liquefaction of the granules.

( X I ) Liver metabolism Yamada ( I 950) reported that the bile capillaries were markedly enlarged with secretory fluid, and in the liver cells granules containing iron decreased after stimulation of the ventromedial hypothalamic nucleus. On the other hand the bile capillaries remained narrow and granules containing iron increased after lateral hypothalamic stimulation. Yamada concluded that the medial hypothalamic area might stimulate bile secretion by the liver cells, and the lateral hypothalamic nucleus might produce the secretion and expel the secretory fluid from the liver by narrowing the bile capillaries in fasted rabbits. After ventromedial hypothalamic stimulation, the acid phosphatase reaction increased in the liver cells and the alkaline phosphatase reaction increased markedly in the bile capillaries (Kurotsu et al., 1951a). The latter is believed to have some relationship to the increase of the secretory fluid in the dilated bile capillaries (Yamada, 1950) in rabbits. The gallbladder contracted, the folds of the mucous membrane increased and the secretion of its cells was cytologically promoted by ventromedial hypothalamic stimulation, whereas after lateral hypothalamic stimulation in the rabbit, the gallbladder became bigger, the folds of mucous membrane decreased and the ordinary epithelium cells appeared cytologically to absorb water (Matsui et a/., 1961). In higher animals, certain hepatic enzymes that metabolize amino acids have been shown to be controlled by the hypothalamus (Shimazu, 1962, 1964a,b). Electrical stimulation of the ventromedial hypothalamic nucleus (20 sec stimulation, every 5 min for 18-20 h) in rabbits, resulted in about an 8-fold increase in activity of tryptophan pyrrolase in the liver homogenate; and lateral hypothalamic stimulation by the same method caused about a 5-fold increase in this enzyme activity. Knox and Auerbach (1955) found the activity of this enzyme was increased by administration of cortisone, and we found an increase of about 43 % of corticosteroid in blood after ventromedial hypothalamic stimulation in rabbits. Studies were made to determine whether the effect of the hypothalamic stimulation on tryptophan pyrrolase was secondary by causing an increase in adrenal activity. Even in bilaterally adrenalectomized rabbits, a 6-fold increase in enzyme activity over the control was recorded after stimulation of either the ventromedial hypothalamic nucleus or the lateral hypothalamic nucleus. These results indicate that the increase in the level of tryptophan pyrrolase observed after electrical stimulation of the hypothalamus is not a secondary effect of increased adrenal activity, but rather a primary effect of hypothalamic activity. To analyze in detail the induction of tryptophan pyrrolase by hypothalamic stimulation, the total amount of apoenzyme and the level of holoenzyme were measured differentially. The activity of tryptophan pyrrolase was assayed on the cell sap fraction of liver homogenate in the presence or absence of excess cofactor. Rat liver microsomes were used as a cofactor preparation. Electrical stimulation of either the sympathetic or parasympathetic zone of the hypothalamus caused a marked elevation in Refirenrer p. 39-43

16

T. B A N

the total amount of apoenzyme. The level of holoenzyme was markedly increased after stimulation of the sympathetic zone (the medial hypothalamic area), but was only slightly increased after stimulation of the parasympathetic zone (the lateral hypothalamic area). Thus, the ratio of holoenzyme to apoenzyme was changed from 1/3 in normal rabbits to 1/2 and 1/5, respectively, in rabbits stimulated in the sympathetic zone and the parasympathetic zone. The activities of tyrosine transaminase and alanine transaminase (Shimazu, 1964a) were likewise elevated about 2- to 3-fold after electrical stimulation of the sympathetic zone. But stimulation of the parasympathetic zone had no influence on these transarninases. Serine dehydratase was affected by stimulation neither of the sympathetic zone, nor the parasympathetic zone.

(XU) Mal$ormation The influence of electrical stimulation or destruction of the mother’s hypothalamus on development of her fetus was studied in rabbits, and the results were as follow (Takakusu et al., 1962). Acute stimulation caused abnormalities chiefly of the central nervous system such as infoldings of the brain and a flexed spinal cord. Besides these, a few cases of herniation of the heart and hypodactyly were found. Malformation of the face was observed in one case whose mother’s ventromedial hypothalamic nucleus had been chronically stimulated during the middle stage of pregnancy. In one case, whose mother’s fornix was destroyed unilaterally, syndactyly and oligodactyly were obtained. A microcephaly (Fig. 6) and herniation of the midbrain were produced by destruction of a large part of the hypothalamus and a part of the thalamus. It was suggested that abnormal proliferation by inhibition of differentiation

Fig. 6. Microcephaly induced by destruction of the mother’s hypothalamus in the rabbit.

SEPTO-PREOPTICO-HYPOTHALAMIC S Y S T E M

17

potency might be a factor in the genesis of malformation in the central nervous system. The mother’s parasympathetic zone of the hypothalamus was thought to be important for the development of the trabeculae in the adrenal gland and thymus, and the sympathetic zone of the hypothalamus was believed to be necessary for their differentiation into parenchymal elements. The effects of electrical stimulation of the hypothalamus on the placenta and uterine vessels were studied in rabbits, and the following results were obtained (Takakusu et al., 1964). Stimulation of the ventromedial hypothalamic nucleus, which belongs to the sympathetic zone, produced dilatation of the uterine vein, narrowing of the villi, withdrawal of fetal blood from the villous capillaries, widening of the maternal blood spaces in the labyrinth, some fragmentation of the syncytium, and bleeding in the intermediate layer. Stimulation of the lateral hypothalamic nucleus, which is the c-parasympathetic zone, induced little change in the uterine vessels, but resulted in contact of the syncytial layers, withdrawal of the maternal blood from the labyrinth and widening of the villi. Accordingly, it was suggested that inhibition of oxygen and nutriment transport from the maternal blood to the fetal blood produced by changes in the placental circulation induced by the hypothalamic stimulation might be one of the causal mechanisms of malformations induced by maternal hypothalamic stimulation or destruction. The specific changes in the placenta mentioned above are similar to the initial feature of the placental change in the toxemia in pregnancy. ( X I I I ) Waking, sleep and emotional behavior

In 1951. it was reported (Ban et al., 1951b) that electrical stimulation of the ventromedial hypothalamic nucleus in rabbits elicited the rage reaction, with which ovulation (Kurotsu et al., 1950) occurred in mature female rabbits, while repeated stimulations of the lateral hypothalamic nucleus (c-parasympathetic zone) induced sleep, and that bilateral destruction of the medial hypothalamic areas (b-sympathetic zone) could produce a state of predominance or super-predominance of the parasympathetic tonus, while bilateral destruction of the c-parasympathetic zones could produce a state of predominance or super-predominance of the sympathetic tonus (Fig. 7). Accordingly, it was thought that sleep could be induced by the state of balance in which the level of the parasympathetic tonus became a little higher than that of the sympathetic after they had conflicted with one another, and that waking was the state of balance in which the level of the sympathetic tonus was a little higher than that of the parasympathetic. Further, it was reported (Ban et al., 1951b) that rage or excitation might be produced in the state of super-dominance of the sympathetic, while lethargy or narcolepsy might be induced in the state of super-dominance of the parasympathetic tonus. These changes could be produced with hypothalamic emotion. It is considered that rage or excitation developing from the waking state belongs to the positive emotional behavior, and that sleep developing from the waking state belongs to the negative emotional behavior (Ban, 1964a). Electroencephalographic changes (Ishizuka et al., 1954) in the hypothalamus in oestrus, anoestrus, pregnant and nonpregnant rabbits also supported these hypotheses on the balance mechanisms. References p. 39-43

18

T. B A N

Fig. 7. Left above: rage reaction induced by stimulation of the ventromedial hypothalamic nucleus, and left below: sedate state induced by bilateral destruction of the same nuclei. Right above: excitatory state induced by bilateral destruction of the lateral hypothalamic nuclei, and right below: sedate state induced by bilateral destruction of the ventromedial hypothalamic nuclei.

Recently, Sano (1962) succeeded in obtaining most marked sedative effects in patients with violent behavior by his postero-medial hypothalamotomy, namely by bilateral destruction of our b-sympathetic zones; and he also demonstrated our aparasympathetic, b-sympathetic and c-parasympathetic zones in the hypothalamus of patients by electrical stimulation before his hypothalamotomy. The sites of electrodes were decided with the help of X-ray photographs. The EEG changes after the posteromedial hypothalamotomy were almost the same as those in the hypothalamotomized rabbits which had been described by Sawyer et al. (1961). It is interesting that our results with rabbits or cats are very similar to Sano’s results with man. ( X I V ) Conditioned reflex induced by hypothalamic stimulation

We produced a conditioned reflex in the pupil, respiration, gastric motility and the general condition of rabbits by using electrical stimulation of the hypothalamus as unconditioned stimulus and sound as conditioned stimulus (Ban and Shinoda, 1956).

19

SEPTO-PREOPTICO-HY POTHA L A M l C SYSTEM

Two forms of the conditioned reflex, namely the sympathetic conditioned reflex and the parasympathetic conditioned reflex, were constructed by using separate reinforcement of electrical stimulation of the ventromedial hypothalamic nucleus or the lateral hypothalamic nucleus, each as the unconditioned stimulus. The reinforcement was given 20 times or so a day at intervals of 5 min. In the sympathetic conditioned reflex, mydriasis, exophthalmos, acceleration of respiration and inhibition of the stomach cs

r. Fronto parietal ~

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Fig. 8. (a) Conditioned EEG response in synipathetic conditioned reflex. CS, conditioned stiniulation (2 c/s). 5th day of the experiment. (b) Conditioned EEG response in parasympathetic conditioned reflex. The gain from the lateral hypothalamic nucleus alone is recorded as well as a quarter of the other three, for its response is extremely peculiar high voltage slow waves. 5th day of the experiment. (c) The same animal that showed the responsive change of (b) showed differential inhibition under the conditioned stimulation of a different rhythm (I c/s). Re/>rmrcs p . 39-43

~

~

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20

T. B A N

movement (not easily produced) were induced by sound from the 3rd or 4th day of the reinforcement. On the 7th or 8th day the response became maximal. In the parasympathetic conditioned reflex, miosis (not easily produced), enophthalmos, depression of respiration and acceleration of stomach movements were all observed to be induced by sound. When the sympathetic conditioned reflex was gradually built up, sham-rage could be induced by such a weak stimulation as could not ordinarily induce the sham-rage, if it had only been applied to the medial hypothalamic area as an unconditioned stimulation that was adopted in the course of reinforcement. Another remarkable observation was that, if any kind of unconditioned reflex was combined in the course of reinforcement, there occurred a special change in the size of the effect of the conditioned reflex of the organ influenced by that unconditioned reflex. For example, mydriasis was more easily produced by reinforcement in a dark room than in a lighted room. Consequently it may be accepted that the hypothalamus has a dynamic adaptability in its functioning. In conditioned EEG responses (Ban and Shinoda, 1960) which were established by electrical stimulation of the hypothalamus in rabbits as unconditioned stimulation, frequency-specific slow waves appeared conspicuously in the hypothalamus. In the sympathetic conditioning, the frequency-specificharmonized slow waves carried superimposed high frequency fast waves and the voltage might be slightly reduced (generalized desynchronization). In the parasympathetic conditioning, on the other hand, high voltage slow waves of 200-300 pV appeared without fast wave activity. Both sympathetic and parasympathetic conditioning established generalization, differentiation and extinction which were confirmed in the EEG responses (Fig. 8). An increase in the blood sugar level and the leucocyte count was observed in the sympathetic conditioned reflex which was formed by using the electrical stimulation of the ventromedial hypothalamic nucleus as unconditioned stimulus (Ban and Shinoda, 1961). On the other hand, a decrease in the blood sugar level and the leucocyte count was observed in the parasympathetic conditioned reflex which was built up through the electrical stimulation of the lateral hypothalamic nucleus as unconditioned stimulus. From these results, we made it clear that the hypothalamic conditioning was also possible in the interoceptive reaction. F U N C T I O N O F THE P R E O P T I C A N D S E P T A L AREAS

The preoptic area belongs to the telencephalon and is closely related to the hypothalamus morphologically (Ban, 1963, 1964b) and functionally. The boundary between the hypothalamus and the preoptic area is not distinct. The preoptic area is divided into 3 cell groups, i.e., the lateral preoptic area, the medial preoptic area and the preoptic periventricular stratum which is in contact with the ependymal layer of the third ventricle (Fig. 1). But their boundaries are not clear. The lateral preoptic area, including the nucleus preopticus magnocellularis, is occupied chiefly by the medial forebrain bundle and the interstitial nuclei of the bundle scattering in this area. The medial

SEPTO-PREOPTICO-HYPOTHALAMIC SYSTEM

21

preoptic area is divided into the pars ventralis and pars dorsalis of the nucleus preopticus principalis. Rostrally the preoptic area continues to the septal region. The stria terminalis originating in the amygdala and partly in the periamygdaloid cortex enters the lateral preoptic area and the medial hypothalamic area from dorsal side (Ban and Omukai, 1959), and these connections were certified physiologically by Yuasa et al. ( 1959). Kurotsu et al. (1950), Kurotsu et al. (1958b), Sakai et af. (1958), Shinoda et al. ( 1958), Ban et a/. (1958) and Yokoyama et a/. (1 960) reported influences of the electrical stimulation of the preoptic and septal areas on ovulation, blood pressure, gastric motility, respiratory movement, milk ejection and urinary bladder response in rabbits. According to these experimental results, the septal region, the preoptic periventric-

Fig. 9. Septo-preoptico-hypothalamicsystem (SPH system) of the rabbit brain. Horizontal section through the septal region, preoptic area and hypothalamus. Area parasympathica A consisting of the septal region (SEP), preoptic periventricular stratum (SPVPI, hypothalamic periventricular stratum (SPVH) and medial niamillary nucleus (MM), and area parasympathica C consisting of the septal region, lateral preoptic area (APL) and lateral hypothalamic area (AHL) are marked by oblique lines. Areas A and C unite in the septal region. The medial preoptic area (APM), medial hypothalamic area (AHM) and lateral mamillary nucleus (ML) belong t o area sympathica B. AH, anterior hypothalamic nucleus; AHIP, anterior continuation of the hippocampus; BOLF, bulbus olfactorius; CAU, caudate nucleus; CE, external capsule; CI, internal capsule; DM, dorsomedial hypothalamic nucleus; F, fornix; HIP, hippocampus; PH, posterior hypothalamic nucleus; PC, cerebral peduncle; PCMS, precommissural portion of the septum; PRM, premamillary nucleus; PUT, putamen; VM, ventromedial hypothalamic nucleus. Rt:firmc,rs p. 3Y-43

22

T. B A N

ular stratum and the lateral preoptic area, including the medial forebrain bundle, showed parasympathetic reactions, and the medial preoptic area showed sympathetic reactions. In accord with these findings, such preopticareas belonging to the telenceph-

Fig. 10. Schematic summary of the courses and terminations of the medial forebrain bundle (MFB) and A-group of fibers. The small black squares show the site of the lesion. (A), A-group of fibers: ACA, anterior limb of the anterior commissure; AD, anterodorsal thalamic nucleus; AH, anterior hypothalamic nucleus; AHIP, anterior continuation of the hippocampus; AHM, medial hypothalamic area; AM, anteromedial thalamic nucleus; APL, lateral preoptic area; APM, medial preoptic area; AV, anteroventral thalamic nucleus; BOLF, olfactory bulb; (C), C-group of fibers; CC, corpus callosum: D, nucleus of Darkschewitsch; EW, nucleus of Edinger-Westphal; HIP, hippocampus; HL, lateral habenular nucleus; H M , medial habenular nucleus; IS, interstitial nucleus of Cajal; LH, lateral hypothalamic nucleus; LT, lateral thalamic nucleus; MD, mediodorsal thalamic nucleus; ML, lateral mamillary nucleus; MM, medial mamillary nucleus; OA(P), pars posterior of the anterior olfactory nucleus; PCMS, precommissural portion of the septum; PT, pretectal nucleus; PTAE, parataenial nucleus; PVA, anterior paraventricular nucleus; RT, thalamic reticular nucleus; SGC, central gray substance; SH, septohippocampal nucleus; SPL. lateral septal nucleus; SPM, medial septal nucleus; SPV, preoptic and hypothalamic periventricular stratum; STLL, stria longitudinalis lateralis; STLM, stria longitudinalis medialis; STM, stria medullaris; TD, dorsal tegmental nucleus of Gudden; TOL, lateral olfactory tract; TUBO, olfactory tubercle; 111, oculomotor nucleus; IV, trochlear nucleus; VI, abducens nucleus.

