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Advances in

Insect Physiology

Volume 15

This Page Intentionally Left Blank

Advances in Insect Physiology edited 4 y

M. J. BERRIDGE J. E. TREHERNE and V. B. WIGGLESWORTH Department of Zoology, The University Cambridge, England

Volume 15

1980

ACADEMIC PRESS A Subsidlaw of Harcourt Brace Jovanovlch, Publishers

London

N e w York

Toronto

Sydney

San Francisco

A C A D E M I C PRESS INC. ( L O N D O N ) L T D 24/28 Oval Road London NW1 2DX

United States Edition publkhed by ACADEMIC PRESS INC. 1 1 1 Fifth Avenue New York, New York 10003

Copyright 0 1980 by ACADEMIC PRESS INC. ( L O N D O N ) L T D

All Rights Re.7ert.t-d

No part o f this book may be reproduced in any form by photostat, microfilm, or any other means, without written permission from the publishers

British Lihrury Cutuloguing in Puhlicution Dutu Advances in insect physiology Vol. IS 1 . Insects - Phy\iology I . Brrridgc. Michael John 11. Treherne, John Edwin I l l . Wigglcsworth. Sir Vincent SV5.7'01 QL4VS 63-1.1039 ISBN 0-1 2-024215-X ISSN 0065-2806

P R I N T E D IN G R E A T B R I T A I N B Y W & J M A C K A Y LIMITED. C H A T H A M

Contri but0 Peter D. Evans

Agricultural Re.yearch Council, Unit of Invertebrate Chemistry and Physiology, Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, U K A. R. Gilby

Division of Entomology, C.S.I.R.O., P.O. Box 1700, Canberra City, A C T 2601, Australia Nancy J. Lane

Agricultural Research Council, Unit of Invertebrate Chemistry and Physiology, Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, U K Stuart E. Reynolds

School of Biological Sciences, University of Bath, Claverton Down, Bath BA2 7AY, U K David B. Sattelle

Agricultural Research Council, Unit of Invertebrate Chemistry and Physiology, Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, U K Helen leB. Skaer

Agricultural Re.rearch Council, Unit of Invertebrate Chemistry and Physiology, Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, U K


Contents Contributors

V

Transpiration, Temperature and Lipids in Insect Cuticle A. R . GILBY

1

Intercellular Junctions In Insect Tissues NANCY J. LANE and HELEN leB. SKAER

35

Acetylcholine Receptors of Insects DAVID B. SATELLE

215

Biogenic Amines in the Insect Nervous System PETER D. EVANS

317

Integration of Behaviour and Physiology in Ecdysis STUART E. REYNOLDS

475

Subject Index

597

Cumulative List of Authors

621

Cumulative List of Chapter Titles

623


Transpiration, Temperature a n d Lipids in Insect Cuticle A. R. Gilby Drvrsion of Entomology,

C S l R 0 , Canberra, Australra

1 Introduction 1 2 Biophysics of cuticular transpiration 3 2.1 Mass transfer of water 3 2.2 Energy budget during evaporation 6 3 Water loss and temperature 9 3.1 Water loss measurements 9 3.2 Critical temperature - dynamic experiments 12 3.3 Energy budget analysis 16 4 Transpiration and cuticular lipids 20 4.1 Insect integument as a limiting membrane 20 4.2 Cuticular lipids and water loss 21 4.3 Chemical composition of cuticular lipids 22 4.4 Hypotheses o n lipid functioning 24 5 Conclusion 29 Acknowledgement 30 References 30

1 Introduction

The water relations of arthropods, and particularly of insects, have attracted the interest of scientists of many disciplines during half acentury. The ability of terrestrial insects to conserve water has been emphasized as one vital basis for the unique success of insects in colonizing a wide range of environments, despite their relatively small size. The penalty of a large ratio of area to volume has somehow been overcome. Given the large interface with the environment, it is inevitable that the insect cuticle should be the focus of attention in attempts to suggest mechanisms by which this has occurred. Early preoccupation with the loss of water from insects has given way to the realization that other factors such as the rapid absorption of water 1

2

A. R . GlLBY

vapour are important in vivo. Recent work has shown that, while some of the structures involved are integumental derivatives, the site of water uptake is not the external integument itself. This has been reviewed recently by Wharton and Richards (1978) and mechanisms are discussed by NobelNesbitt (1977). Furthermore, in the living animal the layer of epidermal cells beneath the cuticle may well be important in controlling the passage of water (Berridge, 1970). Treherne and Willmer (1975) have produced evidence that integumentary water loss from a cockroach may be influenced hormonally. Also, the beetle Cryptoglossa virrucosa was shown by Hadley (1979) to produce in direct response to exposure to low humidity a surface mesh of wax filaments which he associated with lower cuticular transpiration rates. Much of current emphasis in the subject is on aspects such as those characteristic of processesin vivo.However they will not be dealt with here. In a previous article in this series, Beament (1964) gave an extensive exposition of his work over 20 years on active transport and passive movement of water in insects. This represents essentially the current position in the evolution of his ideas and his hypotheses involving detailed models of the functioning of lipids in insect cuticle and the control of cuticular water relations. It follows an earlier comprehensive account (Beament, 1961). Subsequent publications by Beament have not radically changed the viewpoints advanced (Beament 1965, 1967, 1976). An extremely useful and discerning contribution which covers the whole subject is the recent book on water balance in land arthropods written by Edney (1977). Another valuable review is that by Ebeling (1974). These recent publications eliminate the need for any general review and provide an exhaustive reference list. The present article is fairly restricted in its scope. Much of published experimental data on the outward passage of water through insect cuticle is useful in comparative physiology and ecology. However, inferences from the influence of temperature on the permeability of cuticle to water have formed one of the main bases on which have been erected hypotheses on physico-chemical mechanisms involved in water retention. The literature on this topic is in a state of confusion. Much of the experimental data is either inappropriate or too limited for biophysical analysis and the analytical framework for the design and interpretation of experiments is generally unsound, mainly through the neglect of important independent variables. Many authors over-interpret their results and speculate on possible but unsupported mechanisms. 'The effect of these possibly wrong conclusions flowing on into text-books and general articles amounts to proof by repeated affirmation. This paper offers a personal view of current knowledge (or the lack of it) on the interactions of transpiration and temperature in insects and the role of epicuticular lipids. Because of the paucity of soundly based data, it amounts to a critique of the current situation with no possibility of drawing

TRANSPIRATION, TEMPERATURE A N D LIPIDS

3

definitive conclusions on the validity of current or alternative hypotheses except that they lack evidence.

2

Biophysics of cuticular transpiration

Before proceeding to a consideration of experimental data on the effects of temperature on water loss, it is necessary t o set down briefly some biophysical principles. Some of the mathematical expressions depend for their validity on assumptions concerning the mechanism of water loss and the properties of cuticular membranes. Others, however, depend only upon the application of the laws of physics to insects without such assumptions. Some of the parameters have not been experimentally determined but limiting values can reasonably be assigned. Restriction of consideration to passive diffusion and dead insects enables metabolic terms to be ignored.

2.1

MASS TRANSFER O F WATER

2.1.1 Membrane permeability At any point within a membrane, the molecular diffusion of a component, j, in a direction x is described by Fick’s law F.(x)

=

dc . -D.-J dx

where F,(x) is the net flux density of component j at x , g m-’ s-’ D j is the diffusion coefficient of component j, mz s-l and dc,/dx is the concentration gradient of component j, g m-4. Since the actual concentration gradient within the membrane is not known, it is usual to assume homogeneity in the membrane and to replace -dcj/dx by Acj/Ax, where Ac, is the difference in the concentrations of component j in the phases separated by the membrane and Ax is the thickness of the membrane, so that Acj F ~ ( x )= D . JAX It is often preferred to express the flux density by

Acj Rj

Fj(X) = -

where R j is the resistance to diffusion of component j, s m-’.

(3)

A. R . GILBY

4

2.1.2 Permeability of cuticle to water - evaporative resistances When water passes through insect cuticle into an unsaturated atmosphere, the exact place within the integument where the change from liquid to vapour occurs is not known. The dermal and cuticular structure of insects is complex and varied and will not be dealt with here. Between the haemolymph and the cuticle there are several layers, such as the basement membrane and the epidermal cells which contain aqueous components. The implications of such layers acting as the barriers to the passage of water have been proposed by Berridge (1970) and discussed by Edney (1977). An alternative route for the loss of water is provided by the tracheae which pierce the integument through all its component layers. Without at this stage taking into account further subdivisions of the various structures, the major components which can offer resistance to the passage of water are represented in Fig. 1(a), which is not drawn to any scale. The various possible resistances to the flux of water vapour can be incorporated into a resistance network outlined in Fig. l( b ) as an electrical circuit analogue. If water passes from the haemolymph to the surface as vapour, it diffuses through the dermal layers, resistance id, and the cuticle, resistance rc. These two resistances are in series so that their combined integumental resistance, R,, is their sum Rs = rd + rc

(4)

An alternative parallel pathway is transpiration through the tracheae, resistancer,, so that the combined internal resistance to diffusion of water due to the insect, R , , is

In series with the internal resistances is the stagnant layer of air, resistancer,, at the cuticle surface. The total resistance to the diffusion of water, R,, is thus R,

=

Ri

+ ro

(7)

Depending on the relative magnitudes of the various resistances, these expressions can be simplified. Thus if rd can be neglected either because

TRANSPIRATION, TEMPERATURE A N D LIPIDS (0

5

1 Atmosphere

Stagnant oir layer

fa

fC

................ ............... ................

........................................... ..........................................

rd

Trachea

(b)

R, ,integurnental resistance

haernolymph fd

\

fa

fC

V

in atmosphere

,

R,,, total resistance for water vapour Fig. 1 (a) Schematic representation of structures and resistances possibly involved in the flow of water vapour from haemolymph to atmosphere. (b) Resistances involved in transpiration arranged in electrical circuit analogue

liquid water is able to penetrate into the cuticle, e.g. through the pore canals, or because re >> r d , and also if r , >>re, e.g. because spiracles form a small fraction of the area of cuticle or the spiracular valves are efficiently closed, then the total resistance, R,, becomes equal to rc + To. Equation (3) can be applied to the mass transport of water and rewritten

A . R . GILBY

6

E

-

P S

-

P G

(9)

Rw

where E is the water loss rate, g m-2 s-' p, is the water vapour density at the liquid water surface within the insect, g m-3. pa is the ambient water vapour density in the atmosphere, g m-3. and R , is the vapour diffusion resistance of eqn (8), s m-'. The liquid vapour density, ps, may be taken as the saturated vapour density of water at cuticle temperature because the change in vapour density accompanying even large changes in osmotic pressure is small. The solutes in haemolymph have little effect. Because vapour pressure is related to vapour density by the perfect gas law, the driving force for diffusion, P , - P, can be replaced by the saturation deficit of the air if due account is taken of any temperature differences from the cuticle and suitable adjustment is made to the dimensions of R,. The concentration term in Fick's law can be expressed in yet other ways but the utmost vigilance must be exercised in any subsequent derivations to avoid involuntary assumptions concerning the variables concealed in different parameters. Thus, if the chemical potential gradient is considered as the driving force, it follows by the reasoning explained in Noble (1974), pp. 312-313, that the diffusion resistance, R,, thenobtained depends inversely on the water vapour concentration. This is undesirable. Expressions equivalent to eqn (9) are widespread in descriptions of transpiration in biology. As defined in eqn (9), the diffusion resistance R , is &/p,,,RT where p, is the mobility of water in the limiting membrane. 2.2

ENERGY B U D G E T D U R I N G EVAPORATION

Evaporation of water from an insect results in the loss of latent heat of vaporization of water from the animal. The environment interacts with the insect at its surface through the flow of energy. Energy, usually as some form of sensible or radiant heat, arriving at the insect will tend to increase its temperature unless the energy is consumed in metabolic or physiological processes. Because the energy input to an insect less the energy loss must equal the heat stored in the insect, an energy budget equation can be set up. This is based on the principle of the conservation of energy. An energy budget equation contains terms which depend on the physical characteristics of the insect and its environment, but it does not rely on assumptions concerning the architecture of anatomicalstructures or details of physiological mechanisms. The energy balance equation for flux densities into an insect is H + R +(l-a)R, + M - LE-G-q

=

0

(10)


7

where H is the sensible heat flux from the air to the surface, W m-’ R is the net long wave flux to the surface, W m-* R, is the short wave irradiance to the surface, W m-2 a is the short wave reflectivity of the surface M is the metabolic heat per unit area of surface, W m-’ L E is the latent heat flux due to evaporation of water, W m-2 G is the net heat flux by conduction, W m-2 and q is the rate of heat storage in the insect, W m-2. The terms in eqn (10) will be considered briefly as they apply to experimental conditions for the determination of water loss from insects. 2.2.1 Sensible heatflux The sensible heat flux arises from the exchange of heat between the insect and the surrounding air. It is proportional to the temperature difference between the insect and the air and inversely proportional to the resistance to heat transfer. The sensible heat flux is given by

H

= h(T, - T,)

where h is the heat transfer coefficient, W m-’ K-’ and T, and T, are the temperatures of ambient air and of cuticle respectively. The heat transfer coefficient, h , is equal to pcp/rH,the ratio of the volumetric heat capacity of air pcp, to the resistance to heat transfer r,. An increase in the movement of air will cause a decrease in r,, i.e. an increase in h and in the sensible heat flux. If the cuticle is at a lower temperature than the air, such an increase in h will tend to raise the cuticle temperature.

2.2.2 Radiant energy Jtitxes The second and third terms in eqn (10) relate to the exchange of radiant energy. Any insect is always exposed to streams of thermal radiation from surrounding objects and itself contributes to the general radiation flux. The major source of short wave radiation is the sun either directly or by reflection or scattering. In laboratory experiments, R , is likely to be negligible unless the design of apparatus is particularly unfortunate, e.g. by allowing sunlight or other strong radiation to penetrate the specimen chamber. If R, were not negligible, its strongly directional character would impose problems in calculating its effects. For objects near room temperature, the radiation emitted is of long wavelength. An analysis of the net radiation flux is greatly simplified if the insect is surrounded by a radiating surface at a uniform temperature equal to

A. R . G l L B Y

8

the air temperature. This is usually so in quantitative measurementsof water loss which otherwise would have little meaning. Application of the Stephen-Boltzmann law then gives R

= E

m(Ta4 - TC4)

(12)

where E is the emissivity of the cuticle u is the Stephan-Boltzmann constant, W ni-’ K-4 and Toand T, are the temperatures of ambient air and of cuticle respectively. For most natural biological surfaces E is close to unity.

2.2.3 Latent heatflux The evaporation of water from the insect results in latent heat loss as water is removed as vapour from the insect by diffusion and air movement. The energy consumed by transpiration is L E where L is the latent heat of evaporation of water, Jg-’, and E is the rate of water loss, g m-’ s-’. 2.2.4

Metabolic heat, heat lost by conduction and storage

By restricting analysis to dead insects, metabolic heat is eliminated. For an insect supported on a poor conductor with a small temperature difference and with a small area of contact, the heat lost by conduction will be negligible. Under steady-state conditions there is no storage of heat. When these conditions apply, M , G and q in eqn (10) each equal zero.

2.2.5

SimpliJied energy budget equation

Substitution of eqns (11) and (12) in eqn (lo), with the assumptions just outlined, results in the energy balance equation h(T, -- T,)

+ m(Ta4

-

T):

=

LE

(13)

A consequence of eqn (13) is that, in any situation where an insect is enclosed in a chamber of unifrom temperature under subdued illumination, so long as water is evaporating under steady-state conditions then the cuticle temperature will be lower than the air temperature. The cuticle temperature in turn determines the water vapour density or pressure within the cuticle and hence the driving force for transpiration [eqn (9)]. The resistances to evaporation included in R , may also change with temperature and with air speed. The heat transfer coefficient, h , of eqn (13) also depends on air speed. The influence of ambient conditions on rate of water loss and on cuticle temperature is thus very complex.


3

9

Water loss and temperature

Although there is a large body of experimental information on water loss from insects, none of it has been obtained under rigorously controlled conditions where all the data for a complete physical analysis are known. The earliest measurements by Gunn (1933) on the effect of temperature on transpiration were made on live insects. He interpreted an observed increase in the rate of water loss from Bfatta orientalis at about 30°C as due to the onset of active movement of air into and out of tracheae by a muscular pumping action. Such physiological and metabolic reactions, important also in water uptake, complicate attempts to detect biophysical mechanisms affecting transpiration. Although dead insects can exhibit higher transpiration rates than living insects, a relatively small initial rate of respiratory transpiration can assume a markedly increased contribution to the rate of water loss of live insect:, as the temperature is increased (Ahearn, 1970). Fick's Law [eqn (3)J has been applied to calculate the cuticular resistance to water loss in living insects, e.g. by Vannier (1974), but in no investigation on the influence of temperature on transpiration from living insect5 has all the necessary parameters which would enable an analysis, such as both air temperature and cuticle temperature, been reported. Consideration is thus restricted mostly to data from freshly killed insects.

3.1

W A T E R LOSS M E A S U R E M E N T S

The history of the development of experimentation and interpretation of the effects of temperature on transpiration stems from the first systematic investigations by Ramsay (1935a, b). He was concerned with the physics of methods of measuring the evaporation of water and studied the effects of temperature, humidity and wind speed upon the rate of evaporative water loss from living or dead specimens of the cockroach, Periplaneta urnericanu. A wind tunnel was used for experiments up to 35°C. Unfortunately a different apparatus, consisting of a jar with desiccant in a thermostated water bath, was used for experiments between 35-50°C so that the wind speed, induced by a slowly rotating fan, was different and not known. Cuticle temperatures were not measured. From his measurements of the weight loss due to transpiration from freshly killed cockroaches with blocked spiracles, Ramsay plotted the rate of evaporation at a constant saturation deficit against air temperature. Rather than connect the points with a smooth curve, he drew two intersecting straight lines and concluded there was, at about 30°C, a sudden increase in the rate of evaporation of water through the body surface. The interpretation given was equivalent to attributing the

10

A . R. G l L B Y

sudden increase in the raie of water loss to an abrupt decrease in the cuticular resistance to water loss at the particular temperature. Subsequently, Wigglesworth (1945) extended observations to several other species of insect. Groups of dead insects with spiracles occluded were weighed before and after t:xposure to dry, unstirred air over phosphorus pentoxide in flasks immersed in a water bath. Plots of rate of water loss, in mg cm-2 h-’ with no consideration of saturation deficit, against temperature, mostly at 10°C intervals, gave curves increasing with temperature, the increase being steeper at higher temperatures, The curve for each species was interpreted as exhibiting a “critical temperature” at which the rate of water loss was considered to increase abruptly. The critical temperatures differed between species and between developmental stages of the same species. Similar results were obtained by Lees (1 947) with several species of ticks. Holdgate and Seal (1956), using freshly killed pupae of Tenebrio mofitor and nymphs of Rhodnius prolixus and Holdgate (1956), using several species of aquatic insects, investigated the effect of temperature on rate of water loss into dry air by methods similar to those of Wigglesworth (1945). The measurements on the terrestrial insects were conducted in still air and those on the aquatic insects in air that was stirred. All their experimental data fitted smooth and continuous curves. They rejected the hypothesis of a mathematical discontinuity in the previously published insect water loss curves and postulated the apparently sudden rise in the rate of water loss to be an artefact due to the choice of scales for the axes of what were basically exponential curves. With considerable justification, Holdgate and Seal questioned t h e validity of the critical temperature concept. Mead-Briggs (1956) reached similar conclusions from his experiments on several species of insects and emphasized the inconclusive nature of all results to that time. The situation did not remain static for long before the approach to the subject was revolutionized by Beament. First with Peripfaneta americana (Beament, 1958), and later with many other species of insect (Beament, 1959), Beament refined hitherto very crude experimental techniques and published results which appeared to re-establish convincingly the existence of critical temperatures in the water loss-temperature relations of insects. Bearnent used thermistors and thermocouples to measure air temperature and cuticle temperature and emphasis was placed on the necessity for extreme care in handling insects to avoid cuticle damage. Manipulations such as the blocking of spiracles were therefore precluded. The rate of water loss was determined from the weight change of single insects suspended on a beam balance incorporated in the thermostatted apparatus. Attention was paid to the circulation of air and to ensuring that the evaporation occurred into dry air. Air temperature, cuticle-air temperature difference and weight


11

loss could be measured without interference with the specimens. Results for permeability were expressed as mg/animal/mm Hg/h which is consistent with eqn (9) and allows for the saturation deficit of the air as a measure of the driving force for evaporation of water. When plotted against cuticle temperature, Beament's results indicate an extremely sharp, virtually a first order, increase in permeability to water at a critical temperature characteristic of each individual specimen of eleven species of terrestrial insect. Two exceptions were Tipula sp., which is a soil insect with damaged cuticle, and Schistocerca nymphs which both gave smoothly increasing curves. The pronounced discontinuities evident in the curves relating cuticle permeability to water and the cuticle temperature published by Beament have influenced the interpretation of results by most authors publishing on insect water loss in the last 20 years. However, in spite of repeated emphasis, e.g. Beament (1961), on the need for experimental rigour if results are to be interpreted in terms of the permeability of cuticle to water, the majority of investigators have continued to ignore parameters which must be measured, such as cuticle temperature, or have inadequately controlled experimental conditions. The bibliography by Edney (1977) should be consulted for specific references. The simultaneous measurement of water loss and cuticle temperature is experimentally very difficult. Oloffs and Scudder (1965) quoted both air and cuticle temperatures in their work on water loss from Cenocorixa expletu but their measurements were at 5 "C increments in air temperature. This is too coarse for their interpretation of the results in terms of critical temperature to be convincing. In a recent publication, Toolson (1978) advocated the use of the chemical potential gradient as a measure of the driving force for the diffusion of water vapour in transpiration from arthropods. Using results on water loss from a scorpion, he claimed that this procedure resulted in a smooth curve relating the conductivity coefficient, L,, to air temperature as an exponential function, whereas the conductivity coefficient, D,, calculated by the use of saturation deficit as the driving force, exhibited a stepped curve. Similar smoothing effects were claimed when other published data for insects were analysed using the chemical potential equations. However, in the example given on water loss from a scorpion, Toolson fitted an exponential curve to the experimental points for L, but drew a stepped line through the actual points for D,.The subjective impression of a smooth curve in the one case but not in the other is due to the transformations carried out on the data and not to any more fundamental cause. In the equations derived from the use of chemical potentials, the driving force involves the logarithm of the vapour pressure of water. This introduces difficulties in treating transpiration into dry air because the driving force then formally becomes infinite. As mentioned already in Section 2.1.2, the diffusion resistance and hence the

A. R . G I L B Y

12

conductivity coefficient depend on the water concentration as well as the mobility of water in the membrane if they are derived from chemical potentials. In seeking an indicator for water mobility in cuticle, it is preferable to use the diffusion resistance calculated from eqn (9) where R , depends on the water mobility and the temperature but not on water concentration as well. The use of chemical potential, which is not linearly related to concentration, also introduces logical difficulties in the application of the mathematics. Toolson does not take account of the effects of evaporative cooling on cuticle temperature. It will be shown later that errors in published measurements of cuticle temperature are a likely cause of spurious discontinuities in permeability - temperature curves. 3.2

CRITICAL TEMPERATURE - DYNAMIC EXPERIMENTS

An alternative approach to the common method of making measurements on an insect sequentially at a stepped series of fixed temperatures is to record changes as the temperature is varied continuously. Such “dynamic” experiments can result in a significant lag in cuticle temperature in addition to temperature differences due to latent heat of vapourization of water. Choice of a suitable rate of temperature change can minimize any lag and information relevant to the existence of critical temperatures can be gained. 3.2.1 Dynamic experiments on water loss Edney and McFarlane (1974) have studied the effect of temperature on transpiration in two cockroaches, Arenivugu investigatu and PeripEaneta americanu. Although there are unexplained inconsistencies between temperatures quoted in their Table 1 and Tables 2 and 3, they claimed it was possible to estimate approximate cuticle temperatures up to 50°C. With insects suspended from an electrobalance, the weight of each specimen was recorded continuously as transpiration occurred into circulating dry air while the temperature was steadily raised. They analysed their results in two ways. The recorded curve of weight against time was inspected for any abrupt change, and, the rate of water loss was determined graphically from the weight curves at points corresponding to any selected cuticle temperature. From these measurements and with due allowance for saturation deficit, permeability-cuticle temperature curves were constructed. Although the results showed that the permeability of the cuticles increased faster at higher temperatures, both types of curve were smooth and they provided no evidence for a critical temperature. I have carried out similar experiments on P . americana (Gilby, unpublished). Nymphs were from a stock culture maintained in the laboratory.