SEPTO-PREOPTICO-HYPOTHALAMIC SYSTEM

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alon and locating rostrally to the hypothalamus are thought to be continuations of 3 zones of the hypothalamus. Namely, the medial preoptic area is a continuation of the medial hypothalamic area (b-sympathetic zone), and the lateral preoptic area is a continuation of the lateral hypothalamic area (c-parasympathetic zone). The preoptic periventricular stratum is a continuation of the hypothalamic periventricular stratum (a-parasympathetic zone). And rostrally, the preoptic periventricular stratum and the lateral preoptic area are thought to be united with each other at the septal region. From the functional point of view, the septal, preoptic and hypothalamic areas can be united into one system named the septo-preoptico-hypothdamic system or the SPHsystem (Ban, 1963, 1964b), which can be divided into 3 areas longitudinally, namely ( I ) area parasympathica A or area A consisting of the septal region and the preoptic and hypothalamic periventricular layers, (2) area symparhica B or area B consisting of the medial preoptic area and the medial hypothalamic area, and (3) area parasympathica C or area C consisting of the septal region, the lateral preoptic area and the lateral hypothalamic area (Fig. 9). FIBER C O N N E C T I O N S IN T H E SEPTO-PREOPTICO-HYPOTHALAMIC S Y S T E M

( I ) A-group of Jibers (tractus hypothalamicus periventricularis)

In our Marchi’s sections a few fine fibers from the lateral part of the septum pellucidum occupy the medial part of the diagonal band of Broca, proceed caudad in the periventricular stratum of the third ventricle wall, decrease in number and terminate in the subependymal layer of the rostral part of the cerebral aqueduct. On the way, some fibers enter the pars medianus of the medial mamillary nucleus. These fine fibers belong to our A-group of Jibers. A few fine fibers, occupying the medial part of the tract, originate in the medial forebrain bundle which belongs to our C-group offibers and terminate in the subependymal layer of the cerebral aqueduct bilaterally (Fig. 10). So a part of the C-group of fibers joins the A-group of fibers at the rostral border of the midbrain. Megawa (1960) recognized parasympathetic reactions by electrical stimulation of the subependymal layer of the midbrain central gray substance at the level of the superior colliculus. Ascending fibers of our A-group of fibers proceed in the periventricular stratum of the third ventricle wall to the level of the anterior hypothalamus, and on the way, ramify to the pars medianus of the medial mamillary nucleus. Masai et al. (1953) demonstrated fine degenerated fibers from the lesion in the cortical areas 6 and 8 to the subependymal layer. These fibers are also included in our A-group of fibers: they are shown in Fig. 11. ( I / ) B-group offibers

The dorsal longitudinal fasciculus, which originates in the medial hypothalamic area and descends through the central gray substance, corresponds to the dorsales LangsReferenres p. 39-43

T. B A N

24

nucl. nucl. camp zona

infund. \

corp.

Fig. 1 1 . Frontal sections of cat brain of which cortical areas 6 and 8 were destroyed. Degenerated fibers are recognized in the hypothalamic periventricular stratum (above) and subependymal layer of the central gray substance (below). These fibers belong to our A-group of fibers.

biindel of Schiitz (1 891), which was found in the brain of progressive paralysis. Gurdjian (1927) called these fibers the periventricular system of fibers, and divided the system into the hypothalamic and the thalamic divisions. He reported that the hypothalamic division originated in the ventral premamillary nucleus, posterior hypothalamic nucleus, ventromedial hypothalamic nucleus and the posterior hypothalamic periventricular nucleus; on the other hand the thalamic division was closely related to some cells near the nucleus reuniens. Fibers of both divisions could be traced through the central gray substance to the tectum and medulla oblongata. Ascending and descending fibers of the fasciculus obtained by us (Zyo et al., 1962) by the Marchi technique are shown in Figs. 12 and 13. ( a ) Descending fibers of the hypothalamic component. Fibers originating in the ventromedial hypothalamic nucleus, dorsomedial hypothalamic nucleus, posterior hypothalamic nucleus and the dorsal premamillary nucleus, which all belong to the medial hypothalamic area, run dorsocaudad through the third ventricle wall to the central gray substance of the midbrain. The fibers terminate in the central gray substance at the level of the superior colliculus, and partly in the interstitial nucleus of Cajal. If lesion exists dorsally to the supramamillary decussation, degenerated fibers being traced dorsocaudad to the central gray substance reach dorsally to the tegmental nucleus of Gudden decreasing in number, and in part, terminate in the pars dorsalis of this nucleus. The fiber-group sends the sympathetic impulses from the medial hypothalamic area (b-sympathetic zone) to the lower autonomic centers. Therefore, we call this fiber-group B-group of fibers.

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Some descending fibers from the ventromedial and the dorsomedial hypothalamic nuclei are traced to the central gray substance contralaterally through the supramamillary decussation and terminate dorsally to the oculomotor nucleus (Fig. 13). In all our experiments no degenerated fibers were traced to the oculomotor nucleus, nucleus of Edinger-Westphal, nucleus of Darkschewitsch or the trochlear nucleus.

Fig. 12. Schematic summary of courses and terminations of the dorsal longitudinal fasciculus (FLD). The small black round points show site of lesion. AH, anterior hypothalamic nucleus; AL, nucleus dorsalis vagi; AMB, nucleus ambiguus; CA. anterior commissure; CC, corpus callosum; CHOP, optic chiasm; COLI, inferior colliculus; COLS, superior colliculus; CMT, medial central nucleus; CP, posterior commissure; D, nucleus of Darkschewitsch; DM, dorsomedial hypothalamic nucleus; EW, nucleus of Edinger-Westphal; FLD,dorsal longitudinal fasciculus; HIP, hippocampus; HL, lateral habenularnucleus; HM, medial hypothalamic nucleus; IC, nucleus intercalatus Staderini; IS, interstitialnucleusof Cajal; IP, interpeduncular nucleus; LAM, nucleus laminaris (pars anterior); LI, nucleus of locus incertus; ML, lateral mamillary nucleus; MM, medial mamillary nucleus; PH, posterior hypothalamic nucleus; PMD, dorsal premamillary nucleus; PMV, ventral premamillary nucleus; PRH, nucleus prepositus hypoglossi; PVA, anterior paraventricular nucleus; PVP, posterior paraventricular nucleus; RE, nucleus reuniens; RVM, subnucleus reticularis ventralis medullae oblongatae; SG, nucleus supragenualis; SM, supramamillary nucleus; SOL, nucleus tractus solitarii; STPV, hypothalamic periventricular stratum; TD, dorsal tegmental nucleus of Gudden; VM, ventromedial hypothalamic nucleus; 111, oculomotor nucleus; IV, trochlear nucleus; VI, abducens nucleus; VII, facial nucleus; VIIIL, VIIIM and VIIIS, lateral, medial and superior vestibular nuclei ; XII, hypoglossal nucleus.

The dorsal longitudinal fasciculus in which central gray substance was destroyed at the level of the superior colliculus, proceeds caudad ventrally to the cerebral aqueduct as well as the fourth ventricle, and sends fibers to the pars dorsalis of the dorsal tegmental nucleus of Gudden, the nucleus of locus incertus and the nucleus supragenualis (Meessen and Olszewski, 1949). Some fibers of the fasciculus terminate in the medial and lateral vestibular nuclei (Figs. 12 and 13). The dorsal tegmental nucleus of Gudden is divided into two parts, namely the small celled pars dorsalis and the large celled pars ventralis. According to our findings on their fiber connections, the pars dorsalis is connected with the dorsal longitudinal Rt:ferenres p. 39-43

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T. B A N

Fig. 13. Horizontal aspect of the B-group of fibers including the dorsal longitudinal fasciculus (FLD), hypothalamicotegmentaltract (THT), hypothalamiconigraltract (THN)and tegmentohypothalamic tract (TTH). Abbreviations are same as in Fig. 12. VESI, VESL, VESM and VESS, inferior, lateral, medial and superior vestibular nuclei; XDI nucleus dorsalis vagi. TTML shows the lateral tegmentomamillary tract.

fasciculus and the pars ventralis receives fibers of the mamillotegmental tracts and the mamillary peduncle (Ban and Zyo, 1963) (Fig. 14). Degenerated fibers, destroyed in the medial part of the gray substance of the fourth ventricle floor at the level of the facial genu, descend in the gray substance to the level of the nucleus intercalatus (Staderini). On the way, they terminate in the medial vestibular nucleus, nucleus prepositus hypoglossi and nucleus intercalatus. A few fibers entering and penetrating the medial vestibular nucleus terminate in the lateral vestibular nucleus. If the lesion exists in the gray substance of the fourth ventricle floor at the level of the nucleus intercalatus, degenerated fibers terminate in the hypoglossal nucleus and the nucleus intercalatus, and partly in the nucleus alaris (nucleus dorsalis vagi). The majority of degenerated fibers descend laterally to the central canal, and then ventromediad to the subnucleus reticularis ventralis (Meessen and Olszewski, 1949) of the medulla oblongata and the first cervical cord (Figs. 12 and 13). In other cases (Matano et al., 1964) degenerated fibers originating in each part of the vestibular nuclei proceed to the gray substance of the fourth ventricle floor, join the dorsal longitudinal fasciculus and descend laterally to the central canal to reach the rostra1 end of the cervical cord. On the way, some of them terminate in the nucleus prepositus hypoglossi and the nucleus intercalatus (Fig. 15).

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27

3 Fig. 14. Schematic summary of the courses and terminations of the mamillary peduncle (PM) and mamillotegmental tracts observed in our experiments. I , tr. tegmentomamillaris intermedius; 2, tr. tegmentomamillaris medialis; 3, tr. tegmentomamillaris lateralis; 3', tr. tegmentohypothalamicus; 4, tr. hypothalamicotegmentalis; 4, tr. hypothalamiconigralis; 5, tr. mamillotegmentalis lateralis: 6, tr. mamillotegmentalis medialis; 8, tr. tegmentopeduncularis. AHL, lateral hypothalamic area; AHM, medial hypothalamic area; F, fornix; FLM, medial longitudinal fasciculus; FM, fasciculus retroflexus; IP, interpeduncular nucleus; LM, medial lemniscus; ML, lateral mamillary nucleus; MM, medial mamillary nucleus; MT, mamillothalamic tract; PC, cerebral peduncle; PM, mamillary peduncle; PYR,pyramidal tract ;SGC, central gray substance; SM, supramamillary nucleus; SN, substantia nigra; SPVH, hypothalamic periventricular stratum; TD, dorsal tegmental nucleus; TV, ventral tegmental nucleus; 111, oculomotor nucleus; IV, trochlear nucleus.

Rcferenrrs p . 39-43

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T. B A N

Fig. 15. Schematic summary of the courses and terminations of the vestibular component of rhe medial longitudinal fasciculus (FLM) observed in our experiments. The small black round points show the site of the lesion. CHOP, optic chiasm; CNTS, superior central nucleus; CP, posterior commissure; D, nucleus of Darkschewitsch; DSOD, dorsal supraoptic decussation; EP, entopeduncular nucleus; FLD, dorsal longitudinal fasciculus; FUNA, anterior funiculus of the spinal cord; IC, nucleus intercalatus Staderini; IS, interstitial nucleus of Cajal; LH, lateral hypothalamic nucleus; PRH, nucleus prepositus hypoglossi; PRT, pretectal nucleus; RET, reticular nucleus; SGC, central gray substance; SUB, subthalamic nucleus; TEG, tegmentum; VESI, inferior vestibular nucleus; VESL, lateral vestibular nucleus; VESM,medial vestibular nucleus; VESS, superior vestibular nucleus; VS, vestibulospinal tract; VT, ventral thalamic nucleus; ZI, zona incerta; 111, oculomotor nucleus; IV,trochlear nucleus; VI, abducens nucleus.

From these descriptions, it may be concluded that the descending pathway of the dorsal longitudinal fasciculus from the medial hypothalamic area to the caudal end of the medulla oblongata or the rostra1 end of the cervical cord, consists of a t least 3 neurons, and some of the fibers originating in the vestibular nuclei join the fasciculus. ( 6 ) Descendingfibers of the thalamic component. According to Glees and Wall (1946) the fasciculus receives fibers from the medial dorsal nucleus and the centrum medianum of the thalamus. In our cases which were destroyed in midline part of the

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thalamus including the nucleus reuniens and the medial central nucleus (Figs. 12 and 13), degenerated fibers proceed dorsad, occupy the posterior paraventricular nucleus, shift caudad ventrally to the habenula and join the hypothalamic component at the rostral level of the posterior commissure. These fibers occupy the medial part of the B-group of fibers in the midbrain central gray substance and disappear at the level of the oculomotor nucleus. ( c ) Ascending fibers of the thalamic component. The thalamic component runs rostrad ventrally to the habenular nucleus, sends a few fibers to the region ventral and ventrolateral to the lateral habenular nucleus and enters the posterior paraventricular nucleus (Fig. 12). Some fibers of this component terminate in the posterior paraventricular nucleus, and residual fibers proceed ventrad and terminate in the nucleus reuniens, the central medial nucleus and Niimi’s nucleus laminaris (pars anterior). ( d ) Ascendingfibers of the hypothalamic component. Ascending fibers of the hypothalamic component from the midbrain central gray substance terminate most numerously in the posterior nucleus, but also in the dorsal premamillary nucleus, the dorsomedial nucleus and the ventromedial nucleus. A part of these fibers disperses and disappears in the dorsal area to the dorsomedial hypothalamic nucleus. After a lesion has been made in the medial part of the fourth ventricle floor at the level of the facial genu, ascending degenerated fibers can be traced rostrad through the gray substance of the fourth ventricle floor and the midbrain central gray substance as far as the level of the trochlear nucleus. On the way, some fibers enter the nucleus of the locus incertus as well as the pars dorsalis of the dorsal tegmental nucleus of Gudden (Figs. 12 and 13). But ascending fibers of the medial longitudinal fasciculus enter the trochlear nucleus as well as the oculomotor nucleus and the central gray substance dorsal to these nuclei as shown in Fig. 15; hence more rostrally degenerated fibers in the central gray substance include 2 components of the dorsal and medial longitudinal fasciculi. After destruction of gray substance of the fourth ventricle floor at the level of the nucleus intercalatus (Staderini), ascending degenerated fibers are traced in the gray substance of the fourth ventricle floor to the level of the ventral tegmental nucleus. On the way, some fibers enter the nucleus prepositus hypoglossi, the medial vestibular nucleus and the nucleus tractus solitarii, and a few fibers terminate in the lateral and superior vestibular nuclei. More rostrally, some fibers enter the nucleus (supragenualis, the abducens nucleus and the nucleus of locus incertus (Figs. 12 and 13). In other experiments (Matano et al., 1964) (Fig. 1 9 , ascending degenerated fibers originating in the lesions in the superior, medial, lateral and inferior vestibular nuclei could be traced to the rostral level of the pons via the gray substance of the fourth ventricle floor. It is therefore concluded that the ascending hypothalamic component consists of at least 3 neurons and has almost the same connections and courses as shown by the descending fibers. On the other hand (Fig. 15), the ascending fibers in the medial longitudinal fasciculus that originate in the vestibular nuclei penetrate the trochlear and oculomotor nuclei and terminate in the central gray substance dorsally to these nuclei. These Refcsrences p. 39-43

30

T. B A N

fibers may join the dorsal longitudinal fasciculus. The fibers that originate in the vestibular nuclei and mix with the dorsal longitudinal fasciculus may send impulses to the medial hypothalamic area to produce sympathetic responses. The vestibular nucleus receives fibers from the fastigial nucleus of the cerebellum, namely via the fastigiobulbar tract, and sends fibers to the motor nerve nuclei of the eye via the medial longitudinal fasciculus. And some fibers in the medial longitudinal fasciculus could be traced in the Ganser’s commissure (DSOD in Fig. 15) and partly

-

r i g h t truncus

l e f t truncus

sgmpa t h i cue

sympa t h i c u s

before stimul.

stimulation

-

before stimul. 7

etimul a t ion

after s t imul after

. W&Mh v

Left hemisection on t h e l e v e l of t h e decuaaatio nervorum trochlearium before stimul.

before e t imul

W

. r

8 t imulation

stimulation

after stimul. 60 c/a

after 8 t imul

I z z . *. n 1 . 1 n h A 6 1.1

50 FV

.