T

Before use in experiments, individuals were taken from the culture and, with access to food and water, kept isolated in glass jars for at least 5 days to allow recovery from any accidental damage to the cuticle. One day before experiment, food and water were withdrawn. Insects were killed by short exposure to HCN gas about 1h before transfer to the apparatus. The alternative use of H2Sdid not affect results. At all times the avoidance of mechanical damage to cuticle was regarded as of the utmost importance. The apparatus used was similar in principle to that illustrated by Edney and McFarlane (1974) but the air was vented and not recycled. The insect rested on a small copper loop on a terylene thread connected to the electrobalance and reaching into a glass tube immersed in a thermostatted water bath. A stream of dry air was passed through 10 m of copper tube immersed in the water bath to control the air temperature and then passed over the specimen at 100 cmlmin, with the insect head-to-wind. The output of the electrobalance was recorded as the temperature was raised from 20°C to 45°C contindously at a rate which was not linear but averaged a little over 20"C/h. The curves, equivalent to weight-temperature curves, increased smoothly in a fashion similar to those published by Edney and McFarlane (1 974). Alternatively, the recorder output was fed to a Cahn Time Derivative Computer, which enabled the rate of water loss to be recorded continuously. Such a curve, as illustrated in Fig. 2 , does not provide evidence for a step-like break at a critical temperature. However, it should be emphasized that no measurement was made of cuticle temperature, nor was allowance made for the saturation deficit of the air or any alternative indicator of the driving force for the diffusion of water.

3.2.2

Dynamic experiments on cuticle temperature

Examination of Figure 4, and particularly Figure 5, in Beament's (1958) paper where allowance is made for the saturation deficit of the air, reveals that it is the use of experimental values for T,, the cuticle temperature, rather than To, the air temperature, in the presentation of results which produces the abrupt step in the rate of water loss-temperature curve of P. americana. A similar situation applies for Rhodnius prolixus nymphs in Figure 6 of Beament (1959), but To is not shown for other species. Correct measurements of T, are therefore crucial to establishing whether the critical temperature interpretation is valid. Experimentally this is very difficult, much more so than the measurement of water loss. An investigation has been undertaken (Gilby, unpublished) of the cuticle temperature of P . americana nymphs under a regime of steadily increasing air temperature. The apparatus was based on that described earlier (Section 3.2.1). Dry air was passed through 10 m of copper pipe in a water bath and then over the specimen, supported head-to-wind on its legs on a fine nylon

A. R. GILBY

I

45'

z

35°C

1

05

10 Time I h 1

I 5

Fig. 2 Recorder trace of rate of change of weight of a P. americana nymph, weight 44.5 mg, in continuously rising ambient air temperature (indicated). Air flow 100 cm/min

net in a horizontal glass tube in the bath, before being vented. Insects were killed with HCN as described above and placed in position after having been isolated for several days to minimize cuticle damage. Towas measured with a copper-constantan thermocouple made from 6.1 X cm diameter wire placed in the air stream in front of the insect. Identical fine thermocouples were located on the insect, one placed under the posterior edge of the pronotum and another similarly placed on the abdomen under the second tergum. This was done with the utmost care. It was necessary to place the thermocouple wires laterally across the insect to avoid any spearing action and to keep the maximum length of wire in contact with the cuticle to minimize conduction errors. Merely placing the tip in contact with the cuticle gave variable results. The design of the apparatus was such that these preparative procedures were performed in the open before the glass tube was sealed in place and lowered into the water bath. The thermocouples had a common cold junction immersed in ice-water. The output from each

15

TRANSPIRATION. TEMPERATURE A N D LIPIDS

0

1 Trme ( h '

2

Fig. 3 Recorder traces of air temperature (T,) and cuticle temperature, measured at pronotum (T, pronotum) and second tergum (T, tergum), of P . americana nymph, weight 1055 mg, in continously rising ambient air temperature. Air flow 100 cmimin

thermocouple was taken to a multichannel potentiometric recorder and temperatures could be read to 0.1"C. The response of cuticle temperature of a large P . americana nymph to a steady change in air temperature from 25°C to 45°C during slightly more than 2 h is shown in Fig. 3. At this rate of heating there was only a minor lag in cuticle temperature. When the air reached its upper steady temperature of 45"C, the cuticle was within 0.25 "C of its equilibrium temperature, which it reached about 3 minutes later. A slower change in body temperature of P . americana was reported by Coenen-Stass and Kloft (1977) but they did not detect any lowering of body temperature at less than 32°C. With the size of specimen which gave the results in Fig. 3, the cuticle temperatures measured at the pronotum and the tergum were scarcely distinguishable. With small nymphs, differences of up to 1"C were sometimes evident but temperature measurements on small insects were relatively unreliable due to difficulty in placement of thermocouples and other physical causes. The cuticle

A . R . GILBY

16

temperature increased in a smooth curve (Fig. 3). Any sudden increase in transpiration rate should have been reflected in a diminished cuticle temperature because of the higher rate of energy consumption. This was not supported by experiment. In Fig. 4, the differences between air temperature (To) and

Air ternperature,T,,

(“C )

Fig. 4 Differences between air temperature and cuticle temperature of insects at different from ambient air temperatures. 0 ,from data of Beament (1958) for P . antericana nymphs; 0, data of Oloffs and Scudder (1965) for C’. e.rplern adults; A,from Fig. 3 for P. crmericarru nymphs

cuticle temperature (T, tergum) derived from Fig. 3 are plotted against air temperature. After the initial brief lag, a smoothly increasing curve results. This contrasts with the temperature differences shown in Fig. 4 measured from the results of Beament (1958) for large P. americana nymphs. Again, the curve in Fig. 4 derived from the results of experiments with C. expleta (Oloffs and Scudder, 1965) showed a smooth increase in temperature difference with increasing air temperature. 3.3

ENERGY B U D G E T ANALYSIS

3.3.1 Latent heat flux densities The quantitative comparison of results from different publications is almost impossible because of the great variety in the species of insect used and the lack of important information in many papers. For example, several investigators have worked with P. americana nymphs but Beament (1958) does not specify the size of his specimens, information which is needed to calculate flux densities. However a comparison was sought by assuming the reasonable value of 8 cm2for the surface area of a “large nymph” as used in his work. Calculations were made of the latent heat flux from three sets of data on the rate of water loss and these are shown in Fig. 5 plotted against air temperature. The results of Beament and of Gilby were each from single

17


b

10

?

20

A

0 .

30

40

50

Air temperature (TI

Fig. 5 Latent heat flux calculated for transpiration from P. americana nymphs at different ambient air temperatures. 0 ,from data of Beament 1958. Weight of nymph assumed 1 g; 0, from data of Edney and McFarlane (1974); A, from data of Gilby (unpublished). Weight of nymph 115 mg

insects but of disparate size, while those of Edney and McFarlane were an average from ten insects. It is evident from Fig. 5 that there is good agreement between the investigators in the measurements of water loss from P. arnericana into dry air at different temperatures. 3.3.2 Energy budget equation On the basis of the energy budget equation derived earlier, h(T, - T,) + (+(TU4 -T ):

=L E

(13)

it would be possible to calculate the cuticle temperature of any insect if the latent heat flux density, L E , (calculated from the rate of water loss) and the sensible heat transfer coefficient, h, were known for any given air temperature. The value of h , which depends very strongly on environmental conditions such as air movement, is not known experimentally for any insect, let alone under conditions where transpiration has been measured. However, it is possible for certain limiting assumptions to be made which enable existing results on temperature depression to be tested against an extreme estimate. Since the value of a,the Stephan-Boltzmann constant, is known and L E can be calculated as the product of the flux density of water from the insect

A. R . GILBY

18

surface and the latent heat of vaporization of water, the major problem is to provide an estimate of h. Calculations of h for still air conditions gave the results in Table 1. The method used is based on principles described by T A B L E 1 Cuticle temperature depression a n d transpiration. Experimental values a n d values calculated from a n energy budget equation Insect species an d Ref.

27.0 28.3 33.2 37.0 45.7

Beament (1958)

P. americana Edn ey an d Mcfarlane

(1 974)

P . arnericana Gilby (unpublished)

C. expleta

Oloffs an d Scudder (1 965)

Beament (1 959)

LE’ (w m-2) (a)

P. arnericana

R . prolixus

To

2.8 3.3 11.0 30.1 61.8

(b) 2.4 2.9 9.5 26.0 53.4

AT Experimental

2.4 2.4 3.7 7.3 9.4

AT2 Calculated

(a) 0.26 0.31 0.93 2.32 4.34

(b) 0.23 0.27 0.82 2.06 3.84

10 20 30 40 50

1.3 2.0 8.3 30.1 76.8

0.3 0.9 1.4 2.2 6.2

0.14 0.20 0.72 2.26 5.14

30 35 45

4.2 8.3 38.9

0.08 0.20 0.75

0.12 0.24 1.11

20 25 30 35 40 45

23.0 32.7 48.0 73.5 121.5 183.9

0.75 1.09 1.49 2.19 3.93 5.99

1.17 1.60 2.24 3.27 5.13 7.41

6.8 14.3 22.1 33.3 44.2

0.8 2.0 1.8 3.2 4.0

58.6 61.2 63.0 65.2 67.8

(c) 0.50 1.00 1.50 2.17 2.80

(4

0.44 1.88 1.31 1.91 2.46

’

Surface areas used in calculation of LE for P. americana were derived from the insect weight by the empirical relation S = 8.8 w’’~ (,.S cm’, w g) established experimentally.

Results of Edney and McFarlane were averaged from their Table 2 Diameters of equivalent cylinders used in solutions of energy budget equation for P. americana were derived from the insect weight by the empirical relation w = 1. 5 9 d 3(w g,d cm) established experimentally ( a ) Insect assumed to weigh 0.8 g i.e. d = 0.80 ( b ) Insect assumed to weigh 1.0 g, i.e. d = 0.86 ( c ) Equivalent cylinder assumed d = 0.7 approximating fed nymph ( d ) Equivalent cylinder assumed d = 0.4 approximating unfed nymph


19

Gates (1962). In heat transfer by convection, the physics of heat engineering establishes three dimensionless variables, the Grashof number, the Prandtl number and the Nusselt number, which relate basic properties of the air, such as heat conductivity and viscosity, to characteristic dimensions of the object. Using the expression relating the Nusselt number, Nu, to the other two for a cylinder under free convection and the further expression h =Nuk/pc,d, where k, p andlc, are the thermal conductivity, the density and the specific heat of air respectively and d is the diameter of the cylinder, an algebraic expression was established for h for inclusion in eqn (13). For the diameter of the cylinder equivalent to an insect, half the sum of the width and depth was used. An empirical relationship between this and insect weight was experimentally determined for P . americana. Likely dimensions derived from the literature were used for the other insects. The only unknown terms remaining in eqn (13) were then T, and T,. At any air temperature for which the latent heat flux was known, eqn (1 3) could then be solved iteratively for T,. This was done until the error in T, was 0.01 "Cto give the calculated values for AT, the temperature depression in the cuticle, in Table 1. In the calculation of AT from the energy budget equation, the expression used for the sensible heat transfer coefficient, h , was derived for conditions of free convection, i.e. still air. O n the other hand, the experiments which provided the values of the latent heat flux, L E , were all performed in moving air. The inconsistency was deliberately introduced to bias the calculation to yield maximum values for AT. Under the experimental conditions of forced convection, h would be larger than in free convection so that the calculated values of AT represent an upper limit. Comparison of calculated values of A T with experimental values set out in Table 1indicates that the energy budget analysis works well for the results of Oloffs and Scudder for C. expleta and the results of Gilby for P . americana. In both cases, experimental values of AT are less than the calculated values. In the other investigations with P. americana, the data of Edney and McFarlane is equivocal in that the experimental values are generally larger than those calculated. However, examination of their results on individual insects, rather than averages, reveals big variations which are not explained. Furthermore, the temperatures quoted in their Table 2, the source of data for the present calculations, conflict with their Table 1 and their Figure 2. The values of AT found by Beament are all much larger than the maximum values predicted. Some variations in the latter are possible because of unspecified parameters, e.g. size of insect which affects both L E and AT. However the calculations are not sensitive to the size of insect in the 0.8-1 g range as illustrated by the two examples in Table 1. A similar situation exists for R . prolixus where the experimental values of AT exceed the theoretical

A. R. GILBY

20

maxima calculated and the latter are not sensitive to the dimensions of the model. The evidence suggests that several published measurements of cuticle temperature during transpiration are unsound and therefore they are not a valid basis from which to draw conclusions about the existence of critical temperatures deduced from transpiration curves. This is particularly so because too large values of A T compress the temperature scale and expand the permeability scale simultaneously. It is not clear why cuticle temperatures as measured are lower than they should be. The most likely physical errors would produce the opposite effect. However, if the thermocouple probe damaged the cuticle, any damp spot resulting could give rise to exaggerated local cooling. Some evidence for such an effect was noted in the experiments on cuticle temperature measurements described earlier. If thermocouples were aligned towards the head rather than across the cockroach abdomen abnormally low cuticle temperatures were occasionally indicated. In some preliminary measurements of cuticle temperatures of P. americana an infrared thermometer, a non-invasive method of measurement, appeared preferable to thermocouples. However, its use imposed limits to the minimum size of insect which could be studied.

4

4.1

Transpiration and cuticular lipids I N S E C T I N T E G U M E N T AS A L I M I T I N G M E M B R A N E

In Section 2.1.2 and Fig. 1, the various possible resistances to the passive diffusion of water from inside an insect to the atmosphere were enumerated and incorporated into a resistance network in series and parallel. It is not easy to decide the relative contributions of the various resistances in eqn (8) to the total resistance, R,. On available information it is impossible to do so rigorously. Consideration of the energy budget of an insect shows the problem to be complex. Transpiration rate and cuticle temperature are functions of the air temperature, relative humidity, wind speed, net radiation and insect dimensions, as well as the internal diffusion resistances of the integument. The inherent complexity of the system has not been generally recognized. For example, some authors have sought to deduce the contribution of the stagnant air layer to the total diffusion resistance from the effect of air speed on transpiration rate without consideration of the energy flow and its effect on cuticle temperature. Even under circumstances where ro is negligible compared with other diffusive resistances, the alteration to h , the sensible heat transfer coefficient in eqn (13), caused by change in air speed will produce a change in cuticle temperature. This can be quite considerable. For


21

example (Gilby, unpublished), a thermocouple under the tergum of a 1.1 g P . arnericana nymph in the apparatus described earlier registered a cuticle temperature of 38.2"C at an air temperature of 40°C and an air flow of 100 cm/min. When the air speed was increased to 24 mlmin, the cuticle temperature rose to a steady 39.3"C. On resumption of the original air speed the cuticle temperature was again 38.2 "C. Neglect of such effects invalidates any deductions concerning diffusive resistances. However there are a number of indications that the resistance of the integument, R,, is a major component of the total resistance, R,. A thorough and extensive body of work on transpiration from plants, reviewed by Noble (1974), indicates that the leaf cuticular resistance is large compared with the resistance of the stagnant air layer. As pointed out by Edney (1977), a similar situation probably applies even more markedly to insects which are generally much less permeable to water than are plants. Also many of the experiments on water loss from insects have been carried out on insects with the spiractular openings mechanically blocked. Details may be consulted in the review by Edney (1977). Most comparisons of water loss from dead insects with spiracles blocked or unblocked indicate that water loss from the tracheae is of minor importance and sometimes undetectable (Loveridge, 1968). Microscopic examination of cockroaches killed with HCN showed that the spiracular valves normally remain closed. A further indication that the integument is the limiting membrane comes from the results of Beament (1945) on the evaporation of water through isolated cuticles and exuviae of Rhodnius into still air. The permeability to water was of the same order as that from dead insects (Wigglesworth, 1945). As will be discussed later, superficial damage to a cuticle, e.g. by abrasion or washing with water, can cause a big increase in its permeability to water. It is probable that the major barrier to loss of water from dead insects lies in the integument. 4.2

C U T I C U L A R L I P I D S A N D WATER LOSS

The postulate that the epicuticle and its lipids have an important function in transpiration from insects stems from very early work, e.g. Ramsey (1935b). The justification rests on experiments with whole insects and with model systems. Hadley (1978) has found correlations of water loss with epicuticular hydrocarbon composition in several species of tenebrionid beetles. The classical work involving killed or living whole insects is that of Wigglesworth (1945). H e measured the rates of water loss from insects treated superficially in various ways. The application of inert dusts caused a big increase in the rate of water loss. The situation has since been complicated by a controversy over whether mechanical abrasion is necessary to produce the effect (Ebeling, 1974) and the suggestion by Richards (private

A. R . GILBY

22

communication; Wharton & Richards, 1978) that in live insects the epiderma1 cells become involved. However, Wigglesworth associated the altered transpiration with damage he observed to an epicuticular wax. Also, he found that extraction of Rhodnius nymphs with chloroform or detergents increased the rate of water loss by a factor of up to twenty five. In some other insects exposure to chloroform vapour was sufficient to produce similar effects.Although solvents, too, remove more than superficial lipids and may affect other components (e.g. extraction with chloroform makes the cuticle rigid), the cuticular lipids are strongly implicated. These conclusions were supported by companion experiments on model systems by Beament (1945). Lipids were extracted from exuviae of several insects and, when spread on suitable supporting substrates, like tanned gelatin or butterfly wings, formed membranes which behaved in a manner consistent with Wigglesworth’s results on insects when treated with dusts or solvents. Furthermore, the changes in transpiration through the artificially made lipid-membranes with temperature resembled those of intact insects. It should be remembered, however, that this work was before the later refinements in techniques now considered essential. Following earlier observations by Ramsay (1 935b) on the preservation of droplets of water on cockroach cuticles, Beament (1958) reported experiments on the changes in size of droplets of water coated with wax extracted from P. americana exuviae and supported on a silicone treated metal plate in air at different temperatures. His results indicated, even without corrections for surface temperature, an abrupt increase in the rate of water loss from a drop at about 33°C. Whether this can be interpreted correctly as support for the concept of a transition temperature based on lipids in intact cuticle is extremely doubtful. For example, it is possible that at a particular temperature, the lipid on a water drop might spread to the hydrophobic plate due to a decrease in viscosity as noted by Ramsay (1935b) for films of cockroach grease. Further, the conditions of energy flow to the surface of the drop on a conductive plate are so different from those for an insect in air that it would be surprising if they showed similar transpiration - temperature responses. Indeed, on a polystyrene plate (a poor conductor of heat) smooth curves were obtained (Beament, 1958). It has been shown above that experimental support for sharp temperature effects in cuticles is lacking. 4.3

CHEMICAL COMPOSITION OF CUTICULAR LIPIDS

Much of the work on transpiration from insects was done at a time when there was almost no knowledge on the chemical composition of cuticular lipids. From the early 1960s, modern chromatographic and spectroscopic methods of separation and analysis, were applied to the identification of


23

cuticular lipids (Baker el al., 1960, 1963; Gilby and Cox, 1963). There is now a considerable amount of information available on the composition of cuticular lipids from many species of insect. Summaries of the data have been given by Hackman (1974) and by Jackson and Blomquist (1976). Considerable variation in composition is found in the cuticular lipids of different species. However, of the chemical classes of lipid, hydrocarbons and free fatty acids are probably present in the cuticles of all insects. Hydrocarbons predominate overwhelmingly in many species and, for example, often comprise more than 50%, and sometimes over 90%, of the cuticular lipids of cockroaches and grasshoppers. The hydrocarbons are usually n-alkanes, together with methyl branched alkanes or either mono- or di-unsaturated alkenes, and range in chain length from C20-C50.Nelson (1 979) has recently reviewed the surface hydrocarbons, particularly branched compounds of arthropods. Triglycerides and steroids are commonly reported as minor constituents of cuticular lipids and, more rarely, traces of phospholipids. Sometimes, but not always, these compounds might be suspected to be contaminants from the body. Wax esters of long chain acids and alcohols have been found in the cuticles of only a few species, sometimes in considerable proportion, e.g. a recent report of about 65% in a beetle (Baker, 1978). Aliphatic aldehydes have also been detected. Free fatty acids, which appear to be ubiquitous, are commonly of chain length CI4-C2,,, saturated and unsaturated but rarely branched. There are very few reports of alcohols in cuticular lipids. Primary alcohols would be of particular interest because of their known capacity to reduce the evaporation of water. Apart from two reports as a minor constituent, the only substantial occurrence is in the larvae of Samia cynthia ricini (Bowers & Thompson, 1965) where n-triacontanol is the major component. However, this is present as a powder coating the larva and is not laid down on the epicuticle as a discrete layer. Minor amounts of secondary alcohols occur in the cuticular lipids of some grasshoppers (Jackson and Blomquist, 1976). The major part (over 50%)of an insect cuticular lipid is therefore typically made up of non-polar materials which are the hydrocarbons, either saturated or unsaturated. If unsaturated hydrocarbons predominate, as in P. americana (Beatty and Gilby, 1969), the lipid is likely to be a fluid grease. The saturated long chain hydrocarbons are solids. Oxygenated long chain compounds of varying degrees of polarity make up a complex mixture of substances each usually a minor fraction. Phospholipids and sterols, the common polar lipids of the membranes of animal cells and tissues, are minor constituents or absent. It is possible that some, at least, of the oxygenated compounds may derive from chemical degradation of hydrocarbons.

A. R . GILBY

24

4.4

HYPOTHESES O N LIPID FUNCTIONING

Various physicochemical interpretations have been advanced which seek to explain transpiration from insects in terms of specific arrangements, either molecular or crystalline, of cuticular lipids. Most of the hypotheses were made in ignorance of the chemical nature of the lipids and some were made on chemical assumptions now known to be false. An important role is commonly assigned to amphipathic molecules with their dual polar - nonpolar nature. Modern chemical analyses show these to differ so greatly between species that any hope for an explanation common to all insects might well be futile. Furthermore, there is a lack of agreement on the morphological structure of the epicuticle (Edney, 1977; Neville, 1975) which may be due partly to differences between species. It is basic to any understanding of how lipids might function to know whether or not the epicuticular filaments observed by electron microscopy in some insects are wax canals of the kind suggested by Locke (1961), but the nature of which was disputed by Filshie (1970a), and to know what is their relation to the pore canals. The hard, thin, resistant wax layer of the epicuticle of Rhodnius, which is very different in morphology from the mobile grease covering Periplaneta, was stated to comprise a complex of lipids with other materials (Wigglesworth, 1975). Locke (1965) interpreted electron micrographs of Calpodes cuticle as showing a lipid monolayer in the epicuticle, but it is doubtful whether the techniques of the time were capable of detecting such a structure. Further uncertainties regarding the molecular organization of the lipids in insect cuticle arise from the fact that the vast volume of biophysical data which exists on the structure and properties of lipid films is almost entirely for systems based on the lipids of cellular membranes. These are chemically very different from cuticular lipids. Moreover, analogies with films of phospholipids and sterols are entirely speculative and may be quite misleading. It is therefore difficult to apply any definitive tests to existing hypotheses concerning insect cuticular lipids. 4.4.1

The monolayer hypothesis

The monolayer hypothesis, which was first considered tentatively and inconclusively by Ramsay (1935b), owed its principal development to Beament (1958, 1961, 1964). The hypothesis attributed the major influence on the permeability of cuticle to water to an oriented monolayer of lipid molecules underlying a thicker layer of wax randomly arranged on the epicuticle. Much of the argument was based on work with cockroaches, specifically P. americana. From his experiments on cockroaches with much of the lipid


25

removed by a water spray, Beament claimed that the monolayer is five times as impermeable to water as is the randomly arranged grease. Most attention was given to explanations of the so-called critical temperature phenomenon in transpiration. These explanations involved hypothetical transitions in the organization of the monomolecular film either by disruption of the monolayer at the critical temperature so that its organization was destroyed or by a change in phase within the monolayer to a less condensed state of higher permeability (Beament, 1961). Subsequently however, Beament (1964) adopted a very explicit model with amphipathic molecules of lipid in fixed positions on the epicuticle with their hydrocarbon chains oriented at an angle of 24.5" to the vertical and locked together by van der Waals forces so that the zig-zag shape of the methylene groups of the paraffin chains interfit. H e further proposed that, at a critical temperature, thermal energy would overcome the attractive forces so that the hydrocarbon chains assume a mean vertical position with increased interchain space for migration of water molecules through the monolayer. The proposal requires that the polar groups of lipid molecules in the monolayer occupy fixed positions on the surface. This would place extraordinarily specific requirements on the correspondence of the geometry of such sorption sites with the molecular architecture of the lipids to accommodate the model. Aside from arguments concerning any changes which might occur at a particular temperature, it is unlikely that lipids such as are found in insect cuticle would form monolayers substantially impermeable to water. There are compounds such as fatty acids present in the waxes which could, and no doubt do, form an oriented monolayer on the epicuticle surface. However, as pointed out by Gilby and Cox (1963), fatty acid monolayers are effective to control water evaporation only if artificially held in a state of high compression on a film balance and their permeabilities are sensitive to the presence of impurities. The presence of non-polar hydrocarbons characteristic of insect cuticular lipids would disrupt the cohesive forces between the hydrocarbon chains of molecules in a monolayer. Unsaturation and branched chains especially cause expansion in monolayers. Moreover, a monolayer beneath a wax layer is at an interface where the outer phase is an oil rather than air. Such a system is characterised by the formation of expanded monolayers. Experiments on the surface properties of films of cockroach lipid support these expectations (Lockey, 1976). The force-area curve obtained for a film of cuticular lipid from P. arnericana on water showed that a tightly packed monolayer was not formed. The film was readily collapsed and only weakly adherent to the water surface. A more direct assessment of the possible contribution of a monolayer to the restriction of water loss can be made from comparisons with published data on insect systems. Where the rate of water loss per unit area is known, it

A. R. GILBY

26

is possible by the use of eqn (9), with suitable attention to units, to calculate the evaporation resistance (s cm-l) of the system. This was done to give the results in Table 2. Because the measurements on which the calculations are based have been made under different conditions, a close quantitative TABLE 2

Resistances to evaporation of water from insect cuticle and lipid films System

Temperature ("C)

Evaporation resistance (s cm-')

Ref. for permeability data

P. americana nymph'

27

199

Beament (1958)2

Pieris wing coated with 1 p m film of cockroach grease

25

193

Beament (1945)

Water drop coated with film of cockroach grease

30.6

28

Beament (1958)

Condensed monomolecular film of stearic acid at maximum compression

25

2

Barnes and La Mer (1 962)

Weight of nymph assumed to be 1 g for flux density calculation Similar values obtained from results of Edney and McFarlane (1974) or Gilby (unpublished)

comparision is not justified. For example, the evaporative resistance of the stagnant air layer may differ but it is basic to the whole subject that this should be small compared to the resistance of the membrane, as discussed earlier. The permeability data used were those at the lowest temperature available and below any suggested critical temperature. The results in Table 2 show that the resistances to evaporation of water from a cockroach and a 1 p m membrane of cockroach grease are each some 6-7 times larger than the resistance of a film of cockroach grease on a water drop, which is in turn an order of magnitude greater than the resistance of a compressed monolayer of stearic acid. The resistance of the cuticle is equivalent to some 100 condensed monolayers of stearic acid in series. It is conceivable, however, that the net area of cuticle through which water evaporates might be only 1% of the total area. If that were so, the water flux density would be correspondingly greater and the value of the resistance to evaporation from eqn (9) correspondingly less. Nevertheless, this would create new discrepancies between the resistances to evaporation of the insect and those of both the wing coated with lipid and the water drop coated with lipid, whilst the resistance of the film on the water drop would remain equivalent to many condensed monolayers. Consequently it seems that the monolayer hypothesis is scarcely tenable.

TRANSPIRATION, TEMPERATURE AND LIPIDS

27

4.4.2 Crystal structure and other hypotheses Some other hypotheses on the role of lipids in transpiration from insects postulate the existence of crystalline forms in epicuticular lipids. Unfortunately there is very little evidence, so that the suggestions remain highly speculative. Included in this category is the supposition of Gilby and Cox (1963) that cuticular lipids might form a system analogous to the relatively thick duplex films of paraffins and polar lipids, possibly with polymerized structures in the interface, which can reduce water evaporation efficiently. Also, Locke (1965, 1974) attributes a key role to the occurrence of lipidwater liquid crystals in the epicuticular canals to explain changes in transpiration with temperature. Changes in crystal structure might explain changes in permeability to water. The artificial lipid systems from which analogies are drawn with insect waxes differ in chemical composition from cuticular lipids. Direct evidence for any of the postulated structures is lacking. A similar situation exists for the suggestion by Davis (1974) that phase transitions known to occur with phospholipids and sterols may occur in cuticular waxes of ticks. An early hypothesis (Beament, 1945) attributed abrupt changes in cuticle permeability with temperature to changes of crystal form in the lipids from an orthorhombic to a close-packed hexagonal system. Hurst (1950) developed this suggestion and carried out electron diffraction studies on the puparium of Calliphora erythrocephala and on artificial membranes in collodion. If he had used mixtures, his choice of n-fatty acids, unsaturated acids and n-paraffins for the artificial membranes would have proved to be remarkably appropriate as a model for insect waxes but unfortunately he applied the lipids singly and the larval cuticle which becomes the puparium of cyclorapphous flies is probably not typical in that it lacks a discrete lipid layer (Filshie, 1970b). Hurst found that the electron diffraction patterns both of the collodion-lipid films and of the puparium indicated a threedimensional arrangement of orthorhombic microcrystals which were less oriented ior paraffin films. After heating in an intense electron beam, the fatty acid films and the puparium showed a reduction in crystal orientation and a change to a hexagonal packing. It was suggested that such changes in crystalline form were correlated with changes in permeability to water. Where alignment of crystals occurred, it was believed to be due to the orienting effect of a monolayer underlying the wax. Measurements of dielectric constants were made in supporting observations by Chefurka and Pepper (1955) on beeswax, a system which is chemically a poor model. On the other hand, observations by Holdgate and Seal (1956) on wax from the pupa of Tenebrio molitor gave no indication by electron diffraction of any crystalline transition before meltiiig.

28

A. R . GlLBY

Since these attempts to detect microstructure in sheets of insect cuticular lipids, there has been an increase of interest in the study of thermotropic transitions in lipids and biological membrane systems in general. The literature is now very large. The lipids in many membranes are believed to have a liquid crystalline structure (Larsson and Lundstrom, 1976). Various physical methods have been used to detect transitions in lipids, model membrane preparations and biomembranes. They include infrared and raman spectroscopy, dilatometry, X-ray diffraction, calorimetry, nuclear magnetic resonance spectrometry, electron spin resonance spectrometry and fluorescent labelling (Andersen, 1978). Unfortunately, the data available on natural and artificial systems involve phospholipids almost exclusively (Chapman, 1975). The chemical compositions of insect waxes are so different from those of cell membranes that it is not justified to assume they are analogous. Electron paramagnetic resonance (EPR) spectroscopy has been used to study the lipids on the cuticle of a scorpion, Centruroides sculpturatus, by Toolsonet al. (1979). A spin label was applied to the cuticle of living animals which were sacrificed and E P R measurements made between 20-70°C on dissected cuticle. The main contribution to the E P R spectrum was probably in the surface epicuticular lipids. The results indicated a change in the mobility of the spin-labelled molecules at about 35°C but this was not associated with a sudden breakdown of water retention. Conversely, at higher temperatures where the rate of water loss was increasing rapidly, no changes were detected by EPR. Exploratory investigations on some insect cuticular lipids have been carried out using differential thermal analysis (Gilby, unpublished) with a Mettler TA2000. Endothermic processes occurred over the whole range of biological temperatures in waxes extracted from the exuviae of four species. In lipids from P. americana and Periplaneta brunnea, continuous broad peaks were observed from below 10°C to about 40°C. With the lipids from Lucilia cuprina puparia and Austracris guttulosa the peaks extended to over 50°C. Lipid mixtures such as are obtained from natural sources characteristically show broad transitions (Melchior and Steim, 1976). Calorimetry is a sensitive technique to reveal changes in the physical structures of lipids and ordered-disordered transitions (Spink and Wadso, 1976) but does not enable interpretation at a molecular level unless supplemented by other methods. The experiments showed that changes occurred in the extracted cuticular waxes with temperature but there was no sharp transition at any particular temperature. Attempts to detect similar transitions in samples of cuticle cut from cast skins of P. americana failed, possibly because of lack of sensitivity due to the minute quantities of lipid involved. If it should eventuate that mesomorphic structures in cuticular lipids do influence the permea-


29

bility of insect cuticle to water and if the liquid crystals are similar to those in phospholipid membranes, the interpretation of the effects of temperature on transpiration may be further complicated by the effects of changing water concentration in the epicuticle. Small amounts of water affect the melting behaviour of phospholipids (Chapman and Wallach, 1968).

5 Conclusion

The foregoing discussion reveals a distressing lack of well-established information or theory. The relationship of an insect to the physical properties of its environment is a very complex problem. Cuticle temperature and transpiration rate depend on such variables as air temperature and relative humidity, wind speed, the net amount of radiation, the geometry of insect and the diffusion resistances to the passage of water. This is a multidimensional system in which a change in any of the independent variables can alter the influence of the others. Thus, changes in air temperature at one wind speed may affect cuticle temperature and transpiration in quite a different manner from the same changes at a different wind speed because, in addition to any changes in cuticle permeability, there are also changes in the energy fluxes. For a rigorous analysis of experimental results, it is necessary to know physical properties of the insect, such as absorptivities and emissivities to the various streams of radiation and the effective geometry of the structures transferring energy and matter, in addition to environmental parameters. T o analyse the dynamic approach to a steady state after a change in external conditions would also require information on heat conductivity and specific heat of the insect. A complete treatment involving analyses of energy exchange and diffusive resistances has not been made for any insect. Most experimental results in the literature are not useful for assessing and interpreting the effects of temperature on the permeability of cuticle to water because essential parameters have been ignored, usually at least by the failure to measure cuticle temperature. This is very difficult to measure experimentally, especially with thermocouples which themselves interfere with the measurement. The establishment of sharp discontinuities in water permeability-temperature curves at a critical temperature depends entirely on measurements of temperatures in the cuticle. Where the original data are available, an energy budget analysis indicates that the measured values of the cuticle temperature in this work are impossibly low by several degrees. It is therefore not possible to conclude that these critical temperatures are real. The evidence points rather to a smooth increase in permeability with temperature. If there are no abrupt changes in cuticle permeability at a particular

TT

temperature, the incentive to invoke specific physico-chemical phenomena such as changes in crystallinity in epicuticular lipids or the organization of lipid monolayers is greatly diminished. Experimental evidence for such postulated effects in insects is very nebulous. At present there is insufficient known on the distribution of epicuticular lipids, to support detailed hypotheses on molecular mechanisms. Generalizations may be inappropriate because of interspecific differences which exist in morphology and in the chemical composition of lipids. Until soundly based experiments on insects define the phenomena to be explained and until hypotheses can be tested by experiments on model systems of appropriate composition, speculation will remain unproductive. Insect physiologists have not appreciated the biophysical complexities of the problems and even tend to a simplistic view of the integument. A similar confusion existed in plant eco-physiology until about ten years ago. The analytical framework for the study of plant biophysics is now well established and it is to be hoped that a similar prospect is in store for the study of transpiration in relation to cuticle structure and environmental conditions for insects. Acknowledgement

I am indepted to D r A. R . G. Lang, Division of Environmental Mechanics, C.S.I.R.O.,.for his advice on energy budget analysis.

References Ahearn, G. A. (1970).The control of water loss in desert tenebrionid beetles./. Exp. Biol. 53, 573-595 Andersen, H. C. (1978). Probes of membrane structure. Ann. Rev. Biochem. 47,

359-383 Baker, J. E. (1978). Cuticular lipids of larvae of Attagenus negatoma. Insect Biochem. 8,287-292 Baker, G. L., Pepper, J. H., Johnson, L. H. and Hastings, E. (1960).Estimation of the composition of the cuticular wax of the mormon cricket, Anabrus simplex Hald. J. Insect Physiol. 5 , 47-60 Baker, G.L.,Vroman, H. E. and Padmore, J. (1963). Hydrocarbons of the American cockroach. Biochem. Biophys, Res. Commun. 13, 360-365 Barnes, G.T. and La Mer, V. K. (1962).The evaporation resistances of monolayers of long-chain acids and alcohols and their mixtures. In “Retardation of Evaporation by Monolayers” (Ed. V. K. La Mer) pp. 9-33.Academic Press, New York and London Beament, J. W.L. (1945).Thecuticularlipoidsofinsects.J.Exp. Biol. 21,115-131 Beament, J. W.L. (1958).The effect of temperature on the waterproofing mechanism of an insect. J. Exp. B i d . 35, 494-519 Beament, J. W. L. (1959). The waterproofing mechanism of arthropods. 1. The


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effect of temperature on cuticle permeability in terrestrial insects and ticks. J . Exp. Biol. 36, 391-422 Beament, J. W. L. (1961). The water relations of the insect cuticle. Biol. Rev. 36, 281 -320 Beament, J. W. L. (1964). Active transport and passive movement of water in insects. A d v . Insect Physiol. 2 , 67-129 Beament, J. W. L. (1965). The active transport of water: evidence, models and mechanisms. Symp. SOC. Exp. Biol. 19, 273-298 Beament, J. W. L. (1967). Lipid layers and membrane models. In “Insects and Physiology” (Eds J. W. L. Beament and J. E. Treherne) pp. 303-313. Oliver and Boyd, London Beament, J. W. L. (1976). ‘The ecology of cuticle. In “The Insect Integument” (Ed. H. R. Hepburn) pp. 359-374. Elsevier, Amsterdam Beatty, I. M. and Gilby, A. R. (1969). The major hydrocarbon of a cockroach cuticular wax. Naturwissrnschafren 56, 373-374 Berridge, M. J. (1970). Osmoregulation in terrestrial arthropods. In “Chemical Zoology” (Eds M. Florkin and B. T. Schier) pp. 287-319. Academic Press, New York and London Bowers, W. S. and Thompson, M. J. (1965). Identification of the major constituents of the crystalline powder covering the larval cuticle of Samia Cynthia ricini (Jones). J . Insect Physiol. 11, 1003-1011 Chapman, D. (1975). Phase transitions and fluidity characteristics of lipids and cell membranes. Quart. Rev. Biophys. 8, 185-235 Chapman, D. and Wallach, D. F. H. (1968). Recent physical studies of phospholipids and natural membranes. In “Biological Membranes” (Ed. D. Chapman) pp. 125-202. Academic Press, New York and London Chefurka, W. and Pepper, J. H. (1955). On the physical nature of the transition region of insect waxes. Can. Entomol. 87, 163-1 7 1 Coenen-Stass, D. and Kloft, W. J. (1977). Auswirkungen der Verdunstungskiihlung und der Stoffwechselwarme auf die Korpertemperatur der Schnabenarten Periplaneta americana und Blaberus trapezoideus. J . Insect Physiol. 23, 1397-1406 Davis, M. T. B. (1974). Critical temperature and changes in cuticular lipids in the rabbit tick, Haemaphysalid leporispalustris. J . Insect Physiol. 20, 1007-1 100 Ebeling, W. (1974). Permeability of insect cuticle. In “Physiology of Insecta” (Ed. M. Rockstein) Vol. VI, pp. 271-343. Academic Press, New York and London Edney, E. B. (1977). “Water Balance in Land Arthropods” (Zoophysiology and Ecology, Vol. 9) Springer-Verlag, Berlin, Heidelberg and New York Edney, E. B. and McFarlane, J. (1974). The effect of temperature on transpiration in the desert cockroach, Arenivaga investigata, and in Periplaneta americana. Physiol. Zool. 47, 1-12 Filshie, B. K. (1970a). The resistance of epicuticular components of an insect to extraction with lipid solvents. Tissue & Cell 2 , 181-190 Filshie, B. K. (1970b). The fine structure and deposition of the larval cuticle of the sheep blowfly (Lucilia cuprina). Tissue & Cell 2 , 479-489 Gates, D. M. (1962). “Energy Exchange in the Biosphere” pp. 94-142. Harper and Row, New York Gilby, A. R. and Cox, M. E. (1963). The cuticular lipids of the cockroach, Periplaneta americana L. J. Insect Physiol. 9, 671-681 G u m , D. L. (1933). The temperature and humidity relationsof the cockroach BIatta orientalis. 1. Desiccation. J . Exp. Biol. 10, 274-285

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Hackman, R. H. (1974). Chemistry of the insect cuticle. In “Physiology of Insecta” (Ed. M. Rockstein) Vol. VI, pp. 216-270. Academic Press, New York and London Hadley, N. F. (1978). Cuticular permeability of desert tenebrionid beetles: correlation with epicuticular hydrocarbon composition. Insect Biochem. 8, 17-22 Hadley, N. F. (1979). Wax secretion and color phases of the desert tenebrionid beetle Cryptoglossa virrucosa (Le Conte). Science 203, 367-369 Holdgate, M. W. (1 956). Transpiration through the cuticles of some aquatic insects. J. Exp. Biol. 33, 107-118 Holdgate, M. W. and Seal, M. (1956). The epicuticular wax layers of the pupa of Tenebrio molitor L. J. Exp. Biol. 33, 82-106 Hurst, H. (1950). An electron diffraction study of the crystalline structure of the lipids in the pupal exuviae of Calliphora erythrocephala. J . Exp. Biol. 27, 238-252 Jackson, L. L. and Blomquist, G. J. (1976). In “Chemistry and Biochemistry of Natural Waxes” (Ed. P. E. Kolattukudy) pp. 201-238. Elsevier, Amsterdam, Oxford and New York Larsson, K. and Lundstrom, I. (1976). Liquid crystalline phases in biological model systems. A d v . Chem. Ser. 152, 43-70 Lees, A. D. (1947). Transpiration and the structure of the epicuticle in ticks. J . Exp. Biol. 23, 397-410 Locke, M. (1941). Pore canals and related structures in insect cuticle. J . Biophys. Biochem. Cytol. 10, 5 89-6 18 Locke, M. (1965). Permeability of insect cuticle to water and lipids. Science 147, 295-298 Locke, M. (1974). The structure and formation of the integument of insects. In “Physiology of Insecta” (Ed. M. Rockstein). Vol VI, pp. 124-213. Academic Press, New York and London Lockey, K . H. (1976). Cuticular hydrocarbons of Locusta, Schistocerca and Periplaneta, and their role in waterproofing. Insect Biochem. 6 , 457-472 Loveridge, J. P. (1968). The control of water loss in Locusta migratoria migratorioides R & F. 1. Cuticular water loss. J . Exp. Biol. 49, 1-13 Mead-Briggs, A. R. (1956). The effect of temperature upon the permeability to water of arthropod cuticles. J . Exp. Biol. 33, 737-749 Melchior, D. L. and Steim, J. M. (1976). Thermotropic transitions in biomembranes. Ann. Rev. Biophys. Bioeng. 5 , 205-238 Nelson, D. R. (1979). Long-chain methyl-branched hydrocarbons: occurrence, biosynthesis and function. A h . Insect Physiol. 13, 1-33 Neville, A. C. (1975). “Biology of Arthropod Cuticle”. Springer-Verlag, Berlin, Heidelberg and New York Noble, P. S. (1974). “Introduction to Biophysical Plant Physiology”. W. H. Freeman, San Francisco Noble-Nesbitt, J. (1977). Active Transport of Water Vapour. In “Transport of Ions and Water in Animals” (Eds B. L. Gupta, R. B. Moreton, J. L. Oschman and B. J. Wail) Vol. 5 , pp. 571-597. Academic Press, New York and London Oloffs, P. C. and Scudder, G. G. E. (1965). The transition phenomenon in relation to the penetration of water through the cuticle of an insect, Cenocorixa expleta (Hungerford). Can. J. Zool. 44, 621-662 Ramsay, J. A. (1935a). Methods of measuring the evaporation of water from animals. J . Exp. Biol. 12, 355-372


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Ramsay, J . A . (1935b). The evaporation of water from the cockroach. J . Exp. Biol. 12,373-383 Spink, C. and Wadso, I. (1'176). Calorimetry as an analytical tool. Method. Biochem. Anal. 23, 1-159 Toolson, E. C. (1978). Diffusion of water through the arthropod cuticle: thermodynamic consideration of the transition phenomenon. J . Thermal Biol. 3, 69-73 Toolson, E. C. White, T. R.. and Glaunsinger, W. S. (1979). Electron paramagnetic resonance spectroscopy of spin-labelled cuticle of Centruroides sculpturatus (Scorpiones: Buthidae): correlation with thermal effects on cuticular permeability. J . Insect Physiol. 25, 271-275 Treherne, J . E. and Willnier, P. G. (1975). Hormonal control of integumentary water loss: evidence for a novel neuroendocrine system in an insect (Periplaneta americana). J . Exp. Biol 63, 143-159 Vannier, G. (1974). Calcul d e la resistance cuticulaire B la diffusion de vapour d'eau chez un insecta Collembola. C.R. Acnd. Sc. Paris Ser. D. 278, 625-628 Wharton, G. W. and Richards, A. G. (1978). Water vapor exchange kinetics in insects and acarines. Ann. Rev. Entomol. 23, 309-328 Wigglesworth, V. B. (1945). Transpiration through the cuticle of insects. J . Exp. Biol. 21, 97-114 Wigglesworth, V. B. (1975). Incorporation of lipid into the epicuticle of Rhodnius (Hemiptera). J . Cell Sci. 19, 459-485


Intercellular Junctions in Insect Tissues Nancy J. Lane and Helen leB. Skaer A. R. C. Unit of Invertebrate Chemistry and Physiology, Department of Zoology, Cambndge, UK

1 Introduction 36 1.1 Techniques 37 2 Septate junctions 43 2.1 Introduction 43 2.2 Pleated septate junction - structural features 44 2.3 Smooth septate junction - structural features 54 2.4 Occurrence in insects 62 2.5 Distribution in other invertebrates 65 2.6 Occurrence in vertebrates 67 2.7 Functional significance 69 2.8 Formation of septate junctions 73 3 Desmosomes 75 3.1 Introduction '75 3.2 Structural features 76 3.3 Occurrence in insects 79 3.4 Distribution in other invertebrates 8 0 3.5 Functional significance 83 3.6 Development 84 4 Gap junctions 85 4.1 Introduction 85 4.2 Structural features 87 4.3 Model derived from structural evidence 93 4.4 Distribution of gap junctions 94 4.5 Structural differences between arthropod and vertebrate gap junctions 98 4.6 Functions of gap junctions 100 4.7 Dynamics of gap junctional formation and disassembly 109 4.8 Co-existence with other junctional types 118 5 Tight junctions 120 5.1 Historical introduction: d o tight junctions exist in insect tissues? 5.2 Structural features 126 5.3 Model derived from structural evidence 131 35