Fig. 16. Stimulation of right nucleus hypothalamicus ventromedialis.

SEPTO-PREOPTICO-HYPOTHALAMIC SYSTEM

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in the lateral hypothalamic nucleus on the opposite side (Zyo and Ban, 1963) (LH in Fig. 15). Regarding nystagmus and deviation of the eye which are sometimes recorded by the electrical stimulation of the hypothalamus, it may be that the impulses enter the vestibular nuceus via the dorsal longitudinal fasciculus and reach the motor nerve nuclei of the eye through the medial longitudinal fasciculus, or that the impulses enter the abducens nucleus directly via the dorsal longitudinal fasciculus, or that the impulses pass through the hypothalamo-cerebello-vestibularpathway and the medial longitudinal fasciculus to the motor nerve nuclei of the eye. After the ventromedial and dorsomedial hypothalamic nuclei had been destroyed, a small number of fibers was traced to the contralateral side of the central gray substance through supramamillary decussation. But no other decussation of fibers descending or ascending in the central gray substance ventral to the cerebral aqueduct and in tee gray substance of the fourth ventricle floor could be demonstrated. Yuasa (1959) reported that the efferent discharges of both cervical sympathetic trunks were increased by unilateral electrical stimulation of the medial hypothalamic area, and the efferent discharges of the trunk on the operated side were not increased by hemisection at the level of the trochlear or trigeminal nucleus (Fig. 16). These results accord with the results mentioned above. The dorsal longitudinal fasciculus decussates partially at the supramamillary decussation and passes through the central gray substance and the gray substance of the fourth ventricle floor without any further decussation to the cervical cord. When the lateral part of the ventromedial and dorsomedial hypothalamic nuclei was destroyed, some fibers of the descending degenerated fibers were traced through the dorsal part of the mamillary peduncle to the ventral part of the tegmentum. Their course is similar to that of the tractus hypothalamico-tegmentalis of Bodian (1940’1 or the fasciculus hypothalamico-tegmentalis of Shimizu (1948). Some of the fibers enter the caudal part of the homolateral substantia nigra (Fig. 13). We call this group of fibers the tractus hypothalamico-nigralis (Zyo et at., 1962; Ban and Zyo, 1963). The hypothalamico-tegmental tract is also included in our B-group of fibers. (III) C-group of fibers It has been recognized that the medial forebrain bundle originates in the olfactory tubercle and the septal region, and that it reaches the midbrain tegmentum via the lateral preoptic and hypothalamic areas. It seems to correspond to ‘das basale Riechbundel’ of Wallenberg (1901). (a) Descendingfibers. According to our Marchi preparations of albino rats (Ban and Zyo, 1962) and rabbits (Zyo et al., 1963), the course and the termination of the medial forebrain bundle are summarized as shown in Figs. 10 and 17. The fiber group running mediad from the olfactory tubercle, and the fiber group forming the diagonal band of Broca from the septal nuclei enter the medial forebrain bundle and terminate in the rostra1 part of the midbrain tegmentum bilaterally. That is, the medial forebrain bundle is divided into the tubercular and septal groups. Fibers originating in the anterior olfactory nucleus, the olfactory tubercle, and the precommissural portion References p. 39-43

32

T. B A N

Fig. 17. Schematic summary of the courses and terminations of the medial forebrain bundle (MFB) and mamillothalamic tract (MT) observed in our experiments. Abbreviations are the same as shown in Fig. 10. AQ, cerebral aqueduct; CA, anterior commissure; CHOP,optic chiasm; COLI, inferior colliculus; COLS, superior colliculus; CORA, Arnmon’s horn; CP, posterior commissure; IP, interpeduncular nucleus; MT, mamillothalamic tract; PRA, preamygdaloid cortex; TV, ventral tegmental nucleus.

of the septum are followed in the medial forebrain bundle to the midbrain tegmentum. A small number of fibers originating in the olfactory bulb passes through the anterior limb of the anterior commissure as well as the lateral olfactory tract and enters the lateral preoptic area, and a few fibers reach the rostral end of the lateral hypothalamic nucleus. The medial forebrain bundle descends in the lateral preoptic and hypothalamic areas and ramifies to these nuclei. On the way, the bundle which originates in the regions more rostral than the preoptic area does not enter the mamillary body. When our lesions are located in the lateral preoptic area or the lateral hypothalamic area, the medial forebrain bundle sends numerous fibers to the medial mamillary nucleus and some to the lateral mamillary nucleus. Descending fibers in the medial forebrain bundle originating in the septum pellucidum, the olfactory tubercle, and the lateral preoptic area enter the stria medullaris (STM in Figs. 10 and 17) through the inferior thalamic peduncle and reach the habenula. These fibers terminate chiefly in the lateral habenular nucleus but partly in the medial habenular nucleus. On the way, some of the fibers from the lateral preoptic area terminate in the anteroventral nucleus (AV), anterodorsal nucleus (AD), mediodorsal nucleus (MD), parataenial nucleus (PTAE), reticular nucleus (RT) jand anterior paraventricular nucleus (PVA) of the thalamus as shown in Figs. 10 and 17. Fibersprighating in the small lesion of the septum pellucidum terminate in the anteromedial nucleus, the anterodorsal nucleus and the parataenial nucleus (Figs. 10

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and 17). Regarding the fibers traced to the habenula via the stria medullaris, we (Ban and Zyo, 1962) followed fibers to the medial forebrain bundle from the olfactory tubercle, and traced fibers originating in the septum pellucidum to the stria medullaris via the fornix system (Fig. 18). Fibers traced to the medial forebrain bundle from the rostrolateral part of the septum pellucidum enter the stria medullaris via the inferior thalamic peduncle and terminate in the habenula.

Fig. 18. Schematic summary of the courses and terminations of the fornix system recorded in our experiments. The small squares show the site of the lesion. AHIP, anterior continuation of the hippocampus; APM, medial preoptic area; CA, anterior commissure; CC, corpus callosum; CHOP, optic chiasm; CM, mamillary body; COMH, commissura fornicis; DSM, supramamillary decussation; F, fornix column; F contr, contralateral fornix column; FS, superior fornix ; H, habenula; HIP, hippocampus; IP, interpeduncular nucleus; ML, lateral mamillary nucleus; MM(L), lateral part of the medial mamillary nucleus; NDB, bed nucleus of the diagonal band of Broca; NSF, fimbrial septal nucleus; NST, triangular septal nucleus; NSTT, interstitial nucleus of the stria terminalis; PCMS, precommissural portion of the septum; SH, septohippocampal nucleus; SPL, lateral septal nucleus; SPM, medial septal nucleus; STM, stria medullaris.

As Wallenberg (1901), Krieg (1932) and Nauta (1958) reported, we also found that descending fibers in the dorsal part of the medial forebrain bundle ran dorsad along the third ventricle wall and entered the central gray substance. Some of them enter the central gray substance on the opposite side via the supramamillary decussation. In the midbrain central gray substance, these fibers scatter and disappear in the subnucleus lateralis of Olszewski and Baxter. The lateral group disperses ventrolaterad in the dorsal tegmental area of the rostra1 midbrain (Fig. 19). When the caudal portion of the lateral hypothalamic nucleus has been destroyed some fibers on the operated side enter the interstitial nucleus 01 Cajal, the nucleus of Darkschewitsch, the nucleus of Edinger-Westphal and the oculomotor nucleus on the same side. Some fibers, after crossing via the supramamillary decussation, also reach the interstitial nucleus of Cajal, but do not reach the other nuclei (Fig. 10). The medial forebrain bundle is called the C-group offibers, because it is in close relationship Riferenus p. 39-43

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T. B A N

A0

R SN pc

PM

Fig. 19. Degenerated fibers descending from the lesion existing dorsally to the supramamillary decussation are divided into 3 groups: A-group of fibers in the subependymal layer, B-group of fibers in the subnucleus medialis of the central gray substance (fasciculus longitudinalis dorsalis) and Cgroup of fibers belonging to the medial forebrain bundle in the subnucleus lateralis. AQ, cerebral aqueduct; COLS, superior colliculus; FM, fasciculus retroflexus of Meynert ; GM, medial pniculate body; IS, interstitial nucleus of Cajal ; MTEG, medial mamillotegmental tract; PC, cerebral peduncle; PM. mamillary peduncle; R, red nucleus; SGC, central gray substance; SN, substantia nigra.

with our c-parasympathetic zone. Evidently the fibers in the subnucleus lateralis of the midbrain central gray substance also belong to the C-group of fibers. The medial forebrain bundle was generally traced to the tegmentum at the level of the interpeduncular nucleus. But when the caudal portion of the lateral hypothalamic nucleus had been destroyed, degenerated fibers were traced to the ventromedial area of the pontile tegmentum at the rostra1 level to the superior olivary nucleus. These fibers pass through dorsolaterally to the interpeduncular nucleus and dorsally to the medial part of the medial lemniscus. These fibers take almost the same course although their direction is reversible, as the ascending fibers observed in other cases which originate in the lesions of the dorsal pontile tegmentum run ventrorostrad t o mingle with the medial forebrain bundle as shown in Figs. 10 and 17. Our descending fiber-bundle is situated dorsally to the posterior hypothalamo-tegmental tract of Crosby and Woodburne (1951), descends in the ventromedial part of the tegmentum, decreases in number and disappears at the level of the caudal part of the pons, but its terminations are not yet clear (Fig. 17). This fiber-bundle connects the caudal part of the lateral hypothalamic nucleus with the ventromedial part of the pontile tegmentum. So this fiber-bundle may send impulses from the lateral hypothalamic nucleus to the metencephalon. ( b ) Ascendingfibers. Guillery (1957) divided the ascending fibers in rats into 2 groups : the hypothalamo-septa1 group and the mesencephalo-septa1 group. The hypothalamo-septa1 group which originates in the lateral hypothalamic area, terminates mostly in the lateral septal nucleus and partly in the bed nucleus of the anterior commissure and dorsal part of the nucleus accumbens, and the mesencephalo-septa1 group which originates in the midbrain, terminates principally in the medial septal nucleus and partly in the dorsal fornix, the supracallosal striae, the Ammon’s horn and the subiculum. In our Marchi sections (Zyo et al., 1963) of rabbits, ascending fibers of the medial forebrain bundle were traced as follows (Figs. 10 and 17). Ascending fibers from the lateral hypothalamic area run rostrad in the lateral hypothalamic and preoptic areas,

SEPTO-PREOPTICO-HYPOTHALAMIC SYSTEM

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decrease in number and reach’the septum pellucidum. The ventral group enters the olfactory tubercle and partly reaches the rostra1 part of the tubercle. In the septal region, fibers ascending in the lateral hypothalamic and preoptic areas proceed to the diagonal band of Broca. The more thickly myelinated numerous fibers in the medial part terminate in the medial septal nucleus. And thinly myelinated fibers in the lateral part terminate in the lateral septal nucleus. A few fibers enter the nucleus septohippocampalis, and in part, enter the nucleus septohippocampalis of the opposite side after crossing in contact with the corpus callosum (Fig. 20, d and c). Some fibers are traced rostrad to the precommissural portion of the septum. These fibers in the precommissural portion of the septum are divided into 5 groups (Fig. 20b):

d STLM

STLL SPM

U

SPL

Fig. 20. Ascending degenerated fibers originating in the lesion of the rostrolateral part of the lateral preoptic area. ACA, anterior limb of the anterior commissure; AHIP, anterior continuation of the hippocampus; CAU, caudate nucleus; CC, corpus callosum; CI, internal capsule; OA(P), pars posterior of the anterior olfactory nucleus; PCA, posterior limb of the anterior commissure; PCMS. precommissural portion of the septum; SH, septohippocampal nucleus; SPL, lateral septal nucleus; SPM, medial septal nucleus; STLL, stria longitudinalis lateralis; STLM, stria longitudinalis medialis; TOL, lateral olfactory tract; TUBO, olfactory tubercle; VL, lateral ventricle. References p. 39-43

36

T. B A N

Fig. 21. Schematic summary of the courses and terminations of the supraoptic decussations recorded

in our experiments. AQ, cerebral aqueduct; CI, internal capsule; CM, mamillary body; COLI, inferior colliculus; COLS, superior colliculus; EP, entopeduncular nucleus ; F, fornix ; FLM. medial lonpitudinal

fasciculus; FM, fasciculus retroflexus; GL, lateral geniculate body; GM, medial geniculate body; GP, globus pallidus; IP, interpeduncular nucleus; LH, lateral hypothalamic nucleus; LM,medial lemniscus; MT, mamillothalamic tract; PC, cerebral peduncle; PYR, pyramidal tract; RT, thalamic reticular nucleus; SGC, central gray substance; SN, substantia nigra; SOP, supraoptic nucleus; SUB,subthalamic nucleus; TD, dorsal tegmental nucleus; TROP, optic tract; VIII, third ventricle; ZI, zona incerta; 111, oculomotor nucleus; IV, trochlear nucleus.

(I) fibers terminating in the cortex of the medial wall of the precommissural portion of the septum, (2) fibers proceeding dorsad vertically and terminating in the dorsal part of the medial surface of the cortex, (3) fibers terminating in theanteriorcontinuation of the hippocampus, (4) fibers traced to the supracallosal striae, and (5) fibers proceeding rostrad medially to the lateral ventricle and the anterior limb of the anterior commissure and terminating in the pars posterior of the anterior olfactory nucleus (Fig. 20a). The fibers of group ( 4 ) turn dorsad passing rostrally to thegenu corporis callosi and are traced to the striae longitudinales medialis and lateralis at

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the dorsal side of the corpus callosum (Fig. 20, d and c). The fibers in the stria longitudinalis medialis disperse in the cingulum, and partly project dorsad to the dorsal part of the medial surface of the cortex at the level of the rostral part of the septum to the rostral end of Ammon’s horn. The fibers in the stria longitudinalis lateralis disappear in the indusium griseum at the level of the rostral part of the septum. Some fibers of the ascending medial forebrain bundle are followed to the stria medullaris through the inferior thalamic peduncle and terminate in the habenula (Figs. 10 and 17). The ventrolateral group of fibers in the medial forebrain bundle forms the ventral supraoptic decussation (Meynert’s and Gudden’s commissures) at the rostral part of the hypothalamus (Fig. 21). In a case of lesion in the dorsal tegmental area just lateral to the medial longitudinal fasciculus at the level of the dorsal tegmental nucleus of Gudden (Fig. 21), fibers proceeding ventrorostrad in the medial part of the tegmentum and fibers going ventrad along the abducens root and running dorsally to the medial part of the medial lemniscus are united into one group at the level of the inferior colliculus and are traced rostrad dorso-laterally to the interpeduncular nucleus. This fiber-group passes through the region dorsal to the mamillary peduncle and medial to the medial lemniscus and enters the ventrolateral part of the lateral hypothalamic nucleus. These fibers decrease in number terminating in the lateral hypothalamic nucleus and reach the rostral part of this nucleus. A few fibers terminate in the lateral preoptic area. A small number of fibers on the medial group of this fiber-bundle traverses the midline dorsally to the mamillary body, proceeds rostrad in the ventral part of the lateral hypothalamic area and terminates in the nucleus at the level of the ventromedial hypothalamic nucleus (Figs. 10 and 17). Hence it seems that some fibers of the medial forebrain bundle originating in the pontile tegmentum pass through the ventromedial part of the midbrain tegmentum and reach the lateral hypothalamic nucleus (partly on the opposite side) and the lateral preoptic area on the same side. This course is almost the same as the course of the descending medial forebrain bundle from the caudal part of the lateral hypothalamic nucleus, though their directions are reversed. As mentioned above, both descending and ascending fibers compose the medial forebrain bundle and they take almost the same course. These fibers connect the following areas : rhinencephalon, septal area, habenula, hippocampus, thalamus, lateral preoptic and hypothalamic areas and midbrain as well as pontile tegmentum. According to our experiments, it appears that the medial forebrain bundle is closely related to the olfactory or feeding reflexes and t o the parasympathetic responses. This bundleis thought to be the most important main route of the parasympathetic centers. SUMMARY

According to our histological and experimental studies on the central autonomic nervous system, the preoptic area and the septal region are closely related to the hypothalamus from the viewpoints of their functions and fiber connections. The medial preoptic area, which is located rostrally to the medial hypothalamic area (bsympathetic zone), shows sympathetic reactions. The preoptic periventricular stratum Rrfircvwrr p . 39-43

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continues to the hypothalamic periventricular stratum (a-parasympathetic zone) and the lateral preoptic area to the lateral hypothalamic area (c-parasympathetic zone), and they all react parasympathetically. The septum pellucidum, which is situated rostrally to the preoptic area shows, on the whole, parasympathetic responses. Therefore, it is considered that the a- and c-parasympathetic zones of the hypothalamus continue rostrad via the preoptic periventricular stratum and the lateral preoptic area respectively to be united at the septal region (Fig. 9). The A-group of fibers, or the tractus hypothalamicus periventricularis, originating in the septum pellucidum is traced through the periventricular stratum of the third ventricle wall to reach the subependymal layer of the midbrain aqueduct, and ramifies on the way to the medial mamillary nucleus. The tractus corticohypothalamicus periventricularis originating in the cortical areas 6 and 8 to reach the periventricular stratum of the third ventricle wall and the periaqueductal gray stratum is also included in the A-group of fibers. The afferent fibers from the midbrain subependymal layer to the septum are also included in the A-group of fibers. The dorsal longitudinal fasciculus, the hypothalamicotegmental tract with the hypothalamiconigral tract and the tegmentohypothalamic tract (Ban and Zyo, 1963) are included in the B-group of fibers which connects the medial hypothalamic area with other autonomic centers of the brain stem. The dorsal longitudinal fasciculus runs through the central gray substance and the gray substance of the fourth ventricle floor between the hypothalamus and the spinal cord. This fasciculus consists at least of 3 neurons between the hypothalamus and the first cervical cord, and contains both efferent and afferent fibers. Afferent fibers ascend almost the same course as the efferent fibers. The hypothalamicotegmental tract descends caudad to the midbrain tegmentum, and the tegmentohypothalamic tract ascends from the midbrain and pontile tegmentum to the hypothalamus. The sympathetic impulses descend to or ascend from the lower centers through the B-group of fibers, especially via the dorsal longitudinal fasciculus. But some sympathetic impulses can be conducted to the midbrain tegmentum via the hypothalamicotegmental tract. Therefore, these fiber bundles should be considered first when the sympathetic responses of the brain stem are discussed. The C-group of fibers participating in the parasympathetic reactions is the medial forebrain bundle. This bundle includes ascending and descending fibers and connects the lateral preoptic and hypothalamic areas with the rhinencephalon, the septal region, the midbrain and the pontile tegmenta. Some fibers enter the central gray substance of the midbrain and mingle in the A-group of fibers. These fibers are also included in the A-group of fibers. The septal region and the preoptic and hypothalamic areas can be united into one system named the septo-preoptico-hypothalamic system or the SPH-system, based on our studies of the stimulation and destruction experiments and the fiber connections. In conclusion, the septo-preoptico-hypothalamic system is divided into 3 areas: (1) area parasympathica A or area A consisting of the septal region, the preoptic periventricular stratum and the hypothalamic periventricular stratum; (2) area sympathica B or area B consisting of the medial preoptic area and the medial hypothalamic

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area; and (3) area parasympathica C or area C consisting of the septa1 region, the lateral preoptic area and the lateral hypothalamic area.

ACKNOWLEDGEMENT

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Synaptic Interaction at the Mauthner Cell of Goldfish TARO FURUKAWA Department of Physiology, Osaka City University Medical School, Osaka (Japan)

INTRODUCTION

In the spinal cord of teleost fishes a pair of large myelinated nerve fibers runs down closely parallel with each other on the ventral side of the central canal. These fibers constitute a remarkable structure in the fish’s spinal cord, since their diameter can be as large as 50 p. The Mauthner cells, located one on each side in the medulla, are neurons that give rise to these large axons. Because of their big size and peculiar shape, the Mauthner cells have attracted the interest of histologists from early times (Beccari, 1907). Studies of Bartelmez (1915) on catfish and the later work of Bodian (1937, 1952) on goldfish furnished a picture of the structure of the cell with synaptic endings that cover its surface. Especially interesting from the physiological point of view is the fact that synaptic endings of different types are distributed as groups on different parts of the cell’s surface. Furthermore there are types of synaptic endings that are not commonly found in other neurons. Electrophysiological studies disclosed many different kinds of synaptic mechanism that operate on this single neuron. It has been well established that transmission at synapses is ordinarily mediated chemically. In the M-cell, however, there exist excitatory as well as inhibitory synapses that are operated electrically. These electrical synapses coexist with a more common type of synapse where transmission is made chemically. Another interesting fact is that we can distinguish five inhibitory processes each having different sites of action on the Mauthner cell system. Descriptions of these various types of synaptic activity and their possible role in synaptic integration in the Mauthner cell will constitute the main topics of the present communication. Descriptions of some basic properties of the Mauthner cell are based on two papers by Furshpan and the author (Furshpan and Furukawa, 1962; Furukawa and Furshpan, 1963), and descriptions of the excitatory electrical transmission are based on a recent report by Furshpan (1964). The second half of the article is mostly concerned with experiments carried out in the author’s laboratory. METHODS

Anatomy. Fig. lA, taken from Bodian (1952), shows the peculiar shape of the M-cell with various types of synaptic ending that cover the cell’s surface. Of particular

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interest are the large myelinated club endings on the lateral dendrite (see next Section) and the helicoidal feltwork of fine fibers (sp.) that is embedded in the so-called axon cap (Bartelmez, 1915; Bodian, 1952). The axon cap is an approximately spherical structure of glial and nervous elements surrounding the axon of the M-cell from its axon hillock origin to the beginning of its myelination (broken line of Fig. 1A). The axon of each M-cell then expands to about 40 u , in diameter, decussates with its fellow and runs down the spinal cord (Fig. I B). In the spinal cord many fine collaterals come out from the axon. They make contact with motor neurons in the spinal cord with special axo-axonal synapses (Bodian, 1952). This structural feature suggests that M-cells are concerned with synchronous activities of tail muscles. In fact, it has been A

Club endings A

Lateral dendrite

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Cerebellum

Fig. 1. Structure of the goldfish Mauthner cell. (A) Taken from Bodian (1952). A semischematic drawing projected on a transverse plane. Midline is t o the left, dorsal above. Note particularly the thick dendrites and the several types of presynaptic endings. VIII (Xed), the unmyelinated club endings; sp. = fibers which spiral around the axon neck; d = fine dendrites. (B)A schematic drawing of the location of the M-cells within the medulla as seen from above. The cerebellum is shown retracted forward to expose the subjacent medulla. The cells are more than 1 mm beneath the surface of the medulla. Note the decussation of the M-axons.

observed that a single activity of the M-cell evokes a fairly strong tail movement toward the opposite side, i.e. to the same side as the M-axon (Diamond and Furshpan, unpublished observation). Experimental procedures. Experiments were performed upon common Japanese goldfish (Carassius auratus L.), measuring about 12 cm from nose to the tail tip. They were immobilized by intramuscular injection of Flaxedil (ca. 1 pg/g of body weight) and held in position in a fish chamber. The fish were kept alive during the Huferences p. 69/70

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experiment by flowing aerated and dechlorinated tap water through the mouth piece, Because M-cells are located more than 1 mm below the medullar surface, the first problem in working with these cells is to determine their position by blind exploration with the microelectrode. This can be done by taking advantage of the fact that the M-cell, when activated antidromically, generates an extracellular field of action potential around it. By far the most predominant extracellular action potential of the M-cell is a negative potential that can be recorded when the microelectrode approaches within 200p or so of the axon hillock of the cell. The amplitude of this negative spike can be as large as 40mV when the electrode is placed very close to the axon hillock (upper trace of Fig. 2A). In this way it is not difficult to bring the tip of the microelectrode to within 20-3Op from the axon hillock of the M-cell. It is also not difficult to impale the soma or even the lateral dendrite a t various distances from the axon hillock, because these structures are known to extend toward the lateroposterior direction from the axon hillock. Also the axon can be impaled at various points along its course. Although the positional relationship was used mostly as a basis for locating various parts of the cell with the microelectrode, it was also possible to tell from responses recorded whether the structure entered by the electrode was the soma or the axon. For example, the antidromic action potential of the soma is small (30-40 mV) and it is further reduced in size when evoked during the late phase of collateral inhibition (Fig. 5A). On the other hand, when the electrode impales the axon a larger action potential is recorded (60-90 mV) and it is not reduced in size during inhibition (Furukawa and Furshpan, 1963). Stimulation of the M-cell. It is very simple t o activate M-cells antidromically. M-axons can be excited more or less selectively by simply applying electrical pulses to the unopened spinal cord. Presumably M-axons have a much lower threshold than most of other fibers in the spinal cord. Usually two M-axons are excited together, but very often one or the other M-axon is excited selectively when stimulus parameters are finely adjusted. Usually two pairs of stimulating electrodes were embedded close to the unopened vertebral column. This arrangement made it possible to deliver a pair of stimuli at different intensities. Direct stimulation of the M-cell was made by passing a depolarizing current pulse through the intracellular electrode. A bridge circuit was used so that the same electrode served for potential recording and current passing (Araki and Otani, 1955). Orthodromic stimulus to the M-cell was applied t o the 8th nerve root through a very fine bipolar electrode. Activity in the 8th nerve was also evoked by a sound stimulus: a tone pip was applied through a loudspeaker. Action potentials of the M-cell. Action potentials of the M-cell are unique, so that some preliminary account of them may be useful. Fig. 2A shows potential changes produced by antidromic stimulation of the M-cell. In the upper trace, recorded extracellularly from the vicinity of the axon hillock, spike potential appears as a negative deflection of a verp large size. In the lower trace, recorded intracellularly from the soma, the action potential appears as a positive spike. This spike potential, about 40 mV, is much smaller than usually observed in other neurons. Two spikes in Fig. 2A make an almost complete mirror image. These special relationships between

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8

OUTSIDE

MEMBRANE

INSIDE

Fig. 2. (A) Potentials recorded from the M-cell following a single spinal cord shock. Upper trace, extracellular potential recorded from the vicinity of the axon hillock; lower trace, intracellular potential recorded from the soma of the same M-cell. The arrow marks the EHP. Positivity upwards in this and the following oscillographic records. Scales in this applies also to C. (B) A circuit diagram illustrating recording conditions. The left side of the diagram pertains to cell parts which were active during the action potential (axon hillock region); the right side to the inactive region. VI = extracellular recording of action potential; VZ = intracellular recording; E = e.m.f. during the spike; R I = active membrane resistance; RZ = convergence resistance; R3 = tissue resistance around the soma and the dendrite; R.I = membrane resistance of the inactive part of the cell. (C) Block by the EHP of antidromic spike. Extracellular recording; 1 = the EHP produced by the conditioning stimulus; 2 = testing antidromic spike; 3 = 1 and 2, the spike is absent. (D)A schematic diagram illustrating the origin of the EHP. Electromotive forces that are oriented around the initial axon segment give rise to the electrical inhibition.

extra- and intracellularly recorded spikes may be attributable to the fact that the area of M cell membrane that takes part actively in spike generation is limited to a small part in the vicinity of the axon hillock and also to the fact that the membrane time constant of the cell is very short (Furshpan and Furukawa, 1962). In Fig. 2B, E and R1 represent the e.m.f. of action potential and active membrane resistance of the axon hillock region respectively. The action current leaves the cell across the inactive part of the cell membrane (R4), then flows through extracellular space (R3 and Rz) to converge to a small active membrane located in the vicinity of the M-cell axon hillock. Therefore, the intracellular spike potential (VZ)represents the potential drop produced by the action current in the membrane resistance of the soma and the dendrite (R4), while the negative extracellular spike (Vl) represents the potential drop in convergence resistance of the tissue (Rz). R4, when measured as an input References p. 69/70

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resistance of the cell, takes a value of about 105 ohm. The value of Rz is about the same as that of R4. but R3 is much smaller. EXCITATORY RESPONSES

Agerent input to the M-cell. M-cells receive an abundant synaptic input from fibers of the 8th nerve. According to Bartelmez (1915) this direct 8th nerve supply distinguishes the Mauthner neuron from the other cells of the nucleus motorius tegmenti. Both ipsilateral and contralateral 8th nerve fibers innervate the cell but end on different regions of it. The large myelinated club endings (Fig. 1A) are the endings of ipsilateral 8th nerve fibers and are restricted to the distal portion of the lateral dendrite. The proximal portion of the dendrite receives end-bulbs of collaterals from contralateral 8th nerve fibers. The cell body, near the axon hillock, also receives collaterals from contralateral 8th nerve fibers (unmyelinated club endings in Fig. 1A). The ventral dendrite apparently does not receive a direct input from the 8th nerve but is supplied with end-bulbs of axons from the ventral acoustic nucleus and thus has an indirect 8th-nerve input; and the axon cap receives numerous collaterals of secondary 8th nerve fibers. Fiber connections as described above are mostly taken from Bartelmez (191 5) and Bodian (1952). They stimulate our imagination about the role each fiber connection plays in the functional organization of the M-cell system. Although in principle it should be possible to elucidate the function of each fiber group by stimulating it separately and observing the effects thereby produced on the M-cell, our knowledge in this direction is still very incomplete. We know, however, that stimulation of the ipsilateral and contralateral 8th nerves has different effects on the M-cell. Only stimulation of the ipsilateral root normally gives rise to firing of the cell, whereas contralateral stimulation could suppress this firing (Furshpan and Furukawa, 1962; also see Retzlaff, 1957). The origin and properties of large myelinated fibers that terminate on the distal part of the M-cell lateral dendrite will be described. Sound receptor organ and the Mauthner cell. The work of Von Frisch and his school on sound perception in a series of bony fishes has shown that hearing is localized in the pars inferior of the labyrinth which consists of sacculus and lagena, while the pars superior, which consists of utriculus and three semicircular canals, is concerned with equilibrium function (Von Frisch, 1936). Some bony fishes, called Ostariophysi, have notably better hearing ability than other fishes. In this group of fish, in which goldfish is included, the air bladder is connected with the anterior part of the sacculus by means of a chain of three Weberian ossicles which may serve as sound conductors linking the air bladder, acting as a resonator, with the sound receptors in the saccular macula (Fig. 3A). The saccular otolith, the sagitta, has striking wing-like extensions and lies delicately suspended over the saccular macula, while the lagenal otolith is a very massive structure. These findings indicate that the sacculus is the main hearing organ in this group of fishes, though the lagena was also found to be sensitive to sound stimuli. Histological studies showed that there exist two distinct groups of fibers in afferent