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36

6

7

8 9

5.4 Distribution and localization in arthropod tissues 132 5.5 Co-existence with other junctional types 138 5.6 Homo- and heterocellular tight junctions 138 5.7 Comparison with vertebrate tight junctions 138 5.8 Functional significance 141 5.9 Developmental stages in tight junction formation 146 5.10 Tight junction degradation 149 5.1 1 Phylogenetic and evolutionary position 150 Specialized junctions of glia 151 6.1 Introduction 151 6.2 Axo-glial junctions 152 6.3 Tracheo-glial junctions 157 Scalariform junctions 157 7.1 Introduction 157 7.2 Thin-section appearance 159 7.3 Preparations treated with tracer 162 7.4 Freeze-fracture replicas 162 7.5 Model derived from structural evidence 166 7.6 Distribution in insect tissues 168 7.7 Physiological significance 170 7.8 Junctional development 172 Reticular septate junctions 172 8.1 Rectal papillae of dipteran insects 172 8.2 Peripheral retina of dipteran insects 177 Concluding remarks 180 9.1 Development of junctions 180 9.2 Functional considerations 181 9.3 Correlation of insect physiology with junctional structure Acknowledgements 183 References 184

182

GLOSSARY O F TERMS

smooth septate junction = continuous junction zonula adhaerens = belt desmosome fascia adhaerens = discontinuous belt desmosome macula adhaerens = spot desmosome gap junction = nexus = macula comrnunicans = close junction tight junction = zonula occludens = occluding junction terminal bar = zonula adhaerens plus zonula occludens The material illustrated in the plates derives from adult specimens unless otherwise stated

1 Introduction

Over recent years the number of studies on intercellular junctions in invertebrate tissues has enormously increased. Moreover our understanding of the ultrastructure and functioning of these cell-to-cell associations has

INTERCELLULAR JUNCTIONS I N INSECT TISSUES

37

improved due to a variety of technical improvements and ingenious experimental designs. Reviews of insect junctional complexes were last published in 1973 (Satir and Fong, 1973; Satir and Gilula, 1973) although an abbreviated account has appeared more recently (Lane, 1978)*. However, a number of developments have occurred since then. These include more detailed analyses of the regulation and physiology of cell coupling by gap junctions, more precise distinctions between septate and continuous (smooth septate) junctions and the accumulation of evidence that establishes the existence of tight junctions in arthropod tissues. More information on scalariform junctions has also been forthcoming and other membrane modifications, possibly junctional, have been reported (axo-glial junctions and reticular septate junctions). Studies on developing systems have been carried out which elucidate details of junctional formation. Moreover, a fuller understanding of the functional significance of certain intercellular junctions has also been achieved. 1.1

TECHNIQUES

Studies on intercellular junctions generally incorporate one or more of the following four techniques: ( i ) En bloc uranyl acetate (UA) stained thin sections of fixed tissues which show the arrangement of the unit membranes of the adjacent cells with respect to one another and make possible a clear indication of the size of the intercellular cleft; (ii) lanthanum impregnation of the material which stains the extracellular space between the two apposed cells and reveals the details of the true membrane surface as well as any cross-linking structures within the space, and (iii) freeze-cleaving that displays the features of the fractured faces of the cell membranes consisting of E face and P facet, which possess different components characteristic in each case of the junction under consideration. (iv) Some idea of the permeability of the junctional structures may be gained from in vivo incubation of the tissue in physiological saline containing tracers. Each of these techniques will now be considered in slightly more detail, because the first three, at least, are essential in order to categorize junctions correctly. * Since this review was submitted, a review concerned with septate and scalariform junctions in arthropods has appeared, Noirot-Timothbe and Noirot, 1979 t The terminology of Branton et al. (1975) has been used in this review whereby the fracture face overlying the extracellular space is designated by EF, and that adhering to the cytoplasm, PF (see Fig. 1)

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NANCY J. LANE AND HELEN leB. SKAER CYTOPLASM

-/

/-

E FACE

-1

Fig. 1 Diagram to illustrate the way the membranes of two apposed insect cells appear after freeze-fracturing and after lanthanum-incubation. Freeze-fracturing reveals intramembranous substructure, in that the membranes cleave along the mid-line producing an outer membrane half or E face (as shown in the top membrane), and an inner membrane half or P face (as shown in the bottom membrane). Only one of the two fracture faces is revealed in any given area of replica but the plane of cleavage may pass from the PF (or EF) of one cell to the EF (or PF) of the adjacent cell. Since the fracture plane then passes through the intercellular space, some estimate of the size of the cleft as well as the degree of complementarity of intramembranous structures on the two faces can he made. Lanthanum, which infiltrates the space (*) between membranes, will reveal features of this extracellular space only but illuminates very well any structure that is present there, for example, septa or columns. A combination of these two techniques will reveal most features of junctional modifications

1.1.1 Revealing intercellular dimensions b y en bloc U A staining Staining en bloc with uranyl acetate (Farquhar and Palade, 1963) clarifies the membrane image and so allows a more precise assessment of the intercellular space in accurately cut transverse sections. This has been of particular importance in distinguishing gap junctions (2-3 nm intercellular space) from tight junctions (intercellular space occluded) (see Sections 4.2.1 and 5.2.1). 1.1.2

Revealing intercellular structures b y “negative” staining

In order to reveal the fine structural details of the intercellular space and the junctional modifications therein, staining of the extracellular cleft matrix with heavy metals is frequently employed, so that the components of the junctions will be, in effect, “negatively” stained. The junctional elements remain unstained and electron-lucent, but their features, arrangement and distribution are highlighted by the electron density of the stained matrix background in which they lie.


39

This technique was originally employed using colloidal lanthanum nitrate as the negative stain (Revel and Karnovsky, 1967; Goodenough and Revel, 1970). However, any molecule which will bind to the often highly-charged matrix is suitable, and substances such as tannic acid (van Deurs, 1975) and ruthenium red (Luft, 1971), have also been employed. In addition, when material is treated with fixatives including calcium, subsequent treatment with uranyl acetate in the presence of calcium leads to images comparable to lanthanum-treated ones, as the uranium fills the extracellular space, delineating the structures that lie therein. Whether the uranium replaces calcium or binds to a mordant formed by the calcium in the extracellular matrix, is not yet clear, but this method may be referred to as “uranium calcium en bloc staining” (Wallet al., 1975), in contrast to the more conventional “uranyl acetate en bloc staining” (Farquhar and Palade, 1965) for enhancing membrane structure as referred to earlier. Depending on the plane of section, the stained intercellular cleft will reveal the non-opaque intercellular structures either along the longitudinal axis or transverse axis, while tangential sections elucidate the threedimensional structure or interrelationships of these junctional components. Such methods often clarify the detailed features of the junction under consideration because exclusion of tracer by a structure usually gives a clearer impression of its characteristics than positive staining, which cannot distinguish structure from background as easily or effectively. This method, when applied to a junctional region in conjunction with freeze-fracturing, enables the investigator to work out both intercellular and intramembranous structures and at the same time to determine in what ways such structures may be coincident and hence how they might be physically interrelated. From such evidence, three-dimensional models of junctions can be constructed. Without applying both negative-staining by tracers and freeze-fracturing to any given junction, it is not possible to discover all the intricacies of its structure. 1.1.3 Revealing intramembranous structures by freeze-fracture Freeze-fracturing (or freeze-cleaving) is a technique which enables the investigator to see an en face view of membranes in a single unique fracture plane, and hence to observe the relative size and position of any intramembranous components that may be present. Earlier investigations demonstrated that the plane of cleavage passes through the interior of membranes, not along the surface (Branton, 1966; Pinto da Silva and Branton, 1970; Tillack and Marchesi, 1970). Such a procedure exposes two fracture faces which were originally referred to as A and B but are now called the P and E face respectively (Branton et al., 1975). The P face refers

40


to the cytoplasmic half, and the E face to the extracellular half, of the membrane (Fig. 1). The true external surface of membranes can only be revealed by etching, or sublimation of the surrounding ice. When a membrane is fractured, two complementary surfaces are revealed (Fig. 2) so that for every particle seen on, for example, the P face, a

Fig. 2 Freeze-fracture replica from an insect transporting epithelium (Culliphoru salivary gland) to demonstrate the characteristic appearance of the two fracture faces of the cell membranes. The P face (PF) possesses many intramembranous particles (IMPs) which vary in size and have an irregular distribution. The E face (EF) has fewer IMPs which lie scattered through the lipid layer; this interpretation assumes that the membrane structure conforms to the fluid mosaic model formulated by Singer and Nicholson (1972). x 81 300

corresponding pit or depression is to be found on the E face. These pits are often less obvious than the intramembranous particles (IMP), for the angle of shadowing may be too shallow to reveal their presence or they may in other ways become obscured. The smooth areas of the membrane faces are assumed to be the central hydrophobic part of the lipid bilayer, while the IMPs are generally considered to be intercalated protein of some kind (Branton and Deamer, 1972; Pinto da Silvaet al., 1971), possibly enzymatic, structural or antigenic. Recent evidence, however, indicates that lipid by itself can form IMPs and that such particles exhibit complementarity; these may represent inverted micelles of phospholipid (Verkleij et al., 1979). It


41

seems clear, therefore, that care must be taken in the interpretation of the nature of any IMPs. Frequently, the P face of membranes possesses a n abundance of IMPs while the E face is less well endowed; this occurs because the fracture goes along the path of least resistance and particles remain attached t o the PF because they a r e thought to be more firmly bonded t o that face in t h e frozen state (Bullivant, 1974). T h e existence of particles within the plane of the membrane is consistent with the fluid mosaic model of biological membrane structure (Singer and Nicolson, 1972). When intercellular junctional membranes a r e cleaved, it must b e remembered that when o n e sees a P face and E face together, the P face will belong to the cell underneath, and the E face to the cell overlying it, so that the space between these particular two faces represents the intercellular cleft (Figs 1 and 2). This enables o n e t o see any reduction o r expansion in this extracellular space. Hence, when o n e structure o n t h e P F is seen, the comparable structure in the EF is said to be complementary, but it is not in fact in the other half of the same membrane. Complementary replicas a r e required t o see both halves of the same membrane. However, with junctions, the structures within the membranes on either side of the extracellular space are usually symmetrical a n d so the term complementary is still used in these circumstances, and will be throughout this review. For the uninitiated, it is worth noting that with freeze-fracturing, the observer is looking at a replicu of the fractured material. T h e tissue is removed by chemical agents following shadowing and carbon-backing the fractured surface, and the thin metallic replica can then be viewed by transmission electron microscopy. T h e micrographs of freeze-fracture replicas are by convention mounted with the metallic shadow coming from the bottom o r side, a n d all the freeze-fracture illustrations used in this review are mounted in this way. 1.1.4 Permeability studies by in vivo incubation with tracers The presence and distribution of certain kinds of junctions can be determined by the demonstration of a restriction to free permeability. Ions and molecules of various sizes can h e employed as in vivo tracers in fine structural studies t o reveal the sites of any such diffusion barriers. Ionic lanthanum chloride is often used (Machen, et al., 1972; Lane, 1972; Lane et al., 1975a, 1977a) because it is relatively small a n d the hydrated lanthanum ion is 0.92 nm in diameter (Robinson a n d Stokes, 1970); however, since it carries a high positive charge there is a tendency t o associate with structures that are negatively-charged a n d care must therefore be taken in interpreting results. In studies on permeability, however, it is important t o carry out these physiological in vivo incubationsprior to fixation, since fixatives may modify the fine structure of diffusion barriers. I n vivo ionic lanthanum incubations

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N A N C Y J. L A N E A N D HELEN leB. S K A E R

prior to fixation may, therefore, yield different information from the study of tissue fixed in the presence of colloidal lanthanum. Incubation in vivo with solutions of exogenous enzymatic proteins such as microperoxidase (M. W. 1900) (Feder, 1970) and horseradish peroxidase (HRP) (M.W. 40000) (Brightman and Reese, 1969) has shown these substances to be useful tracer molecules but, being macromolecular, their usefulness in determining the accessibility of very small openings or spaces is limited. Caution must also be exercised with respect to dosage and enzymatic activity since these tracers can produce permeability changes at high concentration (see Lewis and Knight, 1977). The movement of ions and molecules through extracellular channels gives an indication of the extent to which they can freely diffuse before junctions interfere with their entry; however, when barriers are demonstrated, they restrict the movement of that particular tracer, and perhaps not necessarily of other molecules of a smaller size or different chemical structure. In this respect, HRP and microperoxidase can be used to give an indication as to whether or not a diffusion barrier exists, and hence whether junctional complexes are likely to be present, forming its morphological basis. A smaller molecular weight molecule such as lanthanum, however, is likely to give more information about the subtleties of the junctional distribution, revealing at greater resolution the finer structural details.

Throughout this review, representative citations are given for the various points raised. In some especially crucial cases, all the known supportive reports will be listed, but ordinarily a range of examples will be presented in order to give the reader an idea of the scope of the papers that touch upon that particular issue. The reasons for this of course are obvious, since in many instances there have been innumerable passing references made to junctions without any further details as to their structure or function. There is one aspect of junctional elaboration that we do not intend to consider in this article. These are the specialized structures developed where three junction-bearing membranes become connected, coined tricellular junctions. Distinctive junctional structures are found in these areas and have not always been recognized as relating to tricellular associations but have been described as novel elaborations in the established structures of various junctional types, These misinterpretations as well as a detailed consideration of arthropod tricellular junctions of the septate and scalariform types has been included in a recent review (Noirot-TimothCe and Noirot, 1979).

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2

2.1

43

Septate junctions INTRODUCTION

Septate junctions, so called because of their ladder-like appearance in transverse section, were first described as septate desmosomes in Hydra by Wood (1959) although Fernhndez-Moran (1958) had previously shown their presence in the insect eye but identified them as “ultratracheoles”. Subsequently junctions having closely similar, though not necessarily identical, characteristics have been described very widely in many invertebrate phyla (for example, Overton, 1963; Wiener et af., 1964; Locke, 1965; Gouranton, 1967; Messier and Sandborn, 1967; Noirot and NoirotTimothCe, 1967; Bullivant and Loewenstein, 1968; Danilova et af., 1969; Gilula et a f . , 1970; Leik and Kelley, 1970; Hand and Gobal, 1972; Flower 1972; Noirot-TimothCe and Noirot 1973; Satir and Gilula, 1973; Flower and Filshie, 1975; Dallai, 1975, 1976; Filshie and Flower, 1977; Wood, 1977; Noirot-TimothCe et al., 1978; Lane and Harrison, 1978; Skaer et af., 1979). Reports of septate junctions in vertebrate tissues (for example, Bargmann and Linder, 1964; Laatsch and Cowan, 1966; Peters, 1966; Hirano and Danbitzer, 1967; 1969; Barros and Franklin, 1968; Lasansky, 1969,1971; Gobel, 1971; Friend and Gilula, 1972; SoteloandLlinBs, 1972; Enders, 1973; Livingston et a f . , 1973; Schwartz, 1973; Aetorfer and Hedinger, 1975; Connell, 1978; Nistal et af., 1978a,b) bear only superficial resemblances to the invertebrate junctions and may in most cases be considered as separate, unrelated junctions (for further discussion see Section 2.6) Berridge and Oschman (1969) and Danilova et af. (1969) independently suggested that septate junctions in invertebrate species could be subdivided into more than one type on the basis of their morphology. Danilova et af. (1969) specified two types, the true “septate desmosome”, found in Hydra, and the “comb desmosome” found in insects. Furthermore they speculated that some other modifications of the septate junction might be found and indeed shortly afterwards Leik and Kelly (1970) carried this subdivision a step further by suggesting a phylogenetic progression from junctions with parallel shelves of septa (hydroids and flatworms) to pleated shelves (leeches and bivalves) to hexagonal or honeycomb arrangements (insect tissues). Flower and Filshie (1975) subsequently suggested that a junction, relatively newly characterized in insects and previously described as a “continuous” junction (Noirot and Noirot-TimothCe, 1967, 1972; Dallai, 1970; Oschman and Wall, 1972; Satir and Fong, 1972; Reinhardt and Hecker, 1973; Noirot-TimothCe and Noirot, 1974), should in fact be recognized as a further variant of the septate type. This junction they called the smooth

44

N A N C Y J . L A N E A N D HELEN leB. SKAER

septate junction to contrast with the previously established honeycomb septate junction (which they termed “pleated” septate junction). The distinction between these two types depends on clearly distinct morphological characteristics rather than phylogenetic occurrence. Green (1 978), in a brief report, enumerated eight morphological types of septate junction which he clamed to be phylogenetically distinct. Green’s published evidence is solely from tissues infiltrated with lanthanum but corroborates previous findings of other workers (e.g. Baskin, 1976). Further evidence (in press) from thin-sections and freeze-fracture (Green el al., 1979a; Green and Flower, 1980) should, when published, shed light on this proliferation of septate junctional types. Until recently, only two types of true septate junction have been described in insect tissues, the original “comb desmosome” or pleated septate junction, as it is now normally called, and the continuous or smooth septate junction. (For a discussion of other variants of septate-like junctions found in insect tissues see Sections 7 and 8). 2.2

P L E A T E D S E P T A T E JLINCTION - S T R U C T U R A L FEATURES

2.2.1 Thin-section appearance In thin section (Figs 3 and 6, insert) pleated septate junctions can be distinguished by the regular intercellular spacing of the membranes which are fairly rigidly separated by 15 nm. The intercellular space is punctuated by septa of uniform thickness (normally 8-9 nm, although Caveney and Podgorski (1975) reported septa only 2-3 nm wide in Tenebrio epidermis) which in some areas may show a periodic separation (the precise spacing Fig. 3 Pleated septate junction from the tracheole investment of the eye of the locust, Schistocerca gregaria showing the regular interseptal distance and prominent septa typical of this junction in thin-section. The circular insert shows an enlargement allowing resolution of the septal substructure (arrow) and a darker spot traversing the membrane at the point of contact with the septum (arrowheads). Insert (lower left-hand corner) shows a septate-like junction that is found between axons in the central nervo,ussystem of the housefly, Musca domestica; this displays certain differences from the conventional pleated septate junction in that the intercellular space is narrower, the septa are of different dimensions, and the junction is limited in distribution, presumably to a macular area between the two apposingaxon surfaces X 158 300; circular insert x 239 100; inset x 142 700 Fig. 4 Thin-section passing obliquely through the pleated septate junctions of the epidermis of Rhodnius prolixus. The honeycomb appearance deriving from the juxtaposition of the pleated septa is clear (see text for further explanation of this phehomenon). The arrow indicates the one region of transversely-sectioned septate junction that is present, straddled by the areas of oblique-section, cutting through the intercellular septal sheets. X 50 600 Fig. 5 Pleated septate junction from the testis of the cockroach, Periplaneta americana. The preparation was fixed in the presence of lanthanum hydroxide which has permeated the intercellular space, revealing the septa in negative contrast. Note the variation in thickness of the septa as the angle of section relative to the orientation of the septum alters. x 164 300

INTERCELLULAR J U N C T I O N S I N INSECT T I S S U E S

45

NANCY J. LANE AND HELEN leB. SKAER

46

appears to vary with species but is in the region of 16-20 nm; NoirotTimothee and Noirot, 1973, reported it to be as low as 9 nm) or which may show a considerable variation in the interseptal distance. Indeed the degree of order in the spacing of the septa can vary considerably. Some of this variation is due to the angle of sectioning relative to the regular undulations of the septa (as explained in the original description, see Danilova et al., 1969, Figure 10; see also Flower and Filshie, 1975, Figure l ) , but even bearing this in mind, the transverse sections of some septate junctions present a much more ordered appearance than others ( c j Figs 3 and 6 insert with insert to Fig. 7). The septate junctions that occur between insect nerve cells (Fig. 3 square insert) appear to be rather different from the normal epithelial ones. They seem likely to be macular rather than zonular, and their structure, for example that of septa, appears to be distinctive (see also Smith, 1967). The septa themselves appear at higher power to have some kind of subunit structure (Fig. 3, circular insert) such as has been suggested previously by Noirot-TimothC and Noirot (1973). They concluded that each septum had two pairs of side arms, projecting at right angles to the septum and on either side of it. This model is consistent with the image arrowed in Fig. 3 (circular insert). The enlarged area in the circular insert also illustrates that the membrane itself may be transversed by an electron dense spot at the point of contact between septum and membrane. Such images have also been seen by Reinhardt and Hecker (1973) after staining with periodic acid-TCH silver proteinate (Thiery, 1967). Fortuitous sections passing tangentially through the junctional membrane sometimes reveal details of the septa in en face view (Fig. 4). It was sections such as these that allowed the original distinction to be made between the insect septate junctions and those of Hydra (Danilova et al., 1969). The septa present an overall honeycomb arrangement but close inspection reveals that individual septa follow a sinusoidal pattern and that it is the alignment of adjacent septa 180" out of phase with one another that produces the overall hexagonal appearance (Fig. 36, insert A) (see Gilula et d., 1970 for optical diffraction of the junction in en face view).

2.2.2

Lanthanum infiltration

After infiltration with lanthanum salts the intercellular space appears electron dense and solid structures in the intercellular cleft appear electron Fig. 6 Freeze-fracture preparation from the testis of Periplanetu americanu to show the rows of particles on the P face (PF) with corresponding pits on the E face (EF). The rows of particles and grooves are fairly regularly spaced in some areas and the junction shows a relatively high degree of order. The insert shows the thin section appearance of a similarly ordered junction from tracheole cells in the tissues of the eye of the locust. X 94 100; insert X 127 000


47

48

N A N C Y J L A N E A N D HELEN I&.

SKAER

lucent. The intercellul:ir s p i c e in the ;ire;i\ ot the junction. in transversc section. is \ l i s h t l y larger ( c t r . I 7 n m ) than in convcnticnally \t;iinecl preparations d u e t o optical f u \ i o n o f the electrov-dense intercellular pace with the osmiophilic outer nienihrane leiitlets on either aide o f it. I he w p t a show up a s electron-lucent b a r s often nar-I-owcr- (4-5 n m ; Satir a n d (iilula. 1973) than i n nci n - in ti It r;i t ecl \ peci ni e n \ . In t;ingenti;il section\ t n c septa are \trikinglj reve;iled ;I\ clear undulating rihtmns set in ; i n electron-de:iw lake ot \tain (Fig. 5 ) . 'l'hc ril,lx)n\ :'un together in more 01- les\ pi-:ilIel t>;ind\ with occ;ision;il iii-cas w h e r e the tight n e \ 4 o f pa c I\ ing 1 ()o\c n \ ;I n d in d iv id 11;I I \c p t ;I ;I cc()ni p I is ti w h (1I- I \ o I- I o o ps alone ( w e . f o r example. illu\ti-ations in N o i r o t - ' l ' ~ n ~ o t h i .acn d Ncirot, 1953). .[his paper ( N o i r o t - I in1othi.e and N o i l - o t . 1073) ;i!so reve;il\ that there i s con\idcral,le \ a r i ; i t r o n in the thickne\\ o f t h e w p t a a n d t h ; i t an individual septum inah var! in thicknes4 a l o n g i t \ Icngth ( f r o n i 3- 1 1 rim. see their Plate IV: \ec al\o Figure 5). T h i \ i\ p i - o t ~ ~ due i l ~ to ~ ~the prcciw angle of wetion w i t h re\pcct to the \epta thc~ni\cl\.c~\. (C'a\cney a n d Podgo:-\ki. 1075) h i i t might. ;I\ Noir-~,t-.l'iriic)thi.ei t n d Noirot \ugge\t. indic;ite ;I v ; i r i i i t i o i i in the width o f i h c \eptum :icro\\ the intcrcellular \piice. .l'he regularity o f the sinii\oid;il u n d u l a t i o n \ of the wpta a r e elearl! w e n after ianthanum infiltrat i o n (Fig. 5 ) : the periodicit! o f the w ; i ~ eha\ twcn reported b y various author4 ; I \ 2 0 - 7 2 n n i ( ( i i l u l a c'i t i / . , 1 9 7 0 ; Noirot-~l'iniottiiie a n d K o i r o t . 197.3: StacLhelin. lO7-4: Noii-ot-.l'iriiottii.c('I t i / , , 1978). 1n ce rt ;Ii n pre p;i i-:it io n \. \c c t io 114 ( ) f I ;I 11t h ;I 11 I I ni -in fi I t ir:it cd ni ;i t e ria I \how sidearms ar-i\ing f r o n i the ci-c\t of tach w a c~(Noirot-~!im~)tlii.e a n d Noirot. 1073; (ireen 1978): i i i \ome c;ise\ the \ide a r m s f r o n i :!dJ:iccnt wpta ,join t o form pattern\ o f complete I:c\agon\ ((ireen. 1978; c/'. ( i i I ~ i l ; i ('I t i / . , 1970). Even without \.i\il,lc \idcar-m\. ;idlacent i-itition\ 180" o u t o f phase with one another give ri\c to the appearance iiescrihed ;I\ honeyconit, b y Loeke (1975) a n d I ) : i n i l o \ ; i ( ' I t r / . ( 1 % 0 ) (Fig, 3 a n d inw1-t A in Fig. 3 6 ) . This honelcomt-,appcai-ancc ill lie enha:iccti if the adjacen! w p t a . r u n n i n g 1x0" out o f ph;i\e. ;II-C clow together and the p;itter:i M i l l he tiirthci- I-cinforccd where the :implit uclc of the \cptiil \v:i\ e i \ regular ;ind o f con\idei-:il~lc:.izc. The rihhons riiay h o \ \ c \ c r aI\o irun together in ph;iw (Figure IV o f N o i r o t Tiniothiie and Noirot. 107.3) 01-cornplctel! o u t o f register. 'l'hii\. in t h i \ I-espect ; i l l the model\ t h ; i t ha\.c heen pi-opo\ed ( 1 3 i i l l i \ , a n t a i ~ d1,oeweristcin. 1968; (iilula ci t i / , , 1970;Yoi:-ot-.l'imotiiiic and Noirot. 197.;; Staehelin. 1073; C'a\cncy a n d Podgoi-\ki. 1075) ( a n d t l i i i t pi-opo\cci i n thi\ revie\\. Figure 10). reprewnt ideali/ed gencr;ili/;ition\ o f ;I \truetiire e\hihiting considcral>lc \ iiri:ition.