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nerves from the saccular macula (Fig. 3B). Large myelinated fibers (diameter 10-15 p ) are distributed over the anterior part of the saccular macula, while small fibers (diameter 5 p), also myelinated, are distributed over a larger area toward the posterior part of the macula. There is a short strip in between the two innervated areas, where fibers terminate only sparsely. Afferent fibers from the lagenal macula are largely similar to those distributed over the posterior part of the sacculus. Action potentials of these fibers were recorded with a microelectrode a t certain points before they enter the medulla. Responses to sound were markedly different between two groups of fibers from the sacculus. Large fibers had their optimal frequency at about 600 c/s; they did not show any spontaneous activity and adapted very rapidly to sound stimuli. On the other hand, optimal frequency of the small fibers from the sacculus lay at about 200 c/s; they usually showed spontaneous activity and adapted only slowly to sound. Small fibers were more sensitive to sound than large fibers. A further difference was found in the relationship between sound frequency and discharge rate in nerve fibers. Some of the large fibers (about half of the total) discharged impulses at a rate twice as frequent as the sound (see Fig. 9A): that is, they fired at a rate of 1000/sec when stimulated by a sound at 500 c/s. But this type of response was not found in the small fiber group. All other large fibers and all small fibers followed the sound with the same frequency of discharge (Fig. 9B). A close examination disclosed that fibers of the latter group were divided into two by the phase of the sound to which they responded. About half the fibers were found to fire in response to the arrival of a certain phase of the sound, e.g. the compression phase, while the rest of the fibers responded at the phase 180" from the former, i.e. at the rarefaction phase. Now turning back to the M-cell again, the existence of a special group of large fibers in the nerve from the sacculus seems to suggest that fibers that terminate with club endings on the lateral dendrite would be a direct extension of these large fibers. This hypothesis was confirmed by recording potentials intracellularly from the 8th nerve fibers in the close vicinity of the lateral dendrite and also from the lateral dendrite itself. It must be mentioned here that the morphology of the M-cell is somewhat different among different species of fish. Otsuka (1962, 1964), who made an extensive survey of the morphology of M-cells in various species of bony fish of non-Ostariophysi type, found only a small number of club endings on the lateral dendrite of M-cells. Catfish (Arneiurus) and goldfish (Curussius uuratus), in both of which large myelinated club endings were found on the distal part of the lateral dendrite, belong to the Ostariophysi. Further studies are needed to clarify the species difference. Since we now know the response pattern of different fiber groups of the auditory nerve, it should be possible to follow their course in the brain by taking these response patterns as an indicator. But trials in this direction are only just beginning. We do not even know definitely the destination of the small fiber group of the saccular nerve. We are sure now that large fibers from the sacculus are connected with the lateral dendrite-of the M-cell with an-electrical synapse (Furshpan, 1964). But electrical stimulation of the 8th nerve produces, besides a short-latency electric EPSP, a large RrScriwrs p. 69/70

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EPSP in the soma of the M-cell that seems to be evoked, a.s judged from the latency and its time course, by the activity of a chemically transmitted synapse. It would therefore be an attractive explanation that fibers of the small fiber group of the saccular nerve end directly on the soma of the M-cell with an ordinary chemical synapse. But the potential change evoked in the soma of the M-cell in response to a sound that gives a maximal response to the small fiber group was disappointingly small. These findings seem to indicate that small fibers of the 8th nerve are connected with the M-cell only indirectly (Fig. 10). Excitatory electrical transmission. Since first described by Bartelmez (1915 ) the large club endings on the lateral dendrites have been known as a special type of synaptic terminal. Here the myelin sheath extends right into the ending and terminates only a few microns from the Mauthner cell surface. Therefore the unmyelinated axon does not extend appreciably at the endings. The synaptic junctions ofthis ending are constructed as an apposition membrane with no intervening material (Bodian,

,, ShL Fig. 3A. For legend see next page.

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Fig. 3. (A) Connection between swimbladder and labyrinth by the Weberian ossicles in Ostariophysi. Black = Weberian ossicles; Sch. = swimbladder; S.i. = sinus impar (perilymphatic space); C.tr. = canalis transversus (endolymphatic space); S. = sacculus; L. = lagena; U. = utriculus; H. r- brain (posterior part being removed); I. = incus; St. :stapes; M. = malleus. (From Von Frisch, 1936). (B) A transverse section of the nerve from the sacculus, fixed with osmic acid. Large fibers that innervate the anterior part of the saccular macula occupy the left half of the bundle, while small fibers that come from the posterior part occupy the right half. A cross section of the saccular macula appears in the lower left corner.

1952). Electron microscopic study by Robertson et a/. (1963) has disclosed that the synaptic membrane complex in cross section shows segments of closure of the synaptic cleft for 0.2 to 0.5 ,u long. These alternate with desmosome-like regions of about the same length in which the gap widens to 150 A. These findings are interpreted as suggestive of electrical transmission in the club endings. Furshpan (1964), who carried out an electrophysiological study quite independently of the electron microscopic study mentioned above, has elucidated detailed processes of transmission at this synapse. As mentioned above, the EPSP evoked in the M-cell in response to a stimulus to the ipsilateral 8th nerve is often constituted of a deflection that starts with a latency of about 0.6 msec. Besides this, however, a very early intracellular potential change is observed (Furshpan and Furukawa, 1962). Furshpan (1964) demonstrated that this early potential change is an EPSP transmitted electrically. The early EPSP appears with a latency of about 0.1 msec, and its over-all duration is only 1.5 to 2 msec, the rising phase being complete in 0.2 to 0.3 msec. lntracellular recordings made at a number of positions along the M-cell disclosed that this short latency EPSP was maximal in the distal half of the lateral dendrite (up to 50 mV), but declined steeply toward the cell body. This regional difference in the size of the early EPSP clearly References p . 69/70

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indicates that the origin of the potential must be located in the distal part of the lateral dendrite. When the EPSP remained above the threshold after having been reduced in size during electrotonic conduction from its site of origin toward the soma, an orthodromic spike was set up from the initial axon segment. I N H I B I T O R Y RESPONSES

Inhibitory input to the M-cell. The 8th nerve on the opposite side constitutes an inhibitory input to the M-cell. As mentioned above, it can easily be demonstrated that stimulation of the ispilateral 8th nerve gives rise to firing of the M-cell, whereas contralateral stimulation can suppress this firing. Moreover, the inhibition is mediated not only chemically but also electrically (Furukawa and Furshpan, 1963). Perhaps reciprocal actions of two 8th nerves are most important in considering the functional organization of the system. However the effect of contralateral stimulation is not purely inhibitory, but partly excitatory to the M-cell, and this prevents further elucidation of the detailed mechanism. Since this confused state seems to be attributable more or less to a crudeness of our technique in stimulating the nerve, it is hoped that clarification would ensue with an improved method. But there is an indication that the reciprocal action of an 8th nerve volley on M-cells, if it exists at all, would be 8 very subtle one. Although we tend to suppose that the 8th nerve on the side closer to the sound source would be stimulated earlier or more intensely than the opposite one, there is some doubt whether this is so in goldfish. As shown in Fig. 3A, the goldfish has only one swimbladder; the Weberian apparatus, which exists as a pair, transmits sound from the swimbladder to the perilymphatic space which is common to both sides; sound is then transmitted to one common endolymphatic space and finally to the sacculus on each side. These structural features of the fish's sound conducting svstem seem to suggest that the activity of the 8th nerve evoked by a sound would be almost identical on both sides. In accord witl' this reasoning is the fact that fishes lack the ability of localizing a sound source accurately (Lowenstein, 1957). Another inhibitory input to the M-cell is its own axon collaterals. Collateral inhibition of M-cells is well developed, and it can be evoked easily and without any mixture of excitatory action. Therefore our analysis of inhibitory actions to be described below was mostly concerned with this type of inhibition. Collateral inhibition of M-cells. Fig. 2A shows potential changes produced by antidromic stimulation of the M-cell. In the upper tract, recorded extracellularly from the vicinity of the axon hillock, an M-cell spike appears as a very large negative deflection. In the lower trace, recorded intracellularly from the soma, the antidromic spike appears as a positive deflection. Now the extracellular spike potential in the upper trace is followed by a positive wave of about 1 msec duration (see arrow). This extracellular positive wave is of maximal size (10-15 mV) in the region of the axon hillock and there is only a very small intracellular potential change that corresponds to this extracellular positivity. This means that most of the extracellular potential is actually impressed on the membrane of the axon hillock as a hyper-

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polarization : hence the potential is called extrinsic hyperpolarizing potential (EHP) (Furukawa and Furshpan, 1963). Fig. 2D is a schematic diagram illustrating the orientation of electromotive forces that give rise to the EHP around the axon hillock of the M-cell. The origin of the EHP is attributable to the activity of nerve fibers that converge to the axon hillock. In the lower trace of Fig. 2A the spike potential is followed by a slow depolarization. This is an inhibitory postsynaptic potential (IPSP) that appeared in a depolarizing direction. In the M-cell the equilibrium potential of the IPSP lies very close to the resting membrane potential, hence the IPSP is not normally recorded as a potential change. In Fig. 2A the IPSP was turned into a depolarization because of a migration of C1 ions into the cell from the microelectrode (Furukawa and Furshpan, 1963; Asada, 1963). Both the EHP and IPSP are evoked through the activity of M-cell axon collaterals. So far as it has been tested with antidromic or direct stimulation of the M-cell, collateral inhibition appears in association with the EHP and IPSP. These points will be described below. Collateral inhibition of M-cells is characterized by the fact that the effects are evoked, so to speak, in all-or-none manner. A single stimulation of M-cells evoked full-sized EHP and IPSP. Not only antidromic excitation, but orthodromic or direct excitation of the M-cell were almost equally effective in producing the EHP and IPSP. Moreover, the EHP and IPSP appeared on both M-cells even when only one of them was activated. Therefore, it was possible to study the inhibitory effects without being disturbed by previous activity if the inhibition were evoked by stimulating the contralateral M-cell only. Another interesting property of collateral inhibition of M-cells is that it is very easily fatigued. The EHP and IPSP appeared in their full size only when evoked at a rate of once per 2 sec, or less. If M-cells were stimulated at a rate of S/sec, for instance, there was no trace of the EHP or the IPSP. Generally, inhibitory effects are assessed by measuring how activities of the target structure are suppressed by inhibition. Upon testing the inhibition of M-cells in this way, we found that different phases of inhibition showed up depending on how the test-stimulus was applied. Three different tests were used : (1) antidromic stimulation; (2) direct stimulation; and (3) orthodromic stimulation. Only the effects of postsynaptic inhibition were detected by tests 1 and 2, whereas inhibitory actions on presynaptic elements could be detected, in addition to postsynaptic inhibition, by test 3. Tests with antidromic stimulation. Both conditioning and testing stimuli were delivered to the spinal cord. It was then found that the EHP and IPSP had quite different effects on the testing antidromic spike (Furukawa and Furshpan, 1963). The testing antidromic spike that was made to arrive near the peak of the EHP was often blocked and could not invade the axon hillock of the M-cell. In the experiment of Fig. 2C, the microelectrode was positioned in the neighborhood of the M-cell axon hillock. The conditioning spinal cord shock was adjusted to obtain the EHP in the absence of the spike, as occurred when only the opposite M-cell fired (CI). C2 shows the testing spike fired by itself. In C3, where the testing stimulus was preceded by the conditioning, the spike is absent. By recording the potential not only extracellularly but also intracellularly from the soma or from the axon, it was confirmed Rcfrrmcvs p. 69/70

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that the block of antidromic impulse during the EHP took place within the axon hillock. A block by the EHP was not always observed, however, because the occurrence of a block depends on the balance between the safety margin of antidromic invasion and the EHP size. Moreover, the all-or-nothing nature of a block made it difficult to analyze the result quantitatively. This difficulty was avoided by applying an artificial electrotonus to the axon hillock region of the M-cell. A microelectrode was positioned with its tip in the axon cap; a recording microelectrode was also inserted into the cap or into the M-cell itself, to monitor the spike. It was then found that the EHP could be imitated by relatively small currents applied by an extracellular electrode. The blocking of the spike took place at the same level of extracellular potential whether brought about by the EHP or by externally applied current pulses. It was also found that critical depolarization needed for an orthodromic firing of the M-cell was elevated during the EHP by exactly the same amount as the size of the EHP. These findings indicate that the EHP suppresses the excitability of the M-cell by an electrotonus extracellularly applied to the initial axon segment (Fig. 2D). A suppression of excitability during the EHP was demonstrated very clearly also with a direct stimulation of the M-cell as will be described later. The effect of the IPSP on the antidromic spike was quite different from that of the EHP. The antidromic spike was not blocked, but it was reduced in size when it arrived during the IPSP. The reduction was observed only with intracellularly recorded spikes (lower trace in Fig. 5A). Antidromic spike size was reduced during postsynaptic inhibition due to the special recording conditions in the M-cell. In the diagram of Fig. 2B, the spike potential recorded inside the soma (VZ)represents a potential drop produced by the action current across the input resistance of the cell (R4).Therefore, spike amplitude is reduced when the input resistance of the cell is decreased by the shunting effect of inhibitory postsynaptic action. The same reasoning explains why the extracellularly recorded spike is not reduced during the IPSP. On the contrary a reduction in the input resistance should be followed by an increase in the action current, and hence an increase in the extracellular spike amplitude. Such an increase was actually observed (Furukawa and Furshpan, 1963). Although the inhibitory synapses here concerned are distributed not only over the soma of the cell but on the base of the lateral dendrite, the reduction of antidromic spike size reflects more sensitively the conductance change that takes place across the soma membrane than that which occurs across the dendritic membrane. On the other hand, the EPSP that originates in the lateral dendrite receive a stronger suppression from the dendritic inhibition (see later). Tests with direct stimulation of the M-cell. The excitability change of the M-cell during inhibition was measured by stimulating the cell directly with cathodal pulses applied through an intracellular electrode (Fukami et al., 1965). This method of testing made it possible to compare the intensity of inhibitory action of the EHP quantitatively with that of the IPSP. Such a comparison was not possible with antidromic testings, for the effect of the EHP and the IPSP on antidromic spike is of a different nature. Nor was it possible with orthodromic testings. There are a few