INTERCELLULAR JUNCTIONS IN INSECT TISSUES

49

2.2.3 Freeze-fracture Freeze-fracture of septate junctions was first carried out on Mollusc tissues by Flower (1970, 1971); Gilula et al. (1970) and Gilula and Satir (1971). The replicas revealed characteristic modifications of the membranes associated with the septate junctions, ranks of more or less parallel rows of particles were found on the PF, corresponding rows of depressions or pits occurring on the E F (as in Figs 6 and 36). Their results have subsequently been confirmed and extended t o the septate junctions of insects (e.g. Flower, 1971; Satir and Fong, 1972; Noirot-Timothee and Noirot, 1973; Satir and Gilula, 1973; Dallai et al., 1977; Noirot-TimothCe et al., 1978). The particles are normally 8.5 nm in diameter (Noirot-TimothCe et al. (1978) give a range of 7-10 nm) and show a somewhat variable separation (although Gilula et al., 1970, examining mollusc tissues, claim a regular centre-tocentre spacing of 21 nm confirmed by optical diffraction). A recent report by Noirot-Timothkeetal., (1978), in which a number of species are considered, gives variations as large as 9.5-20 nm. The variation in spacing between adjacent rows of particles and the degree to which they run parallel to one another is also variable (see Figs 7 and 8); Flower (1970, 1971) g'ives a minimum figure of between 13 and 1 7 nm, and Noirot-TimothCe et al. (1978) a separation of 16 to 20 nm, when the rows are running parallel. Few junctional particles are found on the E F of the junction (Figs 6 and 36) although complementarity of the P F particle rows and the E F depressions can be seen where the fracture passes through the intercellular space and the two membranes of the junction are revealed in adjacent areas (Figs 6 and 36). Chemical fixation has a marked effect on the fracturing characteristics of smooth septate junctions in some tissues (see Section 2.3.3) but this has not been reported for pleated septate junctions. In the latter, the particles fracture into the P F and the depressions appear on the EF whether the tissue has been fixed prior to glycerination and freezing (e.g. Gilulaetal., 1970) or not (e.g. Flower, 1970). The degree of order displayed by the bands of particle rows shows considerable variation both within a single junction but also and to a greater extent between the junctions of different tissues or from different regions of the same tissue. This is illustrated in Figs 6, 7 and 9, which are freeze-fracture preparations from the testis of the locust and the cockroach. Thin sections reveal similar variations in septa1 organization (as shown in Fig. 3 and insert to Fig. 6, in comparison with the insert to Fig. 7). In the testis, septate junctions between cyst cells that envelope the groups of developing germ cells are very loosely organized, with rows of particles that are widely separated and show no indication of lining up in parallel (Fig. 7). In the peripheral layers of cells ensheathing each follicle (the follicle cell layers),

50

N A N C Y J. LANE A N D HELEN leB. SKAER

.

0

.


51

the septate junctions display considerably more complex arrays of particle rows (Figs 6 and 9). Junctions showing an intermediate organization are found where the cyst cell layers merge into the follicle cell layers. A septate junction exhibiting a very loose organization and no E F elaborations in freeze-fracture replicas has recently been described in the rectal sheath cells of termites and cockroaches (Noirot et ul., 1979). Smooth septate junctions are known to show topographical variations in the degree of septa1 and freeze-fracture particle (ridge) organization (Flower and Filshie, 1975; Graf, 1978a; Skaer et ul., 1979; Noirot-TimothCe and Noirot, 1979; Juperthie-Jupeau, 1979). As yet this has not been described for pleated septate junctions although variations such as those illustrated in Figs 7-9 might well be topographically related. There do, however, appear to be genuine tissue differences. Only highly organized junctions are encountered, in our experience, in salivary gland, certain regions of the gut, follicle cell layers of the testis and between epidermal cells. Loosely organized junctions (or those with an intermediate organization) on the other hand, are frequently found in the ensheathing cells within the testis, between the perineurial cells of the central nervous system and in the eye (Skaer, 1979a). 2.2.4

Structural model

Various models have been put forward since septate junctions were first described in insect tissues by Locke (1961). Locke (1965) proposed a possible pattern of orientation of the lipid component of the membrane within the junctions, a facet that does not seem to have been explored further. The other models proposed (Wood, 1959; Bullivant and Loewenstein, 1968; Gilula et al., 1970; Flower 1971; Satir and Gilula, 1973; Noirot-Timothte and Noirot, 1973; Staehelin, 1974; Caveney and Podgorski, 1975) have been concerned with the arrangement of the septa within the intercellular space and the relationship between the septa and the rows of particles seen in freeze-fracture. Common to all models are the septa, either pleated or in honeycomb arrays, traversing the intercellular cleft Fig. 7 Freeze-fracture preparation of the testis of Schistocerca gregaria showing the PF of the loosely organized pleated septate junction, characteristic of the cyst cell layer. Insert shows the thin-section appearance of this type of loose septate junction; the septa are infrequent, irregular and may not lie perpendicular to the junctional membranes. X 47 900; insert X 121 400 Fig. 8 Pleated septate junction from the rectum of Periplaneta americana. The PF particle rows form a slightly more coherent pattern than was illustrated in Fig. 7, joining to run in parallel rows over some distance. However large areas of non-junctional membrane lie between many of the particle rows. x 66 800 Fig. 9 PF of the pleated septate junction found in the inner follicle cell layer of the testis of the locust Schbtocerca gregaria. The particles line up to form broad bands of parallel rows. This junction of intermediate organization should be compared with the more highly ordered type illustrated in Fig. 6. x 60 400

52

NANCY J. LANE A N D HELEN leB. SKAER

perpendicular to the cell membranes on either side. Two areas of controversy remain, firstly the degree to which the septa are inserted into the cell membranes of the junction, prophetically illustrated by Wood (1959, Figure l ) , and secondly the precise structure of the septa themselves. Authors vary in the degree to which they suppose that the septa and the cell membranes are interconnected. Wood (1959), Locke (1965) Gouranton (1967) and Flower (1971) suggested that the septa were not separated from the cell membranes at all but represented out-pushings from the adjacent cell membranes. Gouranton (1967) and Flower (1971) proposed that the septa are made up of two halves, one from each junctional membrane, which meet in the centre of the intercellular cleft. Gouranton (1967) supported this with micrographs of impressive clarity. Flower (1971) furthermore suggested that a dilation occurs in the septum whenever an intramembrane particle was present. If these dilations were offset, the septal undulations could be explained. Bullivant and Loewenstein (1968) and Gilulaet al. (1970), in papers favouring the septate junction as the structure underlying a direct route of intercellular communication, also proposed that the septa were continuous with the membranes. The route of communication proposed in each case was slightly different: Bullivant and Loewenstein (1968) suggested that the septa were arranged to enclose hexagons of high permeability membrane, sealed by the continuity of septa with membrane. O n the other hand. Gilula et al. (1970) on the basis of “negatively stained” oblique sections and freeze-fracturing, arranged the septa in their model as undulating ribbons of regular periodicity and with adjacent septa 180”out of phase, so creating the honeycomb pattern. This latter model forms the basis of subsequent ones (e.g. Staehelin, 1974; Flower and Filshie, 1975). However, Gilula and co-workers (1970) proposed that the septa insert into the membranes at alternate vertices of the pleats, the intramembrane structures responsible for this being the particles revealed by freeze-fracture. The continuity of particle-septum-particle they argued was the route of intercellular communication. Since septate junctions have been superceded by gap junctions as the structures underlying communication (reviewed by Bennett, 1978 and see Section 4.6.3), authors have exercised considerably more caution in assuming that the septa insert into the membrane (as in NoirotTimothCe and Noirot, 1973; Flower and Filshie, 1975; Caveney and Podgorski, 1975). Recently, in a study of several different insect tissues, Noirot-TimothCe et af. (1978) have shown that there is poor correlation between the periodicity of the septal undulations and the separation of the freeze-fracture particles within the rows. From this they conclude that there can be “no structural continuity throughout the thickness of the septate junction”. Their evidence does not show this but does indicate that insertion of the septa at regular intervals along the undulations (as proposed by Gilula


53

etal., 1970; Satir and Gilula, 1973; Satir and Fong, 1973; Staehelin, 1974) is unlikely. Noirot-TimothCe et al. (1978), as well as evidence presented here, show that there is a clear correlation between the topography of particle rows and septal bands and moreover that both fuse to give double structures and end abruptly. It therefore seems plausible to admit some relationship between the two structures (Fig. lo), although the exact nature of their association must await further elucidation. /-CYTOPLASM-/

'-'

FACE -'LANTHANUM IN INTERCELLULAR

Fig. 10 Model of the pleated septate junction to illustrate the way in which the images of the junction obtained by the different methods discussed may be united into a three-dimensional structure. For purposes of simplicity, controversial aspects such as the substructure of the septa and the precise relationship between them and the intramembrane particle rows have not been illustrated although we have assumed that the septa are attached to the membranes at points coincidental with the rows of particles seen in freeze-fracture. The ladder-like appearance is produced by transverse sections through the junction, as in Fig. 3 and insert in Fig. 6 . The E and P face correspond to freeze-fracture images as in Figs 6-9 and the lanthanum-stained intercellular space corresponds to the undulating ribbons seen in Fig. 5

The early models of insect septate junctions assumed the septa to be solid (Bullivant and Loewenstein, 1968; Danilovaet al., 1969; Gilulaetal., 1970) but, on the basis of heterogeneity observed in the intercellular space following lanthanum infiltration, various modifications have been suggested. Indeed the septa may be made up of morphologically distinguishable subunits as shown in Fig. 3 (circular insert). Staehelin (1974) suggested, on the basis of evidence published by Noirot-TimothCe and Noirot (1973), that the septa may consist of thin bands of material, limited to the central regions of the intercellular space, and slung between rows of thin pegs, whose insertion into the membrane is marked by the particles seen in freeze-fracture preparations (see Figure 59 in Staehelin, 1974). An open septal substructure would explain the permeability of the junction to tracers such as lanthanum salts but the evidence for anything other than solid septa is not yet convincing and the junctional permeability to tracers can be explained in terms of

54


the frequent breaks in septal continuity (see for example Noirot-Timothke et al., 1978). Caveney and Podgorski (1975) have extended this idea in proposing structures at right angles to the septa. They suggested that the intercellular space is bridged by narrow septal ribbons (3 nm wide) but that these septal ribbons are connected by two platforms, about 1.5 nm in thickness, which lie in the central region of the intercellular space and are coplanar with the plasma membranes (see Figure 1 5 in Caveney and Podgorski, 1975). The chemical nature of the septa has been investigated in a number of systems. Wood, 1959, Weineret al., 1964, Locke, 1965, Gouranton, 1967, Gilula et al., 1970 and Flower, 1971 suggested that they are an extension of the cell membrane. Noirot-TimothCe and Noirot (1973) on the basis of cytochemical studies suggest that they contain glycoprotein but probably lack lipid. Caveney and Podgorski (1975), consider that both the septa and the interseptal platform, which they suggest link adjacent septa, are formed by elaborations of the glycocalyx. However in the smooth septate junctions of the mosquito Aedes, the septa appear to lack polysaccharides (Reinhardt and Hecker, 1973) unlike those of the pleated septate junction (NoirotTimothCe and Noirot, 1973). Recent observations (Humbert, 1979) on the smooth septate junctions of a collembolan species, demonstrate the glycoprotein nature of the intercellular elaborations. The intercellular material has not been specifically characterized. Gilula et al. (1970) found that the intercellular matrix of a Mollusc pleated junction stained with ruthenium red, indicating the presence of acid mucopolysaccharide. Dallai (1970) demonstrated the presence of glycoproteins in the interseptal space of the smooth septate junction. Djaczenko and CalendaCimmino (1974) report that the “extracellular substance” of septate junctions from the epidermis of a variety of annelid species can be identified cytochemically as predominantly polysaccharide in nature. In view of the possible significance of this extracellular matrix (see Section 2.7) further studies concerning its nature and variation in junctions from different tissues would be of interest.

2.3 2.3.1

SMOOTH SEPTATE J U N C T I O N - STRUCTURAL FEATURES

Thin-section appearance

Continuous or smooth septate junctions are characterized in thin sections both by the regularity of the intercellular separation (14-17 nm wide) and, in general by the uniform electron opacity of the intercellular space (Fig. 11). However in some instances septa can be made out, either indistinctly (e.g. Noirot-Timothke and Noirot, 1967; Hudspeth and Revel, 1971; Dal-


55

Fig. 11 Thin-section appearance of the smooth septate junction from the apical part of the lateral border from the midgut of the housefly, Muscu dornesticu. The regularity of the intercellular space and the featureless electron opaque intercellular material are clear. At the apical extremity of the junction, just below the microvilli (MV), the junction is revealed cut tangentially (arrow) and the presence of fairly straight septa can be resolved. X 65400 Fig. 12 Sub-apical area of the smooth septate junction from Muscu domesticu midgut. This thin section includes an area exhibiting no clear septa and also a finger-like interdigitation cut transversely to reveal a ring of junction indicating clearly the septa1 nature of the intercellular structures. X 106400 Fig. 13 A preparation infiltrated with lanthanum hydroxide duringfixation from the midgut of the horseshoe crab, Limufus pofyphemus. The septa of the smooth septate junction are highlighted in negative contrast (as at arrows). x 77 400

lai, 1975; Reinhardt, 1975; Graf, 1978) or more clearly (see Fig. 12) (Flower and Filshie 1975; Skaer et al., 1979). Where septa are distinct it is difficult, on the basis of thin-sections alone, to distinguish smooth septate junctions from pleated septate junctions. Ancillary techniques such as infiltration of the extracellular spaces with stain (lanthanum salts or ruthenium red), special staining methods (e.g. the uranium calcium method described by Walletal., 1975) or freeze-fracture are necessary to classify the junction with certainty. As a result, various reports in the literature describe septate junctions that may well, on more comprehensive examination, prove to be smooth septate junctions (for example Messier and Sandborn, 1967; Bullivant and Loewenstein, 1968; Smithet al., 1969b; Hudspeth and Revel, 1971; de Priester, 1971; Herman and Preus, 1972; Johnson et aE., 1973;

56


Reinhardt and Hecker, 1973; Reinhardt, 1974; Houk and Beck, 1975; Mills et al., 1976; Hecker, 1977). 2.3.2

Lanthanum infiltration

Transverse sections of the junction, in tissue infiltrated with lanthanum salts, reveal non uniformity in the slightly enlarged intercellular space. Frequent clear bars which exclude the stain traverse the intercellular cleft (Figs 13 and 14). Oblique sections show that these striations or bars are due to septa which run, without the regular undulations familiar in insect pleated septate junctions (see Section 2.2.2), over considerable tracts of the cell surface (Figs 14-18). The septa are fairly uniform in diameter, 4-6 nm wide, (Satir and Gilula, 1973; Flower and Filshie, 1975; Dallai, 1975) or slightly larger, 7-8 nm, (Skaer et al., 1979). The exact width will vary with nonperpendicular alignment to the plane of the section (see for example Fig. 16). Variation in width is also produced by lateral fusion of adjacent septa (thick arrow in Fig. 17). As with pleated septate junctions (Section 2.2.2), in Figs 14-19 All the preparations illustrated in this plate have been infiltrated with lanthanum hydroxide duringfixation and are concerned with the fine structure of smooth septate junctions Fig. 14 Preparation of Limulus polyphemus midgut where the plane of section has cut some parts of the smooth septate junction transversely and some obliquely. In oblique section the intercellular structures are more clearly delineated; relatively straight septa are separated by rows of electron-lucent particles, interpreted as sections through colums. Insert: at higher magnification the septa can themselves be seen to have a particulate substructure. x 140 000; insert x 295 000 Fig. 15 Part of the smooth septate junction from the Malpighian tubule OfRhodniusprolixus. The clear septa are separated by variable distances; some lie closely adjacent to each other forming doublets (thick arrows) and, where they are more widely separated, electron-lucent spots or colums in cross-section are evident. The thin arrows indicate blind-ending septa. X 259 700 Fig. 16 Apical region of the lateral border of the midgut of Musca domestica. The septa are closely packed with only occasional electron-lucent spots between adjacent septa. The junction can also be seen in transverse section, the intercellular space filled with lanthanum and giving only a suggestion of intercellular structure. The arrow indicates a blind-ended septum. X 163 500 Fig. 17 Sub-apical region of the lateral border of Musca domestica midgut. The septa are, for the most part, separated by one or more rows of electron-lucent spots, or transversely-sectioned columns. Septamay, however, fuse (thick arrow) andmay also terminate abruptly(white arrow). X 195 600 Fig. 18 Smooth septate junction from the midgut of Limulus polyphemus. The septa are separated in this region by double rows of electron-lucent particles arranged into a regular Maltese cross pattern. A septa1 termination is arrowed. In some cases septa run side by side in double rows (thick arrow). x 180200 Fig. 19 A slightly oblique section passing through the sub-apical region of the midgut of Musca domestica. The section is more nearly transverse at the top o f the figure where continuous thin clear lines can be made out (arrowheads). As the Dlane of the section deviates from the strictly transverse, the electron-lucent pegs or columns appear as spots or particles (arrow). x 237 800


57

58

N A N C Y J. L A N E A N D H E L E N leB. SKAER

favourable sections, a subunit structure of the septa may be made out (insert in Fig. 14; Lane and Harrison 1978; Skaeret al., 1979). The septa appear to be made up of adjacent subunits which seem to be arranged in groups of 2 , 4 or 6. Adjacent septa may lie close together with only a thin line of stain separating them (2-3 nm interseptal distance) (Figs 15-18) or they may be separated by larger and more variable distances and, in this case, electron lucent spots (2-4 nm in diameter) are normally found in the interseptal spaces (Figs 14-18), although Dallai (1976) reports that in the Malpighian tubules of Periplanetu these transparent pegs are not encountered. In most other tissues studied, however, and in particular the midgut, they seem always to be present, albeit exhibiting variability in distribution and numbers. Where they do occur these spots may line up to form single (Figs 1 6 and 17) or double (Fig. 18) rows between adjacent septa. More commonly, however, the septa are more widely separated with variable numbers of electron lucent spots in the interseptal space (Figs 14 and 15). These spots are thought to represent columns that span the intercellular space as can be seen from sections in which they are cut obliquely (Fig. 19, arrow heads; see also Figure 5, Flower and Filshie, 1975, in which the columns are shown cut obliquely both perpendicular to the septa and in parallel with and between them). The septa run for considerable lengths over the cell surface but are not continuous structures; blind endings are fairly frequently encountered in oblique sections (arrows in Figs 15-18). 2.3.3 Freeze-fracture Freeze-fracture replicas of the smooth septate junction are typified by series of ridges on one membrane with complementary rows of grooves on the other (Figs 20 and 21). The location of the ridges is dependent in some tissues on the treatment prior to freezing. In fixed tissues, the ridges appear on the PF with grooves on the EF; the ridges are approximately 10 nm in width and appear to be made up of short rods (Fig. 20). The EF grooves are overlaid by a variable number of particles (see Flower and Filshie, 1975). In certain tissues a different pattern emerges if the material is frozen unfixed. The ridges, more clearly moniliform in nature (particles about 10 nm in Fig. 20 Freeze-fracture preparation of the fixed midgut of Musca domestica. The apical extremity of the border is identified by the terminal microvilli (MV) and the smooth septate junction can be followed down towards the basal part of the cells. Its ordered appearance loosens as it extends basally. The ridges on the P face (PF) are complemented by E face (EF) grooves. Insert: in this preparation the gut was not fixed before processing for freeze-fracture. In this case, in contrast with the fixed tissue, the more clearly moniliform ridges cleave with the EF and the complementary grooves are found on the PF. Arrows indicate ridge termination. x 22 500; Insert x 65 700


59

60


diameter), are found on the EF, with grooves virtually free of overlying particles on the P F (insert in Fig. 20, and Fig. 21). The influence of fixation has been reported in midgut by several authors (Flower and Filshie, 1975; Dallai et al., 1977; Lane and Harrison, 1978; Skaer et al., 1979) and also in Malpighian tubule (Dallai. 1976; Skaer et al., 1979). However, particularly in the latter tissue, the fracturing characteristics in fixed material are very variable so that in some cases the PF ridges predominate and in other the EF grooves are so decorated with particles as to appear almost ridge-like (cf Figures 11 and 1 2 in Dallai, 1976). In our experience (Skaeret al., 1979), Malpighian tubules show little change in fracturing pattern with fixation. The particle rows are always found on the EF with clear grooves on the PF. There is considerable variation in the packing of adjacent ridges, correlating with findings of diverse patterns of septa1 spacing in oblique sections of lanthanum-infiltrated tissue. Further correlations are found in that ridges or rows of particles may fuse laterally to give double structures (arrows in Fig. 22) and they terminate abruptly (arrows in insert to Fig. 20). Freeze-fracture replicas allow analysis of topographical variations in the organization of the junction (see Fig. 20) in a way that is rarely possible with the relatively restricted regions revealed in oblique sections (see Figs 1 6 and 17 and also Giusti, 1976). In this way it has been possible to show that in the midgut of Musca and Rhodnius the junction is tightly organized apically and loosens as the junction extends basally (Fig. 20) (Skaeret al., 1979; Figure 22 in Lane, 1978a). Similar variations are found in lanthanum-infiltrated specimens in those regions whose position in the apical-basal axis of the border can be analysed. This topographical variation combined with parallel findings in freeze-fracture replicas and lanthanum-infiltrated preparations in terms of double rows (Figs 1 5 and 22) and terminating rows (Figs 15-1 8 and insert in Fig. 20), leads to the suggestion that there is a correlation between the freeze-fracture ridges or rows of particles and the intercellular septa (discussed further in Section 2.2.4). However no authors have reported freeze-fracture correlates of the electron-lucent columns of lanthanuminfiltrated preparations of smooth septate junctions.