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i i i; b io ;2 1’4 1’6 ;i3 r n s u Fig. 4. Excitability change of the M-cell during collateral inhibition as tested with direct stimulation. I direct stimulation of the M-cell through a K-citrate-filled microelectrode; I1 : similar to I, but a KCI-filled microelectrode was used. A and B - extra- and intracellular potential changes produced by conditioning spinal cord shock; C = amplitude of test antidromic spike (per cenf of the control), plotted against intervals between conditioning shock and peak time of antidromic spikes; D = excitability, i.e. reciprocals of threshold current strength. Pulses are shown as solid rectangles the length of which represents the pulse duration. (From Fukami el al., 1965). drawbacks in using orthodromic stimulation for testing the postsynaptic inhibition in the M-cell. ( I ) Results of orthodromic stimulation are modified greatly by an inhibitory mechanism acting also on the presynaptic side (see next section). (2) Excitatory inflow to the M-cell produced by an orthodromic stimulus is not correlated linearly with stimulus strength. This makes it difficult to express the excitability change in terms of change in the threshold. Direct stimulation with brief depolarizing pulses applied through the intracellular microelectrode is apparently free from these obstacles. The test pulse was delivered at various intervals after the conditioning spinal cord shock. The latter fired the M-cell antidromically thus giving rise to collateral inhibition. The excitability of the M-cell is then expressed by the reciprocal of the threshold needed to make the cell fire. Fig. 4 shows results of experiments carried out in this way. In the experiment represented in Fig. 4 I, an electrode filled with 2 M Kcitrate was used for intracellular recording and stimulation. Record A and B show respectively extra- and intracellular potential changes produced by the conditioning spinal cord shock. Since the strength of the conditioning spinal cord shock was so adjusted as to activate only the M-cell on the opposite side, no antidromic spike appeared in records A and B. The positive deflection in A represents the EHPI the References p . 69/70

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small positive notch recorded in B being its counterpart in the intracellular record. Although no IPSP appeared in the record (B) as a potential change, this by no means indicates the absence of the postsynaptic inhibitory action: it only means that the reversal potential of the IPSP is at about the same level as the resting potential. Fig. 4 I-C shows the antidromic test-spike height (expressed as per cent of the control) against intervals between the conditioning shock and the peak of each antidromic test-spike. In I-D the excitability (reciprocal of the current strength required for threshold stimulation of the M-cell) is plotted against time interval after the conditioning antidromic shock. The test-current pulses are shown as solid rectangles the length of which represents the pulse duration. As shown in Fig. 4 I-D, the excitability change of the M-cell took place with a rather complicated time course; there came first a brief depression of excitability having its peak at an interval a little less than 2 msec, then followed a long lasting suppression with peaks at about 2.7 msec and 3.8 msec of intervals. Two factors may contribute to the change in the excitability as measured with this method of direct stimulation: i.e. changes in the transmembrane potential and in the input resistance of the cell. The first peak of decreased excitability is clearly due to the EHP which hyperpolarized the membrane because it took place before any change in the input resistance of the M-cell started. On the other hand, the third peak of suppression seems to be attributable to a decrease in the input resistance of the cell, because the EHP was completed by then and also because there was no change in the inside potential of the M-cell as shown in B. This is also supported by the observation that the excitability was lowered approximately by the same amount as the reduction in the test-spike height. The curve drawn with a dotted line in Fig. 4 I-D represents the part of the lowered excitability attributable to a shunting effect of the inhibitory postsynaptic action. The difference between the continuous and dotted lines, having a time course very similar to the EHP, represents the suppression attributable to the EHP. In working with a microelectrode filled with 3 M KCI, it was usually observed that the IPSP became positive in sign with a lapse of time after the insertion of the electrode. This depolarizing IPSP grew for a while and eventually reached a steady level. An attempt was made to measure the excitability of the M-cell during such a depolarizing IPSP. Fig. 4 I1 shows an example of such an experiment. From the top downwards are shown the antidromic responses recorded extracellularly (A) and intracellularly (B), the antidromic test-spike height (C) and the excitability of the cell measured with direct stimulation (D). The solid rectangles plotted in Fig. 4 11-D are the reciprocals of the threshold current strengths expressed in per cent of the control. The initial brief dip in the excitability curve seen in D corresponds in its time course to the first peak of the E H P (record A). Therefore, the initial dip must be attributed to an increase in the transmembrane potential caused by the EHP.The suppression was then followed by an increase in the excitability. The transition took place very rapidly; the excitability reached its maximum (165 % of the control) within a few tenths of a millisecond. Thereafter it gradually returned to its original level, the en-

S Y N A P T I C INTERACTION OF MAUTHNER CELL

57

hancement of the excitability taking place with a time course similar to that of the depolarizing IPSP. This finding, taken together with the fact that no enhancement was observed when a K-citrate-filled electrode was used, strongly suggests that the increased excitability observed in this instance is attributable to the presence of a depolarizing IPSP. These results with direct stimulation of the M-cell showed that, although its duration was brief, the suppression during the EHP was very marked, being comparable to that during the IPSP, and that excitability changes during the IPSP depended on changes in the membrane conductance as well as in the membrane potential. bome simple formulae were advanced which describe excitability change in terms of conductance and potential changes, and they were found to coincide fairly well with the experimental observations (Fukami et al., 1965). Tests with orthodromic stimulation. So far as the results of antidromic and direct stimulation are concerned, collateral inhibition of M-cells can be fully accounted for as the sum of the effects of the EHP and the IPSP. This is no longer true, however, when the inhibition is tested by orthodromic stimulation. The effect of orthodromic stimulation was found to be suppressed very strongly by an inhibition different in its time course from either the EHP or the IPSP. This additional inhibition will provisionally be called the third type of inhibition. Existence of this third type of inhibition can easily be shown by comparing the time course of inhibitory effects on antidromic spikes and on orthodromic responses. But a much clearer demonstration was obtained after the action of procaine because it abolishes the EHP and the IPSP while conserving the third type of inhibition (Furukawa et al., 1963, 1964). In Fig. 5, potentials were recorded extracellularly from the axon hillock region (upper traces) as well as intracellularly from the soma of the same M-cell (lower traces). Although our analysis was based in the main upon a study of the intracellular records, the extracellular records also supplied useful information. A spinal cord shock was delivered as a conditioning stimulus at the beginning of each sweep, and this stimulus fired the M-cell antidromically. Following this, after various intervals, a test shock was delivered to the spinal cord again in A and B (antidromic test), and to the ipsilateral 8th nerve in C and D (orthodromic test). Control responses to the 8th nerve stimulus are shown to the left in C and D. As shown, stimulation of the 8th nerve gave rise to positive deflection in both upper and lower traces. The intracellularly-produced positive potential is the EPSP. The extracellular positivity in response to 8th nerve stimulation is a potential named by us ‘extracellular orthodromic response (EOR)’. Presumably this potential is produced by the activity of the 8th nerve fibers that converge to the axon hillock. In the lower traces of Fig. 5A and C, one can see the depolarizing IPSP. Both orthodromic and antidromic test responses were reduced in size during the IPSP. The antidromic test spikes showed a maximum reduction near the peak of the IPSP, but the inhibition of the EPSP became very intense suddenly at about the peak of the IPSP and was maintained thereafter. It is to be noted that a marked reduction of the EOR started at about the same time as the reduction of the EPSP. Fig. 5B and D show records 50 min after intramuscular injection of procaine Referenres p . 69/70

T. F U R U K A W A

58 antidromic rest

orthodromic test

Fig. 5. Acomparison of the effects of a preceding antidromic stimulation on the EPSP, EOR, and antidromic spike. Upper traces, extracellular potentials recorded from the vicinity of the axon hillock of the M-cell; lower traces, intracellular potentials recorded from the soma (A-D) and from the lateral dendrite (E). An antidromic stimulus to the M-cell was delivered at the start of each sweep. Testing antidromic stimuli (A and B) or orthodromic stimuli (C and D) were delivered with varied intervals after the conditioning antidromic stimulus. Shown to the left in C and D are control responses to an orthodromic stimulation (i.e. EOR, upper trace; EPSP, lower trace). A and C, before injection; B and D, 50 min and E, 120 min after i.m. injection of procaine (0.3 mg/g of body weight). (From Furukawa et a/., 1964).

(0.3 mg/g of body weight). The effect of the drug in this instance advanced slowly for about 40 min after the injection until it became stationary, the state shown in B and D being maintained almost indefinitely. As shown in these records, the EHP and the IPSP were removed by procaine. There remained a small IPSP which was evoked with a much longer latency than the IPSP in A and C. The size of the antidromic spike was reduced slightly with approximately the same time course as this late IPSP. Despite these rather drastic reductions in the size of the IPSP and its effect on the antidromic spike size, the inhibitory effect as tested by orthodromic stimulus remained almost unchanged as shown in Fig. 5D. The results of this experiment are plotted in Fig. 6 I. The change in amplitude of the antidromic spike (filled circles), the EPSP (open circles), and the EOR (crosses) is plotted against the interval between the conditioning shock and the peak of each

SYNAPTIC INTERACTION O F MAUTHNER CELL

1

0

1

2

% loo

4

1

I

6

8

59

I

10msec

--*TI spike

40

20 0

2

4

6

10 msec

Fig. 6 (I) Plots of results shown in Fig. 5A-D. Amplitudes of the antidromic spike, EPSP, and EOR relative to amplitudes of control responses are plotted against intervals between conditioning stimuli and the peak of each response. A = before injection of procaine; B = 50 min after injection. Note that early reduction of the antidromic spike and EPSP is absent in (B). (From Furukawa et a/., 1964). (11) A comparison of the effect of a preceding antidrornic stimulation on antidrornic spike size. A = recorded inside the soma; B = recorded inside the lateral dendrite of the same M-cell.

test response. In A (before the injection of procaine) the reduction of antidromic spike size started with a latency of 3 msec, whereas the reduction of the EOR started at about 5 msec. The reduction of the EPSP, though it started at about the same time as the reduction of the antidromic spike, became much more intense thereafter in coincidence with the start of the EOR suppression. After the injection of procaine early reduction of the antidromic spike and the EPSP were abolished as shown in B, and all the inhibitory effects started at about the same time with a delay of about 5 msec. The reduction of antidromic spike size was much weaker in B, whereas the reduction of the EOR and the EPSP was maintained almost unchanged. These findings indicate the presence of an inhibition whose effects appear predominantly on the EPSP and EOR. At first we thought that suppression of the EPSP was mostly attributable, as suggested by the suppression of the EOR, to an inhibitory action on the 8th nerve. We also observed that other signs of 8th nerve activity were suppressed during the inhibition. Hence the presence of an inhibitory mechanism similar to presynaptic inhibition in the cat spinal cord (Frank and Fuortes, 1957; Eccles et al., 1961. 1962a, b) or in the crayfish neuromuscular junction (Dudel and Kuffler, 1961: Dudel, 1963) was postulated (Furukawa et al., 1963). We know now, however, that a substantial part of the third type of inhibition can be attributed t o the postsynaptic inhibition that takes placdin the lateral dendrite. Dendritic inhibition. There are grounds for believing that a t least a part of the third type of inhibition is attributable to the postsynaptic inhibition that takes place in the lateral dendrite. According to this interpretation, the discrepancy such as observed in Fig. 5C between the time course of the somatic JPSP and the reduction in the size of theEPSPcan be attributed to the fact that theEPSPis reduced in size by an increasRcfcrencrs p. 69/70

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ed conductance taking place in the soma as well as in the dendritic membrane, whereas the IPSP recorded from the soma represents only the conductance change taking place in the somatic membrane. In the experiment of Fig. 5 , the recording electrode was withdrawn from the soma about 2 h after the injection of procaine and reinserted into the lateral dendrite of the same M-cell at a point about 250 p from the axon hillock (resting potential, 75 mV). A large depolarizing IPSP was then observed in response to an antidromic stimulus (Fig. 5E). The latency and time course of this IPSP coincide well with those of the third type of inhibition. Since this IPSP was recorded from the dendrite at the time when no IPSP was recorded from the soma, it must have its origin in the dendrite. The amplitude of the antidromic spike in the lower trace of E is unduly small (cf. Fig. 6 11-B). The electrical connection between the soma and dendrite seemed to have been impaired in this experiment while we were trying to put the electrode into the lateral dendrite. Usually, antidromic spike in the dendrite at 250-300 p from the axon hillock was 112 to 1/3 as large as the spike in the soma as shown in Fig. 6 11-B. There is also a difference in the spike duration, but it is relatively small. Fig. 6 I1 illustrates another instance in which potentials recorded from the soma (A) were compared with those from the lateral dendrite (B). It can be seen that the dendritic IPSP in this instance also started with a longer latency than the IPSP in the soma, and was maintained for a longer period. An interesting aspect in these observations is that the depolarizing IPSP in each record predominantly reflects only the permeability change taking place in the part of the cell impaled by the electrode. As described before, an elevated intracellular concentration of CI ions that had migrated from the electrode is responsible for the depolarizing IPSP. The observations mentioned above indicate, however, that an increase of the intracellular concentration occurs only in the neighborhood of the electrode: namely, when the electrode impales the soma, for instance, the concentration of C1 ions would be increased in the soma but would remain largely unchanged in the lateral dendrite. Perhaps a rapid leakage from the interior of the cell in comparison with the diffusion velocity along the long axis of the cell would be responsible for this localized change in the CI ion concentration. In the soma, as shown in Fig. 6 11-A, the reduction in the antidromic spike occurs approximately with the same time course as the IPSP. We have already discussed this (see Fig. 41I-B, C and Fig. 5A). But such a parallelism between the timecourse of the IPSP and the reduction in the size of the antidromic spike cannot be seen in Fig. 6 11-B where the potential was recorded from the dendrite. Test-spikes in this record are reduced in size well before the start of the dendritic IPSP. That is, the amplitude of antidromic test-spikes was reduced, when recorded from the dendrite, not only during the dendritic IPSP but also during the somatic IPSP. Another finding which characterizes the dendritic inhibition is the presence of a spontaneous activity. A vigorous spontaneous inhibitory activity was almost always found in the lateral dendrite. The activity often occurred in a form of bursts that came at an irregular interval without any noticeable rhythm. The amplitude not only

61

SYNAPTIC INTERACTION O F MAUTHNER CELL

of the antidromic spike but of the EPSP was markedly reduced (up to 112) during such a burst of activity of dendritic inhibition. This resulted in an irregular fluctuation in the size of the antidromic spike and the EPSP in the dendrite. Model experiment on the dendritic inhibition. In order to confirm the conclusion in the preceding section, it would be desirable to carry out a systematic exploration. A suitable experiment would require that intracellular potentials be recorded at various points along the soma and the lateral dendrite, and that the effects of somatic and dendritic inhibitions be compared at each point. Actually, this experiment is not easy because: (1) chances are large that the dendrite is damaged by repeated insertions of the electrode; and (2) it is usually not possible to evoke either somatic or dendritic inhibition in isolation. In view of these difficulties an experiment on a model neuron was planned. We started by modifying the drawing of the M-cell by Bodian(Fig. IA). The schematized neuron shown in Fig. 7A was drawn as a stretched structure and was made larger than the original drawing by a factor of 10/7 in order to compensate for the shrinkage. It is constituted of 24 sections, each having a length of 50 ,u and a width that matches the average width of the corresponding part in the original drawing. Longitudinal and membrane resistances of each section were then calculated. In making these calculations, specific resistances of the membrane and the axoplasma A

0

ventral dendrite

-
-

RI 14

lateral dendrite

t loterol dendrite

R I 15

R i 16 Ri 17

+

Ri 18 Ri 19

Fig. 7. (A) A schematized M-cell consisting of 24 sections of equal length (50 p). (B) Circuit diapram of a model M-cell designed for elucidating the difference between the effects of somatic and dendritic

inhibitions. All resistanceswere scaled down to one hundredth of the original value. Rm 1-11 = 4.5 kn; Rm 12 and Rm I5 = 10 k n ; Rrn 13 and Rm 14 = 6 klR; Rm 16 and Rm 19 = 17 kR; Rm 17 and Rm 18 = I9 k a ;Rm 2&24 = 4.2kR; Ri 12 and Ri I5 = 0.4kR; Ri 13 and Ri 14 = 0.1 kR; Ri 16 and Ri 19 = 1 k a ; Ri 17 and Ri 18 = 1.2 Rm 12-Rm 15 and Rm 17-Rm 19 may be switched down to 1/2, 1/3. and 1/4 of the values in the above to simulate the shunting effect of inhibitory postsynaptic action. See text for details.

a.