Fig. 21 Freeze-fracturepreparationof the lateral border of the midgut of an unfixed specimen of the moth, Manduca sexta, bearing smooth septate junctions. The particle rows on the EF are complemented by clearly marked grooves on the PF. The highly convoluted nature of the lateral border can be deduced from the complex fracturing pattern of membranes. X 65 600 Fig. 22 Junction from the proventriculusof Rhodnius prolixus, prepared for freeze-fracture without prior fixation. The moniliform EF ridges sometimes run very close together to give a double structure (arrows) (compare with Figs 15 and 18). x 68 800


61


62

2.3.4 Model derived from evidence Few models have been put forward to explain the structure of smooth septate junctions. Flower and Filshie (1975) proposed that the previously named continuous junctions were in fact a form of septate junction, and their model reflects this view. Figure 23 is largely derived from this model; /-CYTOPLASM

-. /

/-E

FACE-I

-

P FACE-\

Fig. 23 Model of a smooth septate junction to illustrate a three-dimensional structure built up from the images obtained by various preparative techniques. The freeze-fracture cleaving pattern illustrated is for unfixed tissues. An idealised situation is shown in which the septa are regularly spaced and separated by a single row of intercellular columns (stippling). Although the evidence is not unequivocal, the septa are shown as inserting into the membrane via the particles therein. The columns seem not to insert into any obvious intramembranous structure

the septa are unpleated and the points of septa1 contact with the membranes are marked by the freeze-fracture ridges or particle rows. However, Flower and Filshie (1975) are non-committal about the degree of correspondence between the intercellular septa and the intramembrane ridges (this point is discussed more fully in Section 2.2.4). The chief difference between the pleated and smooth septate junctions are the intercellular colums, which appear to have no freeze-fracture correlate and so are thought not to insert into the junctional membranes. 2.4

OCCURRENCE I N IXSECTS

The septate junction in insects is associated primarily with epithelial tissues, although not exclusively (as suggested with one exception by Caveney and Podgorski, 1975). In fact septate junctions are found in almost all insect tissues including the epidermis (e.g. Locke, 1965; Hagopian, 1970; Noirot and Noirot-Timothee, 1973; Noirot and Quennedy, 1974; Caveney and Podgorski, 1975; Lawrence and Green, 1975), epidermal glands (e.g. Stuart and Satir, 1968; Crossley and Waterhouse, 1969; Noirot and Quennedy, 1974), imaginal disc (e.g. Poodry and Schneiderman, 1970; van Ruiten and


63

Sprey, 1974), partsof the gut (e.g. Satir and Fong, 1972,1973; Bode, 1977; Credland, 1978; Lane, 1978a,c, 1979; Noirot-Timothte et af., 1978; Skaer and Lane, 1979; Noirot et af., 1979), salivary gland (e.g. Bullivant and Loewenstein, 1968; Kendall, 1969; Burger and Uhrik, 1972; Gaudecker, 1972; Lane et af., 1972; Leslie and Robertson, 1973; Lauverjat, 1973; Skaer et af., 1975), Malpighian tubule (e.g. Bullivant and Loewenstein, 1968; Berridge and Oschman, 1969; Taylor, 1971; Noirot and NoirotTimothte, 1973; Walletaf., 1975; Dallai, 1976; Peacock and Anstee, 1977; Skaer et a f . , 1979), the gonads and associated structures (e.g. testis, Danilovaetaf., 1969; Szollozi and Marcaillou, 1977; Skaer and Jones, 1979; seminal vesicle, Cantacuzene, 1972; accessory collateral gland, Flower, 1972; sperrnotheca, Gupta and Smith, 1969; transiently in ovary, Mahowald, 1972; Lane, unpublished observations on locust and cockroach ovary), the nervous system (reviewed by Lane, 1974); between perineurial cells (e.g. Maddrell and Treherne, 1967; Schiirmann and Wecksler, 1969; Skaer and Lane, 1974; Lane et a f . ,1977a), glial cells (Osborne, 1975; Ribi, 1977), axons and glia (Smith, 1967) and nerve cells (Smith, 1967; Boschek, 1971; Corbikre-Tichant and Bermond, 1972; Sohal and Sharma, 1973), sense organs, for example the eye (e.g. Eley and Shelton, 1976; Shaw 1978; Chiet a f . ,1979; Ribi, 1979; Lane and Skaer, unpublished), antenna (Zachruk et al., 1971), and rnechanoreceptor (Chi and Carlson, 1976), myocardial tissue (Sanger and McCann, 1968), possibly between encapsulating haemocytes (e.g. Grimstone et af., 1967) and between cells of the tracheolar system (e.g. Gupta and Berridge, 1966; Smith, 1968; Laneetaf., 1977a). In view of their apparent ubiquity, perhaps of greater interest is their absence in a few instances. Skeletal muscle does not appear to have septate junctions, although they do occur between myocardial cells (Sanger and McCann, 1968; McCann, 1970). Septate junctions do not appear to have been found in fat body (Clarke, 1973). Moreover, while pleated septate junctions are found in the perineurium in the majority of insect species examined (reviewed by Lane, 1974); they are absent from this tissue in the moth, Manduca sexta (Lane, et al., 1977a; Lane and Swales, 1 9 7 9 ~ ) . Septate junctions have also been observed in in vitro conditions (Poodry and Schneiderman, 1970; Epstein and Gilula, 1977). These latter authors have identified septate and gap junctions between homologous cells in culture. Cells derived from a Homopteran line showed both junctions whereas those from a Lepidopteran line displayed only gap junctions. Pleated septate junctions are found between heterologous cells (Stuart and Satir, 1968; Kloetzel and Laufer, 1969; Satir and Gilula, 1970; Rose, 1971; Noirot and Quennedy, 1974; Flower and Filshie, 1975; NoirotTimothee et al., 1979) as well as more commonly between homologo~lscells. They may also be formed between processes of the same cell and this is

64


particularly well illustrated by the mestracheon system (Smith, 1968; Poodry and Schneiderman, 1970) but occur in other cells as well (Maddrell and Treherne, 1967; Stuart and Satir, 1968; Osborne, 1972; Lane 1974; for further references see Woirot-TimothCe and Noirot, 1979). Smooth septate junctions have been reported only from Malpighian tubules (Dallai, 1976; Lacombe, 1976; Meyran, 1977; Skaer et al., 1979; Green et al., 1979b) and midgut (Noirot and Noirot-TimothCe, 1967,1972; Dallai, 1970, 1975; Andries, 1972; Oschman and Wall, 1972; Satir and Fong, 1972; Reinhardt and Hecker, 1973; Foldi, 1973; Noirot-Timothee and Noirot, 1974; Flower and Filshie, 1975; Ito et al., 1975; Burgos and Gutierriez, 1976; Lacombe, 1976; Houk, 1977; Graf, 1978; Lane, 1978a; Humbert, 1979; Skaer et al., 1979) although junctions closely resembling them, particularly in freeze-fracture appearance, have been found in the eye (Carlson and Chi, 1979). Houk and Beck (1975) claim to have found smooth septate junctions between perineurial cells, although their published micrographs are not at sufficiently high magnification to verify their assertion. Initially it was thought that smooth septate junctions were a substitute for pleated septate junctions (Noirot and Noirot-TimothCe, 1972; Satir and Gilula, 1973); thus pleated septate junctions were found in the posterior regions of the gut (Satir and Fong, 1973; Noirot and Noirot-Timothie, 1977; Noirot-TimothCe et al., 1978), while smooth septate junctions were found in the midgut (Noirot and Noirot-Timothee, 1967). More recently, however, smooth and pleated septate junctions have been found side by side on the lateral borders of Malpighian tubule (Dallai, 1976; Lacombe, 1976; Meyran, 1977; Skaer et al., 1979) although as early as 1969, Filshie is quoted as having found two types of septa in Malpighian tubule (Leik and Kelly, 1970). Pleated septate junctions have also been found in the midgut (Gouranton, 1967; Ito et al., 1975; Skaer, unpublished observations), in some cases coexisting with smooth septate junctions (Itoet al., 1975). Foldi (1973) also reports the coexistence of smooth and pleated septate junctions in the "filter chamber" of an Homopteran midgut. However the junctional identification in this case is based solely on thin-section evidence. Freezefracture and/or lanthanum infiltration is necessary t o confirm this finding. Smooth septate junctions, like pleated septate junctions, are found between heterologous as well as homologous cells (Flower and Filshie, 1975). However, to our knowledge, there do not appear to have been reports yet of smooth septate junctions forming between processes of the same cell.


2.5

65

DISTRIBUTION IN OTHER INVERTEBRATES

Various types of septate junction have been described throughout almost all the invertebrate phyla (see Section 2.1). Pleated septate junctions have however been reported only in the chaetognathes (Duvert et a f . , 1978); platyhelminths (Storch and Welsch, 1977); nemertines (Vernet e f al., 1979); annelids (Coggeshall, 1966; Lasansky and Fuortes, 1969; White and Walther, 1969; Storch and Welsch, 1970, 1972; Boilly-Marer, 1972; Baskin, 1976; Bilbaut, 1980); molluscs (Gilula er al., 1970; Satir and Gilula 1970; Flower 1971 ; Newel1 and Skelding, 1973; Ryder and Bowen, 1977) and other classes of the arthropods in addition to the insects (Crustacea: Lang 1977; Shivers and Chauvin, 1977; Arachnida: Foelix et al., 1975; Lane, unpublished observations; Myriapoda: Juberthie-Jupeau, 1975). Although most authors agree that the septate junctions described in platyhelminths, annelids, molluscs and arthropods are pleated, various distinctions have been made. Baskin (1976) claims that the pleated septate junctions of polychaete epidermis shows unusual characteristics, reminiscent both of the smooth and the Hydra-type of septate junction. The lack of septa in some specimens, more characteristic of smooth septate junctions, might be explained by section angle. In some specimens Baskin found clear septa. An independent examination of the epidermis of another polychaete (Ficopofamus enigmatica) by thin-section, lanthanum staining and freezefracture, clearly reveals pleated junctions (Skaer, 1979b). The Hydra-like elaborations of the intercellular architecture are also described by Green (1978), who claims that these are common to all pleated junctions. Another freeze-fracture study of the epidermal septate junctions of two different annelid species (Welsch and Buchheim, 1977) emphasizes the fundamental similarities between annelid and arthropod septate junctions, while supporting Baskin’s (1976) view that annelid septate junctions may also exhibit distinctive characteristics of their own. Giusti (1976) finds pleated septate junctions in annetids and molluscs but claims that in the molluscs the junctions are characterized by tubular structures scattered randomly between and occasionally within the septa. These tubular structures have also been reported in a cephalopod mollusc by Boucaud-Camou (1978). White and Walther (1969) report similar structures in the photoreceptors of the leech. Pleated septate junctions have been found in the Onychophora with similar tubular structures, which however are not randomly arranged but assembled in clusters (Dallai et al., 1977). The significance of these tubular structures is not clear although Giusti (1976) suggests that they might be isolated gap junctional particles. Such structures are absent in the arthropod classes which show little deviation from the insect pleated septate junction.

66


Smooth septate junctions have, for the most part, been found only in arthropods. Apart from insect species, they have been found in a myriapod (Scutigerella midgut; Juberthie-Jupeau, 1979); crustacea (crayfish hepatopancreas; Gilula, 1972b, 1974; Satir and Gilula 1973; Gammnrus hepatopancreas; Schultz, 1975; Orchestia posterior caeca; Graf, 197th; the midgut of a copepod; Amaud, Brunet and Mazza, 1978; possibly also in the hepatic caecum of Daphnia; Hudspeth and Revel, 1971) and in a slightly unusual form in a chelicerate (midgut and hepatopancreas of Limulus polyphemus; Lane and Harrison 1978). They have also been found in ticks (Binnington, personal communication). Mills et al. (1976) report a septate junction in the midgut of the crayfish Cambarus which exhibits “a very regular array of transverse septa”. Without lanthanum-infiltration or freeze-fracture studies, it cannot be ascertained whether this is a pleated or smooth septate junction . Satir and Gilula (1973) claim to have seen smooth septate junctions in a nematode and in Peripatus (Onychophora); this accords with previous observations in that Lavallard (1967) reported a structure thought to be a smooth septate junction in Peripatus, although Dallai et al. (1977) query this finding. However these authors have demonstrated a smooth septate-like junction in the midgut of Peripatopsis moseleyi (Dallai and Giusti, 1978). These junctions display the typical smooth septate junction appearance on lanthanum-infiltration but freeze-fracture reveals an unusual intramembranous organization; the undulating EF ridges are decorated on their surface with rows of isolated 9 nm particles. Greven (1976) has described a zonuia continua in the midgut of a tardigrade and Mme Auber-Thomay (personal communication in Noirot and Noirot-Timothee, 1967) has found smooth septate junctions in a nematode intestine. Whether further smooth septate junctions will be found in the midguts of other invertebrate species remains to be seen but it seems highly likely given the ubiquitly observed thus far. Green (1978) and Green et al. (1979a) describe anastomosing septate junctions in both echinoderms and hemichordates. Green (1978) considers that these junctions form an evolutionary link between the invertebrate septate junction and the vertebrate tight junction. The lowest vertebrate phylum, the tunicates, display true tight junctions in which the intercellular space is occluded and freeze-fracture reveals an anastomosing network of P face ridges (Cloney, 1972; Lorber and Rayns, 1972). The evolutionary link between junctions displaying an enlarged extracellular space (invertebrate septate junction ca. 14-17 nm) and those with an occluded intercellular space (vertebrate tight junctions) is not clear, $spite their superficial similarity after lanthanum infiltration or freeze-fracture. This similarity of appearance arises from quite separate structures; in the first case the lanthanum-excluding septa and the corresponding rows of particles within


67

the membrane and in the second, the obliteration of the intercellular space by the fusion of ridge-like structures in the junctional membrane. 2.6

OCCURRENCE I N VERTEBRATES

A variety of “septate-like” junctions have been described in vertebrate tissues. As yet they have been identified only in certain tissues, the nervous system (Laatsch and Cowan, 1966; Peters 1966; Hirano and Dembitzer, 1967, 1969; Gobel 1971; Sotelo and Llinhs, 1972; Livingstoner af., 1973; Cabellero et af., 1978), the retina (Lasansky, 1969, 1971; Schwartz, 1973) vascular endothelium (Simionescu et af ., 1976), endocrine tissues (Bargmann and Lindner, 1964; Friend and Gilula, 1972; Enders, 1973), testis (Aetorfer and Hedinger, 1975; Connell, 1974, 1975, 1976, 1978) and transiently in the ovary (Albertini and Anderson, 1974). They have also been described in pathological tissues (Nistal er af., 1978a; Tani et af.,1971) and moreover have been shown to arise artifactually (Bulger and Trump, 1968; Nistal et af., 1978b). Where their existence is established, the septatelike junctions vary both in their structure and in the extent of their resemblance to invertebrate septate junctions. In the adrenal cortex, Friend and Gilula (1972) described a junction with septa formed by 10-15 nm extracellular particles, often circular in profile, spanning the 21-30 nm intercellular space. Freeze-fracture preparations revealed no specialized intramembrane organization associated with these structures. Other descriptions of septate junctions in endocrine tissues (Bargmann and Lindner, 1964; Enders, 1973) have not included evidence from freeze-fracture. In the retina of turtles (Lasansky, 1969, 1971), the adjacent membranes of the septate-like junctions are separated by a uniform 16 nm space, similar to that of the invertebrate junctions. The separation of the septa ( C U . 19 nm) and the observation in oblique sections that they form a regular network is also reminiscent of the invertebrate junctions. Schwartz (1973) has described a similar junction in the retina of the rat. However without freeze-fracture evidence, parallels with the invertebrate septate junction cannot be made. As with other septate-like junctions, the degree to which those in the nervous system parallel the invertebrate septate junction is probably slight. Gobel (1971) and Sotelo and Llintis (1972) have described junctions between axons in the vertebrate cerebellar cortex where 5-8 nm thick septa bridge the 11-18 nm intercellular space at a repeating distance of 16-22 nm. In oblique sections, these septa appear as undulating ribbons sometimes giving a honeycomb appearance of hexagonally arranged “cells” 1 7 nm in diameter (Sotelo and Llinhs, 1932). These features superficially resemble

68

NANCY J. LANE A N D HELEN leB. SKAER

the invertebrate pleated septate junctions, but without freeze-fracture, the existence of a true cell junction, displaying membrane specialization in freeze-fracture replicas, as opposed to a cell contact with no intramembrane specialization (Friend and Gilula, 1972b) cannot be established. Glial-axonal junctions (Laatsch and Cowan, 1966; Peters, 1966; Hirano and Dembitzer 1967,1969; Livingston et al., 1973; Schnapp and Mugnaini, 1975,1976; Schnappet al., 1976) display septa, 10-15 nm wide, and with a repeat distance of 25-30 nm. However, here the intercellular space is only 2-3 nm. Lanthanum infiltration (Hirano and Dembitzer, 1969) reveals straight septa in this intercellular space. Freeze-fracture preparations (Livingstonetal., 1973; Schnapp and Mugnaini, 1975; Schnappetal., 1976) reveal regular convolutions of the glial and axonal membranes, which take the form of complementary ridges and grooves in the glial and axonal P and E faces (ridges on the PF corresponding with grooves on the EF). These regular undulations are separated by 25-30 nm and follow a helical path with respect to the long axis of the axon with its glial wrapping. Both the axon and glial membranes display 8-13 nm particles, associated with the P F ridges in the axon and with either the PF ridges or EF grooves in the glial cells. Schnapp and Mugnaini (1975) and Schnapp et al. (1976) present a model of this axo-glial junction in which straight septa, spanning the narrow intercellular space, insert into the membranes at the point of minimal intercellular distance, where the undulations bring the membranes closest together. This explains the identical spacing of the septa in thin-sections, with that of the ridges and grooves in freeze-fracture. This junction has clear analogies both with invertebrate septate junctions and also with gap junctions. Suggestions as to the functional significance of these junctions reflects this. Livingston et al. (1973) suggest that the particles of the junction could traverse the narrow intercellular cleft and, as in gap junctions, allow the passage of ions between the axonal and glial cytoplasm. Schnapp et al. (1976) propose that a more likely function is adhesive, following the analogy of the junction with a nut and bolt assembly (Petersetal., 1970). Intercellular occlusion is not considered, since these junctions occur in close juxtaposition with tight junctions (Schnapp and Mugnaini, 1975, 1976). Connell (1974, 1975, 1976, 1978) describes a type of septate junction between the Sertoli cells of mammalian testis and claims, on the basis of thin sections, lanthanum staining and freeze-fracture evidence, that these junctions resemble closely the invertebrate septate junction (Connell, 1978). The resemblance is closer to the smooth rather than the pleated type. Her lanthanum infiltrated preparations do resemble the double septa described by Green (1978) from echinoderm epithelium and by Duvert et al. (1978) from the intestine of a chaetognathe (Sagitta). Although there are similarities between the Sertoli cell septate junctions and those in inverte-


69

brate tissues, there are also differences. The uniformity of the intercellular space, a very characteristic and almost diagnostic feature of the invertebrate septate junction, is not clear in the testis (see Connell, 1978, Figures 2 and 3) and the intercellular space, rather than being enlarged (14-17 nm), is slightly reduced (ca. 9 nm). In these two last examples of septate-like junctions (the glial-axonal complexes of the central nervous system and the Sertoli cell junctions of the testis), the junctions are in close co-existence with tight junctions. Moreover, it is suggested that they perform a physiological role distinct from the tight junctions (Schnapp et al., 1976; Connell, 1978), a feature to be borne in mind when considering claims of functional equivalence of invertebrate septate junctions and vertebrate tight junctions (see Section 2.7). 2.7

FUNCTIONAL SIGNIFICANCE

The functional significance of septate junctions is still not entirely clear. The role of intercellular communication assigned to them (Loewenstein and Kanno, 1964; Wieneret al., 1964; Bullivant and Loewenstein, 1968; Gilula etal., 1970; Satir and Gilula, 1970; Rose, 1971) is now generally considered to be fulfilled not by septate junctions (Hagopian, 1970; Poodry and Schneiderman, 1970; Flower 1971; Hudspeth and Revel, 1971;Burger and Uhrik, 1972; Caveney and Podgorski, 1975; Snigirevskaya et al., 1977; Noirot-TimothCeetal., 1978) but by gap junctions (see Section 4.6.1). It is, however, difficult to exclude this possibility with certainty as septate junctions tend to occur in association with gap junctions (see Section 4.8). Intercellular adhesion is generally agreed to be one of the functions of septate junctions (see for example Wood, 1959, 1977; Poodry and Schneiderman, 1970; Berridge and Oschman 1972; Noirot-Timothte and Noirot, 1974, 1979; Baskin, 1976; Noirot-TimothCe et al., 1978; Lane, 1979c) and is a corollary of the structural models proposed for the insertion of the septa into the junctional membrane (see Section 2.2.4.) Although there is little direct experimental evidence for the adhesive function of septate junctions, Filshie and Flower (1977) report a parallel loss of structural stability in Hydra with the disappearance of the septate junctions after glycerol treatment. Correlating with this finding, extensive septate junctions are commonly found in those tissues subject to rapid dilations as for example, the gut and the epidermis in fluid feeders. It has also been suggested that septate junctions function as a transepithelial permeability barrier (Wood, 1959, 1977; Dan, 1960; Berridge and Oschman, 1969; Newel1 and Skelding, 1973; Lord and di Bona, 1976; Filshie and Flower, 1977; Szollosi and Marcaillou, 1977; Noirot-TimothCe et al., 1978; Graf, 1978a; Noirot-TimothCe, and Noirot, 1979; Greenet a/.,

70


1979a). Evidence conflicting with this supposition has been presented by several authors (Treherne et al., 1973; Ryder and Bowen, 1977; Lane 1978c, 1979a, c) who have shown penetration of septate junctions by tracers. Authors vary in the methods they employ to test the penetrability of junctions by tracers but in a number of cases, tracer molecules are added to the fixative solution and conclusions drawn about the permeability of the intercellular space by the depth to which the tracer penetrates the junction in moribund‘tissue (e.g. Hudspeth and Revel 1971; Hand and Gobel, 1972; Newel1 and Skelding, 1973; Gilula, 1974; Baskin, 1976; Filshie and Flower, 1977; Graf, 1978a). Physiological conclusions based on the movements of tracers are always difficult to make since the properties of the tracer may interfere with the physiology of the system and at best can indicate only the permeability of the tissue to the particular tracer molecule under consideration. However the value of such studies is considerably enhanced if the tracer molecule is applied in conditions as near to the in vivo state as possible. Such conditions were first defined for the use of ionic lanthanum by Machen et al. (1972) and have subsequently been used in the study of invertebrate tissues (e.g. Lane, 1972; Lane and Treherne, 1972; Treherneet al., 1973; Leslie, 1975; McLaughlin, 1974b; Ryder and Bowen 1977; Szollosi and Marcaillou, 1977; Lane and Swales, 1978a, b, 1979a, c; Lane, 1978c, 1979a, c; Lane and Harrison, 1979b). Of thesein vivo incubations all except Szollosi and Marcaillou have demonstrated complete penetration of the septate junctions by the applied tracer. Szollosi and Marcaillou (1977) show septate junctions in the basal stain-excluding compartment of the locust testicular follicle. In this tissue, however, both septate (Figs 6 , 7 and 9) and simple tight junction-like ridges are found (Fig. 47 and Skaer and Jones, 1979 and Szollosi, personal communication). Thus until further analysis of junctional position and tracer penetration is completed, the identity of the sealing junction cannot be made with certainty. Lord and di Bona (1976) demonstrated that blisters are produced between the septa of planarian epidermal junctions when subjected to osmotic stress (the normal external < 6 mosmol fluid was enriched with NaCl, KCl, urea, mannitol or urea to levels in excess of 300 mosmol). They drew an analogy with similar blistering behaviour previously demonstrated in vertebrate epithelial tight junctions and so concluded that both junctions can be functionally classified as “limiting junctions” constituting “ratelimiting barriers to the passive, paracellular flow of water and small molecules”. The significance of blister-production by elevating the osmolarity of the external environment to completely unphysiological levels is questionable. A similar criticism could be levelled at a parallel study on the midgut of crayfish (Mills et al., 1976). The existence of a potential difference across an epithelium has been