References p. 69/70

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T. F U R U K A W A

were taken as 53 Q/cm2 and 63 Q/cm2 respectively. The results of these calculations were used in making an equivalent circuit of the M-cell that is constituted of 24 sections of resistance network. The input resistance (at the soma) of this equivalent circuit was 1.1 x 105 Q and was very close to values actually observed on the M-cell ( I .2 x 105 Q, Furshpan and Furukawa, 1962). This in turn legitimated the use of specific resistance values mentioned above. The model neuron actually constructed with electrical resistors and rotary switches consisted of 10 sections as shown in Fig. 7B. In this figure, Rm 12-Rm 19 and Ri 12Ri 19 represent respectively the membrane resistance and the longitudinal resistance of each section that bears the same number in Fig. 7A. The input resistance of the ventral dendrite viewed from section 12 is represented by Rm 1-1 1, and that of the lateral dendrite viewed from section 19 by Rm 20-24. All resistances used in this actual model were scaled down to one hundredth of the original values (see legend of Fig. 7). Membrane capacitance was not taken into consideration in the present model. Because the membrane time constant of the M-cell is very short (about 0.5 msec), omission of the capacitative component should not produce any serious error. In order to simulate an increased membrane conductance during the somatic and dendritic inhibition, provisions were made that values of Rm 12-Rm 15 and Rm 17-Rm 19 may be reduced to 1/2,1/3 and 1/4 of their normal values by changing the positions of rotary switches. Measurements on this model neuron were made as follows. Brief electrical pulses were injected through a lo5 Q resistor at either of two points: in order to simulate an antidromic spike it was injected at the point marked 0 p, whereas to simulate the EPSP generated in the distal part of the lateral dendrite it was injected at the point marked 300 p. In either event potentials were measured at 7 points along the network as shown in the figure. Results of such measurement are summarized in Fig. 8. In this figure, potential sizes are plotted against the distance from the center of the soma as abscissae. Electrical pulses were injected into the soma in A and C and into the dendrite in B and D (see arrows). Therefore curves in A and C show decrements in the antidromic spike in spreading electrotonically from the soma toward the lateral dendrite, while B and D represent electrotonic decrement of the EPSP in the reversed direction. Open circles plot the decrement under a normal condition, while filled circles, crosses, and small dots plot decrements observed when membrane resistances were decreased to '12, and of the normal value in the region indicated by a thick base line. Therefore, A and B correspond to the somatic inhibition, and C and D to dendritic inhibition. Results shown in Fig. 8 can be summarized as follows. (1) Dendritic inhibition, as shown in C, has only a negligible effect on the antidromic spike recorded in the soma. (2) The EPSP recorded in the dendrite receives only a slight influence from the somatic inhibition. (3) The antidromic spike, recorded either from the soma or from the dendrite, is reduced in size by the somatic inhibition. (4) The EPSP, recorded either from the soma or from the dendrite, is reduced in size by the dendritic inhibition. Thus it is clear that situations observed in the M-cell under various conditions were fairly faithfully reproduced on the model e x p e r i m ~ ~ t .

SYNAPTIC INTERACTION OF MAUTHNER CELL

63

0 10 -

0

I

.O

r

P

100

D 10

08 -

08

06 -

06

-

0.4

04

02

-

2 200 =P 0

100

Fig. 8. Results of the model experiment. Potentials recorded on the model are plotted against the distance from the axon hillock. Current pulses were injected at 0 ,LA in A and C, and at 300 p in R and D (see arrows), hence the former simulates electrotonic decrement of the antidromic spike from the soma to the dendrite, whereas the latter simulates electrotonic decrement of EPSP in the reversed direction. Open circles, control; filled circles, crosses, and small dots, effects of various intensities of somatic inhibition (A and B) and dendritic inhibition (C and D).

Frank and Fuortes (1957) produced convincing evidence of spinal presynaptic inhibition, evidence that was thereafter greatly developed by Eccles and others (Eccles, 1964). But Frank (1959) proposed an alternative explanation to his finding. He argued that if an inhibitory action were exerted far out on the dendrite of the motor neuron, the EPSP depression could occur without any trace of the inhibitory influence itself as detected by a microelectrode in the motor neuron soma. He designated the phenomenon ‘remote dendritic inhibition’. It has been shown above that the dendritic inhibition in the M-cell produces the kind of inhibition expected by him. Inhibition at the receptor site. Afferent discharges in the 8th nerve fibers were found to be suppressed during collateral inhibition of M-cells. Fig. 9A and B show two instances in which unit discharges were recorded from large fibers that innervate the sacculus (CJ Fig. 3B). In either instance, a stimulus sound of about 600 c/s was used. I t was recorded with a dynamic microphone placed close to the animal, and was displayed on the oscilloscope. In A, the upper trace shows afferent impulses that are set up at a rate exactly twice that of the sound, monitored in the lower trace. About half of the large fibers in the sacculus showed this type of response. B1 shows impulses that were set up at the same rate as the sound and B2 shows that the discharge was absent when the sound was preceded by a conditioning antidromic shock to M-cells. References p. 69/70

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Fig. 9. Unitary action potentials of 8th nerve fibers. (A) Spikes set up at twice the frequency of the sound; lower trace, stimulus sound (about 600 c/s) recorded with a microphone placed beside the animal. (B) 1 = spikes set up at the same rate as the sound (4); 2 = suppression of spike discharge during collateral inhibition of M-cells; 3 = monitor of the M-cell spike.

In this instance, a second microelectrode was placed in the vicinity of the axon hillock to monitor the action potential in the M-cell (B3). A suppression such as shown in B2 was observed only when the spinal cord shock was strong enough to fire the M-cell antidromically. Small ripples appear in B2. A similar suppression was observed in small fibers. Judged from its latency and duration, it seems appropriate to regard the inhibition at the receptor site as a part of the third type of inhibition. An inhibitory action of the olivo-cochlear bundle on sensory fiber terminations and on the hair cells of the organ of Corti is well known (Galambos, 1956).The presence of a similar efferent control mechanism in the fish’s ear was anticipated, for endings possessing properties of efferent fibers have been demonstrated in the vestibular neuroepithelium of fishes (Lowenstein et al., 1964; Flock, 1964). Our experiments showed that this was indeed so. Efects o j strychnine on collateral inhibition of M-cells. One of the remarkable effects of strychnine at a very low concentration is its ability to block postsynaptic inhibition in the vertebrate central nervous system (Eccles, 1964). Although some

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65

exceptions to this generalization have been reported (e.g. Andersen et al., 1963), it is still an interesting drug to be tested on M-cells. When a small amount of strychnine (2-5 pg/g of body weight) was administered intramuscularly into the tail of a goldfish, responses of the M-cell to ipsilateral 8th nerve stimulation were augmented. The time course of the EPSP became much more prolonged, and a repetitive firing of the M-cell occurred superimposed on such a sustained depolarization. Periodic firing of the cell then ensued during progressive action of the drug. A block of inhibition occurred along with these changes. A test of inhibition was made in a way similar to that employed in testing the effect of procaine (see Fig. 5 and 6 I). It was found that the inhibitory effects as detected by antidromic stimulation (reduction of antidromic spike size) and those as detected by orthodromic stimulation (reduction of the EPSP and EOR) were removed with approximately the same time course after an injection of strychnine: namely strychnine removed the IPSP and the third type of inhibition in parallel. On the other hand the EHP was less susceptible to the effect of strychnine than the two other types of inhibition (Furukawa et al., 1964). As to the mechanism of action of strychnine, it is postulated in analogy with the action of curare at the cholinergic junction that the drug would block postsynaptic inhibition by combining with the receptor in competition with the inhibitory transmitter substance (Eccles. 1964). It is therefore suggested that the IPSP and the third type of inhibition in M-cells may be mediated by chemical mechanisms similar to those involved in postsynaptic inhibition in cat spinal motor neurons. It is also explicable from the same reasoning that the EHP, and hence the electrical inhibition, are more resistant to the action of strychnine. DISCUSSION

We have described various types of synaptic action that impinge on the Mauthner cell. There exist excitatory and inhibitory synapses that are based on electrical mechanisms in addition to those based on more conventional chemical mechanisms. To add to the complexity of the system these different types of synapses all have different sites of action. As to the excitatory synapses, electrical synapses are on the distal part of the lateral dendrite, while chemical synapses cover wider areas of the cell (cf. Diamond, 1963). Inhibitory synaptic action is mure complicated, for we know that it takes place at 5 different loci : ( I ) electrical inhibition that takes place at the axon hillock; (2) chemical postsynaptic inhibition of the soma (somatic inhibition); (3) chemical postsynaptic inhibition of the lateral dendrite (dendritic inhibition); (4) inhibition of 8th nerve activity taking place at the receptor site; (5) inhibition of 8th nerve activity taking place inside the medulla. Inhibitions of ( I ) and (2) suppress the activity of the M-cell as a whole, while inhibitions of (3)-(5) specifically suppress the excitatory input from the 8th nerve, leaving other excitatory inputs unobliterated. Fig. I0 shows a hypothetical diagram of neuronal organization subserving collateral inhibition of M-cells. The inhibitory effect is exerted upon the M-cell and upon Referenres p. 69/70

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66 blocked by fat iauc

.

Vest ibular

Sacculus halr cell

Iefr M-axon

Fig. 10. Supposed neuronal organization for collateral inhibition in the M-cell. E = excitatory junction; I = inhibitory junction. Two kinds of 8th nerve fibers, and 4 kinds of interneurons are shown. Three possible terminations (a-c) of N4 are indicated. It is assumed that, besides releasing chemical transmitter substances from their terminals, Nz axons and secondary 8th nerve fibers enter into some close relationship with the axon cap and subserve the generation of the EHP and EOR respectively. See text for further details.

the 8th nerve. There are two kinds of fibers in the 8th nerve: large fibers terminate directly on the distal part of the lateral dendrite of the M-cell with club endings, and small fibers are distributed, after being relayed in the vestibular nucleus, over the axon hillock and the soma. On the peripheral side these 8th nerve fibers are connected with hair cells of the saccular macula. In Fig. 10, the large fiber is joined with the hair cell by a chalice. Tt was drawn by analogy from the structure in the vestibular sensory epithelia of mammals such as guinea-pig, rat and cat (Wersall, 1960). No detailed morphological study has been made on saccular macula of the Ostariophysi. We assume that collateral inhibition is mediated by four interneurons, N1 to N4. The role of generating the EHP and postsynaptic inhibition of the M-cell soma is, for convenience, attributed to a common inhibitory neuron Nz. The axon of N2 is assumed to reach the axon hillock region and enter into some close relation 'with the axon cap before ending on the soma of the M-cell. N4 is another inhibitory neuron to which is allocated the role of exerting the third type of inhibition. Three possible sites of termination for the axonal branches of N4 are drawn in the diagram. Branch a, that terminates on the base of the lateral dendrite, is associated with the dendritic inhibition. Branch b, that terminates on the nerve chalice and hair cell, is associated with the inhibition at the receptor site. Branch c ends on the soma of the secondary 8,h nerve cell. As indicated by the suppression of the €OR (Fig. 5C), some part of the third type of inhibition in the M-cell must be attributed to an inhibition acting on the presynaptic element. There is a possibility that a mechanism similar t o presynaptic inhibition in the spinal cord (Eccles, 1964) or in crayfish neuromuscular junction (Dudel, 1963)

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67

is involved in the third type of inhibition. But we know that this third type of inhibition is totally blocked by strychnine. This clearly indicates that inhibition of the presynaptic element of M-cells is different in nature from presynaptic inhibition in the cat spinal cord. There are at least two possibilities. It might be supposed that the activity of the 8th nerve fibers is suppressed by the inhibitory synapse of axo-axonal type that exerts its action through a mechanism similar to the usual postsynaptic inhibition. Another possibility is that presynaptic inhibition of M-cells might simply be an inhibition in the vestibular nucleus where many of the 8th nerve fibers synapse. Fig. 10 was drawn according to the latter view. Therefore activity of non-synapsing 8th nerve fibers does not receive an inhibitory effect except a t the receptor site. N3 is interposed between N1 and N4 in order to explain the longer latency of inhibition exerted by N4. Finally, N1 is the interneuron which receives fibers from M-cell axon collaterals on both sides. Now the effects of some drugs, such as strychnine and procaine, and of repetitive stimulation may be explained by allocating different susceptibility t o each synapse. Strychnine would block the postsynaptic inhibitory action at synapses marked I in the diagram. On the other hand, the action of procaine is most conveniently explained by a block at the E-synapse between N1 and N2. The E-synapse between N1 and N3 and that between N3 and N4, however, are more resistant to the action of procaine. The fatiguability is shared by all three types of collateral inhibition of the M-cell. Therefore, this property is best attributed to the E-synapse between axon collaterals and N1. Possible functional meaning of various types of synaptic activity in the M-cell. We have succeeded in elucidating to a certain extent various types of synaptic activity in the M-cell. It now remains to discuss the possible functional meaning of this complicated system. By microelectrode recording from Mauthner axons of Protopterm (lungfish), Wilson (1959) showed that a spike was obtained only as a startle response elicited by a severe jar to the aquarium. This was followed by a sudden flip of the tail. I t seems then that the Mauthner cell system functions as an escape mechanism. Certain structural features of the M-cell system seem t o aim at a very fast response which is required for the escape. To begin with the input side, myelinated fibers of a specially large size (I 0-1 5 p, see Fig. 3B) are employed to connect the hair cells in the anterior part of the sacculus to the lateral dendrite of the M-cell. The distance connected by these fibers is very short; in fishes such as those used in the present study it is perhaps not more than 5 mm. This corresponds to a conduction time of only a few tenths of a millisecond if a velocity of 20-30 m/sec is assumed (Tasaki, 1953). Moreover, as mentioned in the results, impulses in these fibers are set up with a short delay of 0.5 msec or so after the arrival of sound. Some of the fibers can respond to both compression and rarefaction phases of the sound : this prevents an additional delay from occurring due to the phase relation of the sound (Fig. 9A). On the output side, Mauthner’s large myelinated axon (d,40 p ) conducts impulses at a velocity as fast as 80-100 m/sec (Furshpan and Furukawa, 1962). Impulses are then transmitted to spinal motor fibers via special axo-axonal synapses (Bodian, 1952). From these considerations it would be reasonable to assume that the electrical excitatory synapses R ~ f e r e n c ~p. s 69/70