71

taken to indicate that the intercellular junctions seal the paracellular pathway (e.g. insect salivary gland, Loewenstein and Kanno, 1964; and see Noirot-TimothCe and Noirot, 1979). In tissues where polarized pumps have been shown to be active such as insect midgut (Harvey et al., 1968), Malpighian tubule (Maddrell, 1971) and salivary gland (Berridge et al., 1976a), the maintenance of a transepithelial potential need not depend on sealed intercellular spaces but is a function of the relationship between the electrical resistance of the epithelium and the rate of ion transport. The resistance of the epithelium moreover, will be a function not only of intercellular leakiness bur also the permeability of the cell membranes and will therefore also depend on the relative area presented by each of these surfaces. This latter ratio is variable but in the case of Malpighian tubule, the relative permeabilities in different species can be related to the geometry of the cells which determines the area of basal (or apical) cell surface, relative to that of the lateral borders (Maddrell, 1979a). Lane and Swales (1978) have shown the absence of septate junctions in the nervous system of a moth, Manduca sexta, where a physiological bloodbrain barrier has been demonstrated (Pichon et al., 1972; Lane, 1972). Furthermore, the upper Malpighian tubule of Rhodnius in which smooth septate junctions are found (Skaer et al., 1979) apparently offers little restriction to the transepithelial movement of a range of organic solutes which include such substances as sucrose and inulin, that could scarcely d o other than cross the epithelium by the intercellular route (Maddrell and Gardiner, 1974). However, as calculated by Filshie and Flower (1977), the presence of complex arrangements of septa, even if they are relatively short with frequent blind endings, dramatically reduces the cross-sectional area available for intercellular permeation and increases the transepithelial path length. Thus the rate of passive flow through the junction is bound to be reduced and, if the molecule is large enough, its entry might be blocked (as with ferritin (Zylstra, 1972; Ryder and Bowen, 1977)). Moreover, the interseptal space may contain electron-dense material, especially in the smooth septate junction (Noirot and Noirot-TimothCe, 1967,1979; Dallai 1970). In the midgut of several insects this material has been shown to be glycoprotein in nature (Dallai, 1970). Some specificity in the otherwise unselective retardation of passive flow through the junction, imposed by the architecture of the septa, might therefore be conferred by the chemical nature and charge characteristics of the intercellular matrix trapped between the septa. Some evidence to support this comes from a study by Gupta et al. (unpublished data, 1972) in which the septate junctions of Calliphora Malpighian tubule were shown to be permeable to the anion, sulphate, but not to a heavy metal cation, barium. A similar suggestion relating to the septate junctions of

72

NANCY J . LANE A N D HELEN leB. SKAER

Hydra has been made by Staehelin (1974) and the idea of the intercellular space and cement as an ion-exchange resin, molecular sieve, gel filter and chromatographic column is considered by Oschman (1978). The situation in Hydra and other cnidaria, in which a structurally distinctive type of septate junction has been described, is rather different from the insects. In this phylum, tight junctions have not been described. Moreover, several investigators have concluded, on a variety of grounds, that the septate junctions act as permeability barriers (e.g. Wood, 1959,1977; Leik and Kelly, 1970; Hand and Gobel, 1972; Staehelin, 1974; Flower and Filshie, 1977; King and Spencer, 1979). Although some of the criticisms cited above can be levelled at permeability studies carried out on these tissues, it may be that these “Hydra”-type septate junctions restrict transepithelial permeability. Finally Dallai (1 976) has suggested that septate junctions (both pleated and smooth) might confer greater rigidity on the intercellular membranes, preventing the occlusion of the intercellular space and so facilitating the rapid flow of water through the intercellular cleft. In view of the considerations mentioned above concerning the effect of the septa on path lengths and cross-sectional area of the intercellular space, as well as the finding that the septa normally run perpendicular to, rather than parallel with, the direction of fluid flow, this suggestion seems rather unlikely. Moreover in a tissue where bulk transport of fluids take place, a specialised junction (the reticular septate junction) has been described, which, it is suggested, is involved in ensuring a ready paracellular flow (Lane, 1979c, and see Section 8.1.6). Throughout this discussion it has been assumed that the roles of smooth and pleated septate junctions are identical. It was originally thought that they represented analogous structures and that smooth septate junctions might be associated with regenerating tissues or those of endodermal origin, whereas pleated septate junctions were associated with non-regenerating tissues or those of ectodermal origin (Noirot and Noirot-TimothCe, 1967). The results described by Dallai (1967), Lacombe (1 976), Meyran (1 977) and Skaer et af. (1979) conflict with this interpretation since smooth septate junctions occur in Malpighian tubules which are thought to be ectodermal in origin and in which constant cell turnover does not occur. The embryological origin of Malpighian tubules is, however, open to discussion (see Wall ef al., 1975). Both Wall et af. (1975) and Dallai (personal communication) consider it possible that some parts of the Malpighian tubule may be of endodermal origin. This however still leaves unexplained the occurrence of smooth and pleated septate junctions side by side in Malpighian tubules, which argues against the two types of junction being analogous structures.

I NTERC E LLU LAR J U N (:TI 0 NS I N I NSECT TI SSU ES

2.8

73

FORMATION O F SEPTATE JUNCTIONS

Gilula (1972a, 1973) has studied the formation of septate junctions in sea urchin embryos. The assembly of the junctions can be followed from the detection of the presumptive junctional site at 9-12 hours to its maturation at 72 hours. In thin-sections, the development can be followed by the appearance of electron-dense septa spanning the intercellular cleft. In parallel with this, the 8.5 -9 nm particles, revealed by freeze-fracture (EFcf. PFin insects), arise in the presumptive region and gradually become aligned. Initiaily they line up into simple rows and subsequently into swathes of parallel rows, possibly in association with a nucleation furrow, and finally the multiple rows form a continuous belt around the cells with the P F displaying furrows complementary with the particle rows. The development of pleated septate junctions in insects has been followed during the embryonic and larval development of Calliphora (Lane and Swales, 1978a). The junctions in thin-section show incomplete formation of the septa in early stages of development. As the junctions mature, the septa develop and join the junctional membranes to give the regular appearance in the fully-formed larva. In freeze-fracture, the PF particles are disorganized in their arrangement in the early larva. They gradually line up, initially into short, abruptly terminating rows and subsequently into the extensive meandering rows, typical of the mature junction between Calliphora perineural cells. To date, the development of smooth septate junctions has not been investigated, though it has been mentioned that in certain insect species the formation of the junction must occur during embryonic life since the mature junction is present at each instar (Rhodnius midgut; Lane 1978a). However during the remodelling of the gut of Manduca sexta during pupation, the smooth septate junction reforms and developmental stages can be found (Fig. 24, and Lane 1979e). Short, moniliform ridges are found on the E F in unfixed tissue, often collected together into disorganised stacks (insert, Fig. 24), while meandering P F grooves or E F ridges can frequently be seen, not aligned, and often terminating abruptly (arrows, Fig. 24). Even in mature tissues, such as the gut, where cell replacement occurs, the reformation of cell junctions is inevitably involved. One might therefore expect to find stages in this cycle of breakdown and re-establishment of smooth septate junctions. Lane (1978a, 1979b) considers this possibility and shows short, isolated PF ridges (Lane, 1979b, Figure 28) in the proventriculus of Rhodnius which could represent a developmental stage in the formation of the continuous junctions that are found in this tissue. In this context, of interest also are the macular continuousjunctions of Graf (1978a) in Orchestia midgut. These macular junctions are situated below and

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75

contrast with the belt of septa representing the apical zonular junction. It is possible that they could be developmental stages, although Graf does not comment on this possibility. Houk (1977) however suggests that the smooth septate junctions of the flea (Culex tarsalis) are able to dissociate and reform after the junction has reached a mature state. He cites evidence (Milesetal., 1973) to indicate that the passage of virus particles from the gut lumen to the haemocoel occurs sufficiently rapidly to preclude the transcellular route. Furthermore, the morphology of the flea gut alters very greatly during ingestion of the blood meal, the columnar epithelium becoming squamous. Houk suggests that this alteration may entail breakdown and reassociation of junctions. Howard (1962), also working with flea, showed that after bloodmeal ingestion, polystyrene spheres of 5 p m diameter could penetrate the intercellular cleft to within a few microns of the basal lamina. The spheres are too large to pass through the septate junctions and so, discounting a transcellular route, this observation would imply the temporary dissolution of the junction.

3 Desmosomes 3.1

INTRODUCTION

The majority of work published on desmosomes is concerned exclusively with vertebrate material. Thus our knowledge and interpretation of desmosome-like structures found in insect tissues is set against the background of a wealth of information on the various desmosomal types characteristic of vertebrate tissues. Some of the information about these vertebrate junctions will be summarized first so that similarities and differences of the insect junctions can be assessed. Desmosomes in vertebrates can be distinguished on morphological grounds into two distinct types; the macula adhaerens or spot desmosome and zonula (and fascia) adhaerens or belt desmosome (Farquhar and Palade, 1963). The junctions are characterized not only by their geometry on the cell surface but also by their appearance in thin-section and by the degree of elaboration they display in the intramembrane planes revealed by freezefracture. Hemi-desmosomes have an appearance identical with half a macula adhaerens and so will be described with them. Fig. 24 Freeze-fracture preparation of midgut from a fixed Manduca sexta pupa. The smooth septate junction, in the process of reforming between the cells of the potential adult midgut, displays a very loosely ordered appearance with frequent terminations, both of the PF ridges and EF grooves (arrows). This is particularly clearly seen in the insert of unfixed tissue, which shows an array of very short EF ridges, apparently becoming aligned. x 73 700; insert x 65 900


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3.2 3.2.1

STRUCTURAL FE.ATURES


(a) Vertebrates The spot desmosome is typified by its shape (oval, 0.2-0.5 p m in length) and by an enlarged intercellular space (25-35 nm) which contains material often formed into a central dense stratum lying parallel to the cell membranes, and whose subjacent cytoplasm is elaborated into dense fibrillar plaques. The junctional intercellular space is continuous with the non-junctional intercellular space but is distinguished from it by the electron-dense stratum which in some cases is a double structure, 4 nm separating the two dense lines, and which appears to be connected to the two junctional membranes by a complex array of connecting 4 nm filaments. These, in some cases, (Rayns et al., 1969) show a square array (7-8 nm centre-to-centre spacing). This latter observation was made after infiltration with lanthanum hydroxide to which the stratum and interconnecting filaments are impermeable. The intercellular material has been shown to stain with ruthenium red (Kelly, 1966; Luft, 1971) and amixture of chromic acid and phosphotungstic acid (Rambourg, 1969) and this, combined with its susceptibility to trypsin treatment (Overton, 1968; Berry and Friend, 1969), suggests that it contains glycoprotein. The cytoplasmic plaques have a filamentous appearance in thin-section and are separated from the membrane surface by a slightly less electrondense zone, The plaques are the sites of attachment of the numerous tonofilaments, 10 nm in diameter. Kelly (1966) concluded, on the basis of studying stereo-pairs, that the tonofilaments, rather than terminating in the plaques, loop through them and terminate either in the tonofilament bundle in the cytoplasm or, if there are further desmosomes arranged in series, the filaments may course through the cytoplasm linking one plaque with the next (Fawcett, 1958; Lentz and Trinkaus, 1971). It has recently been suggested that secondary filaments, derived from the 10 nm tonofilaments, traverse the membrane and anchor on particles making up the intercellular midline (Leloup et al., 1979). In vertebrates, a second type of desmosome has also been described, the zonula adhaerens. In shape, it can either be belt-like (zonula)or have a more interrupted topography Cfascia) but beyond this they are distinguished from maculae adherentes on the basis of several structural features. In thinsection, the separation of the membrane is smaller (15-20 nm), no central intercellular stratum is seen, although the intercellular space stains to reveal fine filamentous material. The cytoplasmic plaque is closely applied to the membrane but itself is more loosely organized and the filaments associated with it have a diameter of 7 nm as compared with 10 nm for the spot desmosomes. These filaments appear to have some actin-like properties


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since they bind heavy meromyosin (Ishikawa et al., 1969; Tilney and Mooseker, 1971). ( b ) Arthropods The foregoing structural features have been summarized by Campbell and Campbell (1971), McNutt and Weinstein (1973), Overton (1974), Staehelin (1974) and Staehelin and Hull (1978) who deal largely with vertebrate material. The desmosomes of arthropods are not as readily classified, for example, some authors claim that spot desmosomes are very infrequent (Satir and Gilula, 1973) or absent (Noirot-Timothte and Noirot, 1979) whereas their presence is asserted in a number of individual reports. Moreover, insect desmosomes exhibit some distinct differences from their vertebrate counterparts. The intercellular separation is variable as is the organization of the intercellular material. The most striking difference in thin-section is the association of the cytoplasmic plaques with microtubules rather than with tonofilaments (Fig. 25). This is especially well established for the much studied sites of muscle attachment where both hemidesmosomes and more extensive desmosomes (sometimes termed fascia) are found (Fig. 25) (Auber, 1963; Shafiq, 1963; Lai-Fook, 1967; Beaulaton, 1968; Caveney, 1969; Hagopian, 1970). However the association with microtubules is also found in other tissues (Satir and Stuart, 1965; Stuart and Satir, 1968; Moulins, 1968; Smith, 1968; Ashhurst, 1970; Friedman, 1971; Corbitre-Tichant, 1971) although not universally (Gupta and Rerridge, 1966; Stuart and Satir, 1968; Reinhardt and Hecker, 1973; Reger, 1974; Noirot and Noirot-Timothte, 1976). Where they occur, the microtubules appear to run parallel with, rather than insert into, the cytoplasmic plaque and a role of skeletal stiffening of the junctional membrane has been attributed to them (Ashhurst, 1970). In the majority of cases the blanket term “desmosome” appears to be used in describing these junctions in insects. Where a distinction is made between different types it seems to be solely on the basis of junctional shape. Thus the junctions involved in muscle attachment in cockroach epidermis are termed zonulae adhaerentes (Hagopian, 1970), the apical desmosome region of the columnar cells of the sternal gland is called the terminal bar (Stuart and Satir, 1968), the myoepidermal connections of Calpodes and Rhodnius are designated fasciae adhaerentes (intermediate junctions) (Lai-Fook, 1971), whereas the spot desmosomes found between cells are called maculae adhaerentes on the basis of their distinctive, restricted shape rather than a detailed analysis of their structural characteristics. Both zonular and macular desmosomes are described by Noirot and Noirot-Timothte (1976) in the rectum of various cockroach species where according to the authors, these junctions are comparable with their vertebrate equivalents though structurally simpler. Cytochemical investigations of insect desmosomes appear to be limited to


78

Fig. 25 Numerous hemi-desmosomes at the site of insertion of skeletal muscle in the locust Schistocerca gregaria. The hemi-desmosomes occur in pairs, occupying opposite sides of the dilated extracellular spaces, which are filled with an electron-opaque material. Note also the cytoplasmic plaques of electron-dense fibrillar material and the high density of microtubules. Insert: Macular desmosome joining glial cells in Calliphora nervous system. The elaboration of the extracellular material into striations, the relative uniformity of the intercellular space, cytoplasmic fibrillar thickening and microtubules lying close to, but not intimately associated with the junction are all demonstrated. x 60 100; insert X 90 000

the study of Reindardt and Hecker (1973), who found that maculae adhaerentes and hemidesmosomes of the mosquito midgut did not stain with periodic acid-TCH-silver-proteinate (specific for polysaccharide) but that the cytoplasmic mat stained strongly with phosphotungstic acid (specific for basic amino acid-rich protein)

3.2.2

Freeze-fracture appearance

Studies on vertebrate tissues have shown that spot desmosomes (maculae adhaerentes) are associated with intramembranous particle aggregates.


79

These particles fracture onto both the EF and the P F although the P F is normally more richly endowed than the EF. This however can alter with fixation or developmental stage (see Staehelin, 1974). The particles are of an irregular, normally elongate shape, about 12 to 20 nm in diameter. The intercellular stratum is evident as an etch-resistant line 5 to 7 nm in width, which in some preparations can be seen to be made up of small particles (e.g. Leloup et al., 1979). In contrast, the zonulae and fasciae adhaerentes reveal very little in the way of intramembrane elaborations. Freeze-fracture shows no differentiation of the membranes in the area of these junctions. Friend and Gilula (1972b), Satir and Gilula (1973) and Satir and Fong (1973) point out that structures such as these, having a clear intercellular and cytoplasmic differentiation but no intramembranous specializations should be called intercellular contacts and not junctions. O n this basis, the majority of insect desmosome-like junctions should be designated contacts and not junctions. There are very few examples of freeze-fracture preparations of insect tissues that show any intramembrane elaborations and these are of spot desmosomes (e.g. Skaer and Lane, 1974; Baerwald, 1975; Chi et al., 1979). In the rare instances where freezefracture images are obtained, the membranes show symmetrical fracturing characteristics, plaques of small irregularly organized particles being found both on the E F and PF. More commonly, in tissues where spot desmosomelike junctions are very common, no freeze-fracture evidence for their presence can be found (Lane and Swales, 1978a, b). Freeze-fracture preparations of tissues, in which fasciar or zonular junctions have been described, also show no specialized intramembrane features (e.g. Graf, 1978a; cf. Figures l A , PIId; Chi et al., 1979, Figure 13).

3.3

OCCURRENCE I N INSECTS

Desmosomes are found almost universally in insect tissues (see Smith, 1968) and only selected examples of their occurrence will be given in this review. Zonular junctions are found at the extremities of lateral borders in epithelia (Locke, 1965; Stuart and Satir, 1968; Oschman and Berridge, 1970; Noirot and Noirot-Timothie, 1976; Lane, 1979c), although Flower and Filshie (1975) claim that they are found only in ectodermal epithelia where cuticle is present external to the cellular layer, and hence are absent from Lepidopteran midgut. Further Noirot et al. (1979) have noted the absence of an apical belt desmosome in the rectal sheath cells of a termite and cockroach, where a cuticular lining is found. Spot desmosomes are found in more varied positions along intercellular borders and may be found in very

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80

large numbers in certain tissues such as muscle where desmosomes are involved in skeletal attachment via myoepidermal junctions (Bouligand, 1962; Auber, 1963; Shafiq, 1963; Lai-Fook, 1967; Caveney, 1969; Hagopian, 1970; Kuo, McCully and Haggis, 1971) and in an analogous “connective-epidermo-cuticular” connection found in the hypopharyngeal cavity of Blabera craniifer (Moulins, 1968). Desmosomes may also be found in the nervous system both between glial cells (Fig. 25, insert and Schurmann and Wechsler 1969; Laneetal., 1977a; Ribi, 1977; Lane and Swales, 1978a, b, 1979a) and also in the perineurium (Skaer and Lane, 1974, Lane et al., 1977a; Lane and Swales, 1978a). Structures termed interfibrillar junctions and resembling desmosomes are also found associated with the intercalated disc of insect myocardium (Sanger and McCann, 1968; McCann, 1970). The intercellular space containing “basement membranelike material” varies from 20-55 nm in width and the junctional membranes display cytoplasmic plaques of increased electron density into which the muscle thin filaments insert. Hemi-desmosomes, although found in regions of intercellular dilation on the lateral borders of epithelia, are found in their greatest numbers in positions where the cell membrane abuts onto connective tissue (basal lamina) (Fig. 25). Desmosomes appear to form between homologous cells, heterologous cells and between different regions of the membrane derived from the same cell, when an autodesmosorne is formed (Smith, 1968; Ishizaki, 1973; and for examples of all three types see Noirot and Noirot-Timothte, 1976).

3.4

DISTRIBlJTlON I N OTHER INVERTEBRATES

3.4.1 Arthropod tissues Desmosomes are also found widely in the other classes of arthropods. A slightly unusual type of desmosome, exhibiting marked asymmetry and not associated with microtubules has been found in the myoepidermal attachments in an Acarid mite (Kuo et al., 1971). The attachment of copepod muscles is via similar desmosomes, which however are associated with fine tubules designated “tonofilaments” by the original author (Bouligand, 1962) but which Lai-Fook (1 967) suggests are microtubules. Desmosomes in crustacean muscle are discussed by Komuro (1970) and Anderson and Smith (1 971), and muscle attachment in the venomous spider Latrodectus mactans is also associated with desmosomes between cells packed with microtubules (Smith et al., 1969a). Graf (1978a) has shown the presence of an apical “zone of adhesion” in the


81

midgut of an amphipod Orchestia carimana. In thin-section the junction shows a regular, but reduced intercellular space of 11-12 nm, with electron-dense cytoplasmic plaques and intercellular material that is sometimes organized into broad septa and which does not stain with lanthanum. In freeze-fracture preparations, very little alteration of the intramembrane regions are found although there is a slight diminution in the population of intramembrane particles. Contrasting with this situation, a recent investigation using freezefracture has revealed the most highly organized intramembrane specializations so far reported as being associated with hemidesmosomes (Smith et al., 1979, working with crayfish muscle). Each hemidesmosome consists of a “stripe” of 7-8 nm particles on the P F with complementary EF grooves. “Stripes” up to 3 p m in length are found lying parallel at regular intervals (80 -90 nm centre-to-centre spacing). This elaboration resembles those reported in molluscs by Prescott and Brightman (1976), who suggested that they might represent desmosomes. However, Franzini-Armstrong (1979) describing similar freeze-fracture images from the striated muscle of a spider, does not regard them as junctional structures. Shivers and his co-workers have applied freeze-fracture techniques t o a variety of crustacean tissues. They have described in some detail the structure of both mature and forming hemi-desmosomes (Shivers and Brightman, 1977; Shivers, 1977). However, their images are much more closely comparable to the arthropod gap junctions (see Section 4.2). The striking similarities to gap junctions are in the fracturing characteristics ( E F particles), the enlarged size of these particles, the linear particle configurations and loose plaques associated with developing junctions (see Section 4.7.1) and the involvement of two membranes, which would be inconsistent with a designation of hemidesmosome for these junctions (see Shivers, 1977, Figures 5 and 6; Shivers and Brightman, 1977, Figures 3, 6-8, and 18). A similar interpretation is made in the antenna1 gland where junctions are described, which in both thin-section (upper circles Figures 10 and 11 in Shivers and Chauvin, 1977) and freeze-fracture (EF particles of enlarged size Figure 9, PF pits with occasional particles Figures 13 and 14 in Shivers and Chauvin, 1977) appear more similar to gap junctions than maculae adhaerentes.