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that interpose between 8th nerve fibers and the Mauthner’s dendrite may aim at a transmission with a minimal synaptic delay. We know now that a difference of a tenth of a millisecond counts in this system. It may then be supposed that the electrical inhibition of M-cells has its meaning in the fact that it can be effected without any synaptic delay. An inhibition must act rapidly in order to be in time because of a very fast excitatory action. It is to be noted in this connection that the EHP can be evoked not only by collateral inhibition but also by stimulation of the contralateral 8th nerve. We cannot fully appreciate the functional meaning of collateral inhibition of the M-cell. One may suppose, however, that its function will be to limit the duration of activity of M-cells that were initiated as a startle response. It is not difficult t o suppose that a startle response consists of a disorganized activity of M-cells. Hence it would be profitable for the animal should it stop immediately after a few initial actions. It is interesting in this connection that the duration of the third type of inhibition is much longer than inhibitions that act on the axon hillock and the soma. This means that the acoustic input that causes a startle response is obliterated for a little while even after the M-cell becomes responsive again to excitatory volleys that impinge on the soma or on the ventral dendrite. Then the excitatory influences that are associated with an organized activity of the M-cell would be transmitted on the soma and on the ventral dendrite. Such orderly activities of M-cells would not be suppressed by collateral inhibition because of a fatigue in the neuron chain (Fig. 10). SUMMARY

1. Results of neurophysiological studies on the goldfish Mauthner cell are described. Emphasis was placed on description of various types of excitatory and inhibitory synaptic actions that impinge on this fish neuron. Since synapses of different structure are distributed on different parts of the M-cell, it presents a suitable material for elucidating the function of synapses in relation to their structure and spacial location in the cell. Another interesting aspect with the synapses in the Mauthner cell is the presence of excitatory and inhibitory synapses that are mediated electrically besides those mediated chemically. 2. Large direct 8th nerve fibers terminate on the distal part of the lateral dendrite with club endings. Excitatory action at this synapse is transmitted electrically. The origin of these large fibers was traced to the anterior part of the saccular macula. 3. Collateral inhibition is very well developed in the M-cell. Inhibitory effects were tested by delivering antidromic and orthodromic stimuli to the cell, by stimulating the cell directly with electrical currents, by delivering sound stimuli to the animal, and finally by observing the drug effects. Such analyses led us to conclude that the inhibition comprises 5 different inhibitory processes, which include electrical inhibition at the axon cap, chemical inhibition that takes place separately at the soma and at the lateral dendrite, inhibition at the sound receptor, and so forth. 4. Dendritic inhibition is perhaps a new type of inhibition. It suppresses only the

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excitatory actions that impinge on the dendrite on which the inhibition acts, while excitatory actions that impinge on the soma or other dendrites suffer almost no effect. 5. Neuronal organization subserving these various types of synaptic activities and their possible functional significance are discussed. REFERENCES ANDERSEN, P., ECCLES, J. C., LBYNING,Y., AND VOORHOEVE, P. E., (1963); Strychnine-resistant inhibition in the brain. Nature (Lond.), 200, 843-845. ARAKI,T., AND OTANI,T., (1955); Response of single motoneurons to direct stimulation in toad’s spinal cord. J. Neurophysiol., 18,472-485. ASADA,Y.,(1963); Effects of intracellularly injected anions on the Mauthner cells of goldfish. Jap. J. Physiol., 13, 583-598. BARTELMEZ, G. W., (1915); Mauthner’s cell and nucleus motorius tegmenti. J. comp. Neurol., 25, 87-128. BECCARI,N., (1907); Ricerche sulle cellule e fibre del Mauthner e sulle lor0 conessioni in Pesci ed Anfibii. Arch. ital. Anar. Etnbriol., 6, 660-705. BODIAN,D., (1937); The structure of the vertebrate synapse. A study of the axon endings on Mauthner’s cell and neighboring centers in the goldfish,. J. con~p.Neurol., 68, 117-159. ~ D I A N D., , (1952); Introductory survey of neurons. Cold Spr. Harb. Symp. quanf. Biol., 17, 1-13. DIAMOND, J., (1963); Variation in the sensitivity to GABA of different regions of the Mauthner neurone. Nature (Lond.), 199, 773-775. DUDEL, J., (1963); Presynaptic inhibition of the excitatory nerve terminal in the neuromuscular junction of the Crayfish. Pflugers Arch. ges. Physiol., 277, 537-557. DUDEL,J., AND KUFFLER,S. W., (1961); Presynaptic inhibition at the crayfish neuromuscular junction. J. Physiol. (Lond.), 155, 543-563. ECCLES,J. C., (1964); The Physiology of Synapses. Berlin, Springer-Verlag. ECCLES,J. C., ECCLES,R. M., AND MAGNI,F., (1961); Central inhibitory action attributable to presynaptic depolarization produced by muscle afferent volleys. J. Physiol. (Lond.) , 159, 147-1 66. ECCLES,J. C.,MAGNI,F., AND WILLIS,W. D., (1962a); Depolarization of central terminals of Group I afferent fibres from muscle. J. Physiol. (Lond.), 160, 62-93. ECCLES,J. C.,SCHMIDT,R. F., A N D WILLIS,W. D., (1962b); Presynaptic inhibition of the spinal monosynaptic reflex pathway. J. Physiol. (Lond.), 161, 282-297. FLOCK, A., (1964); Structure of the macula utriculi with special reference to directional interplay of sensory responses as revealed by morphological polarization. J . Cell Biol., 22, 413-431. FRANK,K.,(1959); Basic mechanisms of synaptic transmission in the central nervous system. I R E Trans. med. Elecrr., ME-6, 85-88. FRANK,K.,AND FUORTES,M. G. F., (1957); Presynaptic and postsynaptic inhibition of monosynaptic reflexes. Fed. Proc., 16, 39-40. FUKAMI, Y., FURUKAWA, T., AND ASADA,Y., (1965); Excitability changes of the Mauthner cell during collateral inhibition. J . gen. Physiol., 48, 581-600. FURSHPAN, E. J., (1964); ‘Electrical Transmission’ at an excitatory synapse in a vertebrate brain. Science, 144, 878-880. FURSHPAN, E. J., AND FURUKAWA, T., (1962); Intracellular and extracellular responses of the several regions of the Mauthner cell of the goldfish. J. Neurophysiol., 25, 732-771. T., FUKAMI, Y., AND ASADA,Y.,(1963); A third type of inhibition in the Mauthnercell FURUKAWA, of goldfish. J. Neurophysiol., 26, 759-774. FURUKAWA, T., FUKAMI, Y., A N D ASADA,Y., (1964); Effects of strychnine and procaine on collateral inhibition of the Mauthner cell of goldfish. Jap. J. Physiol., 14, 386399. FURUKAWA. T., AND FURSHPAN, E. J., (1963); Two inhibitory mechanisms in the Mauthner neurons of goldfish. J. Neurophysiol., 26, 140-176. GALAMBOS, R., (1956); Suppression of auditory nerve activity by stimulation of efferent fibers to cochlea. J. Neurophysiol., 19, 424-437. O . ,(1957); The acoustico-lateral system. The Physiology ofFishes, Vol. 2. M. E. Brown, LOWENSTEIN, Editor. New York, Academic Press (pp. 155-186). LOWENSTEIN, 0.. OSBORNE, M. P., A N D WERSALL, J., (1964); Structure and innervation of the sensory

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epithelia of the labyrinth in the Thornback ray (Raja clavata). Proc. roy. SOC. B, 160, 1-12. OTSUKA,N., (1962); Histologisch-entwicklungsgeschichtliche Untersuchungen an Mauthnerschen Zellen von Fischen. Z . Zellforsch., S8,33-50. OTSUKA. N., (1964); Weitere vergleichend-anato:nische Untersuchungen an Mauthnerschen Zellen von Fischen. Z . Zelvorsch., 62, 61-71. RETZLAFF, E., (1957); A mechanism for excitation and inhibition of the Mauthner's cells in teleosts. A histological and neurophysiological study. J. comp. Neurol., 107, 209-225. ROBERTSON, J. D., BODENHEIMER, T. S., AND STAGE, D. E., (1963); The ultrastructure of Mauthner cell synapses and nodes in goldfish brains. J. Cell Biol., 19, 159-199. TASAKI, I., (1953); Nervous Transmission. Springfield, Thomas. VON FRISCH,K., (1936); Uber den Gehorsinn der Fische. Biol. Rev., 11, 210-246. WERSALL, J., (1960); Electron micrographic studies of vestibular hair cell innervation. Neural Mechanisms of the Auditory and Vestibular Systems. G . L. Rasmussen and W. Windle, Editors. Springfield, Thomas (pp. 247-257). WILSON,D. M., (1959); Function of giant Mauthner's neurons in the lungfish. Science, 129, 841.

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Neural Mechanism of Hearing in Cats and Monkeys YASUJI KATSUKI Department of Physiology, Tokyo Medical and Dental University, Tokyo (Japan)

I. INTRODUCTION

When the endocochlear mechanism of hearing was disclosed by Von Bektsy (1943) and later confirmed by Tasaki et a/. (1952), further information on the neural mechanism of hearing was desired, because both Von Helmholtz’s (peripheral) theory ( I 862) and Rutherford’s (central) theory (1 886) were shown not to be completely valid. Just at that time the superfine capillary microelectrode technique enabled us to explore the unitary activity of nerve cell in the brain of higher animals. The author therefore planned to study the functional organization of nuclei at different levels in the classical auditory system step by step, by single neuron analysis. The single neuron analysis in that field had already been performed at the cochlear nuclei and other regions by Galambos and his collaborators (Galambos, 1952; Galambos et a/., 1943, 1952, 1959; Rose and Galambos, 1952; Rose et a/., 1959). Tasaki (1954) also succeeded in recording the response of primary auditory neurons to sound stimulation in guinea-pigs. The present author mainly used cats as experimental animals; under light anaesthesia with nembutal various regions of the skull were opened to expose a part of brain which it was desired to study. For some experiments monkeys were used. Guinea-pigs were used only for the study of microphonic potential. The electronic apparatus for recording the responses of neurons to sound stimulation included a cathode follower preamplifier and a high-gain main RC or DC amplifier. Most records were photographed on a running film through a conventional oscilloscope, e.g. Tektronix 502, while an automatically driven sound producing apparatus was working. For the determination of thresholds of a neuron for sounds repetitive photographs were taken once per 1 or 2 sec. The sounds used were mostly short ones, tone bursts with ditrerent durations, different frequencies ranging from 30 to 20,000 c/s or higher and different intensities, 0 dB being 80 dB above the average human threshold (0.0002 dyne per sq cm) except where otherwise indicated. Long continuous pure tones were also used. When the effect of interaction of two or more sounds was studied, a pure tone was mostly used as a background sound. In order to finish successive experiments of measuring thresholds of a neuron for sounds with various frequencies within as short a time as possible, an automatic sound producing apparatus was designed, using a rotatory switch driven by a motor, because the recording time of responses from one and the same neuron was limited. Rr/rrmr.cr p. 94- 97

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The animal was usually put in a sound-proof room, where the temperature was regulated at around 28" and the sound stimuli were usually delivered to it in a free field, though the sound was, if necessary, sent separately to each ear through headphones. Since all the experiments conducted by our group during several years have been performed under the same physical conditions, all results shown below are comparable with each other, except where otherwise indicated. II. M E C H A N I S M O N T H E C O C H L E A R N E R V E

( 1 ) Coding in the cochlea

As described above the movement of the basilar membrane in the cochlea observed by Von BCkisy (1943) was a traveling wave elicited by a sound wave, by which Corti's organ on the membrane is vibrated. It has generally been accepted that the deformation of hairs on the top of hair cells caused by the hair cell movement produces the cochlear microphonic (CM) potentials. Recent electron microscopical studies by several authors (Engstrom et al., 1962; Flock et al., 1962) suggest a characteristic orderly arrangement of two kinds of hairs, many stereocilia and a single kinocilium on the outer hair cell, though this situation has not yet been clarified on the inner hair cell of the cochlea. The exact mechanism of the CM production in the cochlea remains obscure. The mechanism of the initiation of impulses at the ending of the cochlear nerve fiber which makes contact with the base of the hair cell, is so far not clear; there are two hypotheses, one is electrical and the other chemical. According to the former hypothesis the electric current flow due to the change of membrane potential of the hair cell initiates impulses at the dendritic ending of the primary cochlear neuron, whereas the latter hypothesis proposed by electron microscopists (Engstrom and Wersall, 1958; Schuknecht et a/., 1959; Smith and Sjostrand, 1961) insists on a chemical transmission between the hair cell and nerve endings where they make synaptic contact. The electron microphotographs of hair cells represent the structure which is similar to the ordinary synapse observed in neurons of lower as well as higher animals. They also distinguish two different types of dendritic endings, small and large. The former contains vesicles sparsely whereas in the latter they are distributed densely. Furthermore at the region of hair cell that faces the large ending there is often found a thickening of the cell membrane with a synaptic ribbon. In large endings synaptic vesicles are seen to be concentrated close to the cleft having a width of several hundred A. Histochemical studies of the endings have also been made on guinea-pigs. Acetylcholinesterase has been found distributed at the nerve endings, especially at large endings whereas at small endings little or none has been found (Schuknecht et al., 1959). By cutting Rasmussen's bundle (Kimura and Wersall, 1962) which is thought to be efferent, at the facial colliculi in the pons, it was confirmed that most of the large endings were the terminals of the crossed olivo-cochlear bundle and efferent in nature. Other large endings which did not degenerate at all are considered the ter-

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minals of the homolateral fibers coming from the superior olivary complex, or of fibers of some other origins. After section of the auditory nerve at the internal auditory meatus most small nerve endings showed degenerative changes which indicates that the small endings are afferent terminals. Low contents of vesicles in small nerve endings do not oppose this conclusion. From these experimental results it may be reasonably predicted that acetylcholine is a chemical transmitter released from the large endings of efferent fibers. There is, however, another finding contradictory to that prediction. Desmedt and Monaco (1962) discovered that the intravenous injection of strychnine opposed the effect of crossed and uncrossed efferents. It is generally thought that strychnine has antagonistic action to inhibitory substances and not to acetylcholine in other parts of the nervous system. The present author with his collaborators (Tanaka, 1964) tried to obtain a conclusive answer to this problem: they designed an experiment to deliver acetylcholine directly arld close to the synaptic region by the electrophoretic method. The guinea-pigs were preliminarily so operated upon that the cochlear microphonics (CM) and the neural component N1 were recorded by means of vestibulo-tympana1 leads from the basal turn of the cochlea. According to Tasaki’s method, nichrome wire electrodes were inserted into both vestibular and tympana1 scalae through tiny holes and another into the scala media, namely the former two electrodes were in the perilymph and the latter in the endolymphatic space. The electrode in the endolymph was used for the electrophoresis. A capillary microelectrode with 2 p tip diameter, filled with various ionic solutions was inserted into the cochlea through the transparent thin membrane of the round window. The electrode was further advanced through the basilar membrane while the DC potential between the capillary electrode and the perilymph was being measured. The position of the tip of the electrode was invisible, so the negative resting potential obtained was taken as an indication of the tip position of the electrode in the organ of Corti. Ionized chemicals contained in the capillary were administered in the vicinity of the hair cells by means of a 10-6 A current in the square wave form of 500 msec in duration applied at a frequency of 1 per sec for 8 to 10 min. The application of inorganic ions such as sodium, potassium and chlorine had no effect on the CM and N1 response. Fig. 1 indicates that the CM and N1 were not influenced by applied potassium ions in 8 x 10-6 A current. In contrast with the results for inorganic ions, administration of acetylcholine (ACh) or prostigmin elicited marked changes in the cochlear response. The CM decreased abruptly in amplitude during or shortly after ACh administration using the same anodic current as used for the potassium or sodium ions. Other drugs have been similarly tested but with negative results. Application of GABA, which is known as a blocking agent on the synapses in the cat brain, produced no effect. Administration of strychnine showed no consistent change in the CM and N1 responses, though Desmedt and Monaco reported its inhibitory effect on the olivo-cochlear efferent inhibition. Local application of adrenaline and atropine had no effect on the cochlear responses. The effect of ACh on the nerve response was examined by unitary responses of the primary auditory neurons. The firing rate of fibers with characteristic frequencies R