3.4.2 Non-arthropod tissues Desmosomes and desmosome-like structures occur so widely in the invertebrates that they are reported at least in passing in almost any ultrastructural study of a particular tissue. As a result a comprehensive survey of their

82


occurrence is beyond the scope of this review and an indication of the variety of organisms in which they are found is all that will be covered. Structures not altogether unlike desmosomes have been reported between prokaryotic and eukaryotic cells (Wagner and Barnett, 1974) in that intercellular filaments apparently derived from both cells traverse a regular and enlarged (30-40 nm) “intercellular space”. Hemidesmosomes and desmosomes have been found in flagellates (autodesmosome) (e.g. Brooker, 1970) and rotifers (e.g. Koehler and Hayes, 1969) and miniature autodesmosomes have been reported in trypanosomes (e.g. Vickerman, 1969; Smith et a f . , 1974). This latter study includes evidence from freezefracture but suggests that there is no intramembrane specialization associated directly with the desmosomes. In coelenterates, the situation appears confused as to whether desmosomes comparable in structure with those of insects exist (e.g. Roosen-Runge and Szollosi, 1965). Areas have been found where the intercellular space is very regular and the membranes are closely associated with cytoplasmic vesicles and these have been suggested as possible sites of adherence. However specialized cells termed desmocytes are also found in Cnidaria (Mackie, 1962; Chapman, 1969; Knight, 1970; Bouillon and Levi, 1971;Marcum and Diehl, 1978) and these cells are richly endowed with filaments ( 7nm in Cordyfophora;Marcum and Diehl, 1978) and, by their attachment to the perisarc and mesoglia, are thought to act as skeletal structures (for further details see Chapman, 1969). Wood (1 977), however, claims the occurrence of intermediate junctions beween myoid processes in Hydra; these junctions resemble fasciae adhaerentes and like them reveal little intramembrane specialization in freeze-fracture replicas. Cells from echinoderm tissues may be linked by desmosomes (e.g. Holland, 1971; Pentreath and Cobb, 1972) and desmosome-like junctions are found in planarians (e.g. Oaks, 1978) and chaetognathes (Duvert, 1977; Duvert et a / , 1978). Desmosomes of the macula adhaerens type as well as terminal bars have been described in the Pogonophora (Gupta et a f . ,1966; Gupta and Little, 1970). Zonulue adhaerentes are reported in the epidermis of annelids (e.g. Baskin, 1976; Storch and Welsch, 1970, 1972; Knapp and Mill, 1971; Boilly-Marer, 1972; Michel, 1972; Bilbaut, 1980; Skaer, 1979b) and both desmosomes (homo- and heterocellular) and hemidesmosomes have been found in the nervous system of earthworms (Hama, 1959; Coggeshall and Fawcett, 1964; Coggeshall, 1965; Giinther, 1976), the leech (Zimmerman, 1967) and ragworms (Baskin, 1971); these junctions are also found in many other annelid tissues. Similarly in molluscs, desmosomes are commonly found (e.g. Guptaetal., 1969; Lane and Treherne, 1972; Newel1 and Skelding, 1973; Kataoka, 1976; Ryder and Bowen, 1977).


3.5

83

FUNCTIONAL SIGNIFICANCE

The adhesive role of desmosomes, both macular, fasciar, zonular, and hemi-, appears t o be generally agreed. This adhesion is apparently achieved by the attachment of fibres projecting from the cell surface either to an extracellular matrix (as in the case of hemidesmosomes) or to similar fibres of another cell. In this latter case, the fibrillar attachments may be mediated by other intercellular structures, seen in thin-section as the stratum or midline (see Leloup et al., 1979 for a detailed model), Underlining the adhesive significance of desmosomes, they are found in insects in those tissues or in those positions in tissues subject to mechanical stress. Thus they are found at the attachment sites of insect muscles and in tissues subject to volume changes such as the epidermis (especially of fluid-sucking bugs, e.g. Rhodnius prolixus), the midgut of certain blood-sucking insects (where the intercellular spaces decrease in size during tissue distention after a bloodmeal (Reinhardt and Hecker, 1973)), salivary gland, Malpighian tubules and rectal tissues. They are also found in the ensheathing structures of the nervous system but especially noteworthy is their abundance in the glia of Manduca sexta where the nervous system, by virtue of its attachment to the musculature of the body wall, is in a state of constant agitation (Lane, 1972; Lane et al., 1977a). Desmosomes are also found where the relative position of neighbouring cells is physiologically critical; thus, for example, they are found in the rhabdomeres of the eye (e.g. Eley and Shelton, 1976; Shaw, 1978; Schinz, 1978; Nickel and Scheck, 1978). In vertebrate desmosomes, cytoplasmic tonofilaments are found in association with the electron dense plaques and, in the case of zonulae and fasciae adherentes, these filaments appear to be actin-like in composition. Moreover filaments may run through the cytoplasm of some cells forming an interconnected system, locating on the cell membranes at the sites of the desmosomes (see Staehelin and Hull, 1978 for a diagrammatic representation). This type of system would allow the transmission of tension through the cell. Where the filaments are contractile (7 nm), they may also be involved in controlling cell shape. On the other hand, the non-contractile filaments (1 0 nm) appear to form a structural framework for the cytoplasm and may also be involved in the passive distribution of shearing forces within the tissue. Whether such a network exists in insect cells is not known but clearly few desmosomes are associated with systems of tonofilaments as complex as in vertebrates and, where microtubules are present, the intimacy of their relationship with the junction is not clear.


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3.6

DEVELOPMENT

Little detailed work seems; to have been done on the formation of desmosomes in invertebrates, in contrast to the situation in vertebrate tissues. Considerations of the morphology of formation, timing in development of the appearance (and disappearance) of desmosomes, the stability of the forming and mature junctions and the involvement of the two cells in the process and symmetry of development are discussed and summarised by Campbell and Campbell ( 1971), McNutt and Weinstein (1973), Staehelin (1974) and Overton (1974). Within the invertebrates, Shivers and Brightman (1977) have followed the development of hemidesmosomes in the regenerating nerve root sheath of crayfish by means of freeze-fracture techniques. They show the alignment and clustering of 12 to 13 nm EF particles as the junctions develop. However there are doubts as to the classification of these junctions (see Section 3.4.1 and Lane, 1978a), and the evidence indicates that these clustering particles could be forming gap junctions (see Section 4.7.1) which are known to be present in the sheath of the crayfish nerve cord (Lane and Abbott, 1975). Lane and Swales (1978a) describe the formation of gliallperineurial and glial/glial maculae adhaerentes in the development of Calliphora larva. The development of cytoplasmic plaques appears to precede the intercellular elaborations. The intercellular separation appears regular and the electron density of the plaques is striking at a stage when the intercellular material is still amorphous. This material becomes at first faintly striated and then, as the junction elongates to its mature dimensions, the intercellular substance becomes clearly fibrillar (Fig. 25, insert). An even earlier stage in the development of desmosomes has been described in the pupal nervous system of Manduca sexta (Lane and Swales, 1979c). Here the membranes become aligned, showing a very regular intercellular space. However, at this stage, there are no cytoplasmic electrondense plaques nor is the intercellular material structured in any way. It is of interest to note that the precise alignment of membranes is achieved in the absence of observable cytoplasmic o r intercellular superstructures of filaments or microtubules. Although desmosomes are found in many mature tissues, they may be formed only transiently at some stages in development, and may be lost as soon as the junctions of the mature tissue are established (Poodry and Schneiderman, 1970; Reinhardt et al., 1976). A similar situation has been described in vertebrate tissues (see Overton, 1974) where, however, the transient desmosomes are found to differ in structure when compared with their mature counterparts, being simpler, smaller and with no fibrous attachments.


4

4.1

85

Gap junctions I N T R O D u CT I O N

Gap junctions were originally described as such (Revel and Karnovsky, 1967) because the cell membranes in the regions in question were separated by a narrow gap of only 2 to 4 nm. They had been earlier referred to as a “nexus” (Robertson, 19F3); Dewey and Barr, 1964), and more recently have been termed macula communicans (Simionescu et al., 1975), because they appear to be the sites of cell-to-cell communication. These sites of communication are in the form cf special low resistance pathways between cells, through which the direct exchange of substances can occur; that which is transferred may be ions and/or metabolites and the cells are then said to be ionically or metabolically coupled. The importance of arthropods as experimental material in the elucidation of the function of gap junctions was evident from the beginning in that the first ionically coupled cells to be described were neurones of the crayfish (Furshpan and Potter, 1957). Subsequent studies on such coupling between excitable cells were made in crustacea (for e.g. Asada et al., 1967; Payton, Bennett and Pappas, 1969; Peracchia, 1973a, b, 1974) and other excitable tissues (see Bennett, 1977, 1978; Sotelo, 1977) where intracellular microelectrode techniques demonstrated low resistance pathways between the cells. Transfer of tracers indicated the degree of their permeability and fine structural studies elucidated their characteristic thin-section and freezefracture appearance. These specialized electrical synapses are now referred to as electrotonic, low-resistance or gap junctions. Low-resistance pathways have also been found to occur between nonexcitable cells in epithelia and other tissues, as well as as between cultured cells. Again, arthropod material has featured significantly since the salivary glands of dipteran flies (Loewenstein and Kanno, 1964; Loewenstein, 1976, 1977; Loewensteinet al., 1978b) have been used as an elegant test system to study such parameters as the molecular weight range of substances capable of being transferred between coupled cells and the effects of cations on junctional permeability. Cultured insect cell lines have also been employed to study uncoupling and the factors which affect this phenomenon (Gilula and Epstein, 1976). Gap junctions have also been implicated in metabolic coupling or cooperation, by which is implied the cell-to-cell transfer of metabolites (SubakSharpe et al., 1969). This phenomenon has been little studied in arthropod systems and has tended to be most frequently analysed in cultured mammalian cells where it has been shown that metabolically coupled cells may also be ionically coupled (Gilula et al., 1972). Exchange of regulatory molecules between coupled cells in the coordination of development has

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also been suggested in a variety of different systems, but in insects, thus far, gap junctional communication has been found to play no clear-cut role in differentiation, for example, of cuticular pattern (Warner and Lawrence, 1973; Lawrence and Green, 1975). There is suggestive evidence in the insect nervous system, however, where interglial gap junctions are present throughout almost the entire larval and pupal development (Lane and Swales, 1978a, b, 1979a, c), to indicate that exchange of regulatory substances may take place throughout larval and pupal growth. The actual disappearance of gap junctions has often been thought to be the developmental cue for cell determination (see Section 4.7.2) and here again relatively few arthropod systems have been studied, save, for example, grasshopper embryonic CNS (Goodman and Spitzer, 1979) and the locust eye (Eley and Shelton, 1976) where it is possible that molecules exchanged between cells, prior to the uncoupling or junctional disappearance, may provide the signals to trigger off the next stage of development. In the arthropods, as well as many other invertebrates, nonexcitable tissues are found to be coupled by gap junctions, which often co-exist with septate junctions (for example, Hudspeth and Revel, 1971;Gilula and Satir, 1971; Rose, 1971; Hand and Gobel, 1972; Skaer and Lane, 1974; Caveney and Podgorski, 1975; Lane et al., 1977a; King and Spencer, 1979). This originally led to difficulties in determining which junction was the actual site of cell-to-cell communication (see Section 4.6.3). The septate junction was the more obvious structure, indeed the only one seen in early investigations, and hence seemed the likely candidate (Wiener et al., 1964; Loewenstein and Kanno, 1964; Gilula et al., 1970; Loewenstein, 1973). However, the fact that in crayfish septate axons (Asada et af., 1967) and also in many vertebrate tissues, cells known to be coupled, apparently possessed only one type of junctional specialization, the gap junction (for example, Revelet al., 1971; Pinto da Silva and Gilula, 1972; Gilulaetal., 1972; Pinto da Silva and Martinez-Palomo, 1975; Larsen, 1975), helped clarify the situation. The gap junction has now emerged as the single intercellular structure which is organized in such a way as to permit coupling by allowing interchange of ions and small molecules between cells via the channels present in their component particles. X-ray diffraction studies have enabled the molecular architecture to be analyzed so that models can be produced (Caspar et al., 1977; Makowski et al., 1977). Although the mere presence of gap junctions cannot alone be taken as proof that molecules are being exchanged between the cells thus coupled, there is evidence from a variety of sources to verify the fact that different substances can by physically interchanged from cell to cell via the gap junctions. For example, low resistance connections have been found to be associated with the capacity for cell-to-cell transfer of fluorescent dyes such


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as Procion yellow or fluorescein (Kanno and Lowenstein, 1964; Payton et al., 1969), labelled nucleotides (Subak-Sharpe et al., 1969) and labelled amino acids and small peptides (Simpson et al., 1977). (For tables giving details of examples of coupled cells and permeant molecules, see Bennett, 1978; Loewenstein, 1976; Loewenstein et al., 1978b). In recent years several reports have appeared which review the structural features found in gap junctions in vertebrates (Bennett, 1977,1978; Gilula 1977, 1978; Larsen, 1977a; Revel, 1978) but the structural features in the invertebrates may show some deviation from these (Flower, 1977; Lane, 1978a). A description of those found in arthropods follows.

4.2 4.2.1

STRUCTURAL FEATURES


In thin-sections, gap junctions are characterized by a reduction of the usual intercellular cleft between the membranes of apposing cells down to 2 to 4 nm (Fig. 26). Because of this they have also often been referred to as regions of “close” membrane apposition or close junctions in insect tissues (Hagopian, 1970; Burger and Uhrik, 1972; Chi and Carlson, 1976b). There is often a cross-striated appearance across this gap (Figs 26 and 27) which is no doubt due to the component junctional particles which bridge the cleft between the cells. This very narrow gap is usually only visible when en bloc uranyl acetate staining is employed (Farquhar and Palade, 1965); without this additional membrane enhancement the adjacent membranes of such junctions seem to be directly apposed o r fused. Such membrane apposition was mistakenly referred to as being a tight or occluding junction by earlier investigators of insect material (for example, Locke, 1965; Osborne, 1966; Berridge and Gupta, 1967; Grimstone et al., 1967; Maddrell and Treherne, 1967; Smith, 1968; Stuart and Satir, 1968; Oschman and Wall, 1969; Smith et al., 1969b; Schurmann and Wechsler, 1969; Lane and Treherne, 1969, 1970; Treherne e f a/., 1970; Zacharuk et al., 1971; Leslie and Robertson, 1973; McLaughlin, 1974a; Peacock and Anstee, 1977b) because they had not en bloc stained the tissues and the intercellular space could not be resolved. The appearance of a gap junction is often referred to as being septilaminar (heptalaminar or 7-layered), where the true tight junction (and indeed the less highly resolved image produced without en bloc staining) is a pentalaminar (or 5-layered) structure. Gap junctions being extensive regions of close membrane apposition are distinguishable from tight junctions which usually take the form of tiny punctate appositions. Recognizable septilaminar gap junctions were first seen in insect tissue in epithelial cells (Hagopian, 1970). In this context, however, it is of interest that as early as

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Fig. 26 Thin-section of a lengthy gap junction, from the perineurium of the cockroach, Periplanera americana,en bloc stained with uranyl acetate. Note the close apposition of the two unit membranes separated by a reduced intercellular cleft of 2-4 nm, with the occasional appearance of cross-striations. x 181 500 Fig. 27 Thin-section of a lanthanum-impregnated gap junction from the rectal pad cells of a cockroach. The normal intercellular clefts (C) can be seen to narrow where the gap junction begins; as the section starts to cut tangentially across the gap junction, through the plane of the membrane, the gap junctional sub-units can be resolved as non-opaque particles in negative contrast against the lanthanum-stained background. In this preparation the sub-unit packing is fairly regular and the central channel can be seen in some of the particles. The central pore is more clear-cut in the insert, which is from the primitive arthropod, Limulus, after lanthanum staining. x 146 400: insert x 289 400

1965, in examining the so-called zonulae occludentes of the caterpillar Calpodes, Locke commented that “the intercellular material in the tight junctions is frequently discontinuous with a repeat pattern like the junctions in synaptic disc membrane complexes”. Since he refers here to Robertson’s (1963) work which first described the nexus or gap junction, it is clear that what Locke was actually seeing, although he did not appreciate the fact, were gap junctions! Equally Trujillo-Cenoz (1965) showed a “synaptic contact” in fly eye which is undoubtedly a gap junction and similarly,


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Landholt and Ris, in 1966, studying soma-somata interneuronal junctions in the wood ant, described electrical junctions, which were “characterized by a diminished width of the (synaptic) cleft as well as by a typical crossdirectional ‘subunit pattern’ in the junctional membranes”. 4.2.2

Lanthunum staining

After treatment with lanthanum, either colloidal (Revel and Karnovsky, 1967) or ionic (Machen et al., 1972; Lane et al., 1977a) the gap junctional cleft becomes heavily stained. This intercellular space associated with gap junctions can also be negatively stained with tannic acid, ruthenium red and phosphotungstate or uranyl salts (van Deurs, 1975; Zampighi and Robertson, 1973). The extracellular space becomes, in these circumstances, indistinguishable from the dense outer leaflets of the membranes of the two adjacent coupled cells, producing a pentalaminar appearance with a gap of about 7.5 to 8.5 nm (Fig. 27) instead of the usual ca. 3 nm. Again, cross striations may be seen (Fig. 27), while, in tangential sections, when the junction is viewed en face, a lattice is apparent, with the component subunit particles revealed as electron lucent spots or particles lying in an electrondense network-like background (Fig. 27). The en face images demonstrate the relatively loose packing of the particles in the arthropod macular gap junctions. These non electron-dense spots are presumed to be the points where the intra-membranous junctional particles of the two apposed and coupled cells lie opposite each other, with particle subunits in contact, and the junctional channels of the two opposed particles in register; the central channels frequently take up the lanthanum and appear electron-opaque (insert, Fig. 27). This feature provides evidence for the presence of a central pore through which the exchange of ions and small molecules is thought to occur. 4.2.3 Freeze-fructured uppeurar1c.e Evidence for gap junctions by freeze-cleaving in insects was first put forward by Flower in 1972 and the differences they exhibited from those of vertebrates were mainly in terms of their cleaving characteristics; the junctional particles showed preferential fracturing onto the E face (then termed the B face) so that they were termed B-type or inverted gap junctions. The particles were about 13 nm in diameter, and left complementary pits in the P face (then termed A face) (Fig. 28). The reduced intercellular cleft is obvious when the fracturing plane cleaves across the junction (insert to Fig. 28) and most subsequent reports reveal that the tendency for the particles to fracture onto the E F (although some may remain on the PF- see Fig. 29 and

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INTERCELLULAR J U N C T I O N S I N I N S E C T T I S S U E S

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Section 4.4.1) and to measure approximately 13 nm (the range is about 12 to 14 nm) is fairly universal in arthropod tissues. Although a range of smaller subunit sizes has been reported in lanthanum-stained insect gap junctions, for example 6.5 to 9 nm (Caveney and Podgorski, 1975) or larger, it must be remembered that the shadowing that occurs when replicas are made produces an increase in the actual size due to the heavy metal deposition (see discussion in Skaer and Lane, 1974). Particle separation is irregular, or, if uniform, is usually about 12 nm (Flower, 1977) and occasionally fused aggregates of two of more junctional particles occur (double arrows in Fig. 28). However, here the possibility of shadowing artefacts should be borne in mind. The junctions appear as irregular macular aggregates of intramembranous particles; each junction may vary from about 30 nm to several pm in diameter and may contain only a few particles or many 100s of particles (Fig. 30). In some cases, linear E F arrays of 13 nm gap junctional particles are observed in mature tissues (Fig. 31) (see Section 4.5, point 4). Central channels may be observed in the individual gap junctional particles after shadowing (as in Fig. 33, insert). The size of these pores in insects has been calculated on the basis of the molecular weights of molecules permeating these ,junctionalchannels (Simpsonetal ., 1977) and ranges from about 1 to 2 nm Comparable pores have been seen in replicas of such arthropod tissues as the moth (Lane and Swales, 1979a), collembola (Dallai, 1975),Lirnulus (Johnsonet al., 1973; Lane, 1978b) and the crayfish (Peracchia, 1973b). Although some earlier investigators claimed that the channels seen in replicas are artifacts due to decoration effects or plastic deformation (Plattner et al., 1975), the more recent X-ray diffraction studies tend to corroborate their existence, at least in mammalian tissues (Casper et al., 1977; Makowski et al., 1977). In some cases, the resolution may be sufficiently good to discern the subunits which compose each particle and surround the central pore; originally, in crayfish tissue, there appeared to be 6 of these hexamers (Peracchia, 1973b) but recent studies suggest that there may in some cases only be 4 tetramers (Peracchia and Peracchia, 1978). Structural diversity may occur in the gap junctions within a single organism, even within the same tissue. Thus in the caecal epithelium of the crustacean Orchestia two types occur that differ in size, spacing and polarity of their intermembranous particles (Graf, 1978b). Also, in crayfish giant Fig. 28 Freeze-fracture replica showing typical circular arthropod gap junctions with E face (EF) particle aggregates and P face (PF) pits. Some particles lie so close to one another as to appear fused (double arrows). Note the presence of PF particles, occasionally aligned into linear ridges (PF arrow) with complementary EF grooves (EF arrow). There is a reduction in the intercellularcleft at the gap junctional regions (* and in insert). x 44 100;insert x 79 200 Fig. 29 Replica of gap junctions with E face (EF) particles and complementary PF pits with a number of particles which fracture onto the P face. x 45 400

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INTERCELLULAR JIJNCTIONS I N INSECT TISSUES

93

axon gap junctions, the component globules are organized into two different arrangements, both hexagonally packed but with different unit “cells” (Peracchia, 1973a, b); these, however, have been shown to be due to changes in junctional membrane permeability, from high to low coupling resistance (Peracchia and Dulhunty, 1976) (for a further discussion of this, see Section 4.6.3b). Morphologically distinct kinds of gap junctions with regard to particle distribution, size and spacing in freeze-fracture replicas have been found to occur between different cell types in the same organism as well. For example, in locust heart muscle, linear gap junctions occur (Fig. 31) while other locust tissues possess typical macular plaques. In Planaria, three such types exist; these are thought to relate to the specialized functions of the different tissues in which they are found, so that, perhaps, they have differing permeabilities, one shuttling food reserves, another ionic currents, and a third, exchanging metabolic or development signals (Quick and Johnson, 1977). Although such structural differences between the gap junctions in insect tissues are not common, subtle distinctions in function need not necessarily be reflected in obvious ultrastructural differences, since a small change in channel diameter, for example, would undoubtedly not be recognizable (Pappas et al., 1971; Bennett, 1978). Whether tissues are glycerinated before or after fixation and whether they are fixed or unfixed, may affect the fracturing properties of the gap junctional particles (Flower, 1977). For example, the anastomosing particulate network appearance of unfixed B-type gap junctions is artifactual and induced by glycerol (Flower, 1977). 4.3

MODEL DERIVED FROM STRUCTURAL EVIDENCE

After a thorough analysis of the information gleaned about gap junctions from thin-sections, lanthanum-impregnated material and freeze-fractured replicas, it is possible to construct a model to explain their three-dimensional structure insofar as the evidence permits (Fig. 32). Although a number of reconstructions have been proposed for vertebrate gap junctions (for example, McNutt and Weinstein, 1973; Staehelin, 1974; Pappas, 1975; Makowskietal., 1977; Bennett, 1977; Staehelin and Hull, 1978) there seem to have been only very simplified versions put forward for those of arthropods (Pappas et al., 1971; Satir and Gilula, 1973; Peracchia, 1973b; Fig. 30 Freeze-fracture replica from the moth, Manduca sexfa, showing the EF particle aggregatestypical of insect gap junctions. Note the variability in size and number of component particles in these gap junctions, their relatively loose packing, and the frequency with which they are to be found. x 79 600 Fig. 31 Freeze-cleave replica from the dorsal vessel or heart of the locust, Schistocerca gregaria. These 13 nm EF particles have the features characteristicof gap junctions but occur in a linear instead of a macular configuration. x 74 000

NANCY J. LANE AND HELEN IeB. SKAER

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Loewenstein, 1976). The one presented here (Fig. 32) attempts to incorporate information from all possible perspectives; it draws attention to the EF fracturing position of the junctional particles and the complementary pits produced in the P face as well as indicating the appearance in both /-

CYTOPLASM-/

Fig. 32 Model of an arthropod gap junction showing the junctional particles which lie in apposition so as to straddle the extracellular space with the central channels aligned. In replicas the junctional particles fracture onto the E face and the central channel can be resolved; complementary pits are to be found on the P face. Lanthanum infiltrates the intercellular space as well as the pores within the junctional particles, Although depicted as being regularly packed, the particles are in fact usually irregularly arranged. Each particle is subdivided into sub-units which may be 4 or 6 in number (not shown on diagram). These particles are seenen face in tangential sections as non-opaque spots against an electron-dense background

lanthanum-infiltrated sections and en bloc uranyl acetate-stained thinsections. The lanthanum sits in the extracellular space around the junctional particles and also frequently, presumably artefactually (see Bennett et al., 1972) enters the central channel that links opposing particles; this gives rise to the striations seen in transverse sections and to the lattice or network effect around the unstained particles in en face views. In Fig. 32 the particles that comprise the junction are depicted as single globules. The X-ray diffraction evidence thus far available, in support of 6 hexameric sub-units making up each particle, is limited to vertebrates (Casparet al., 1977; Makowski, et al., 1977). Freeze-fracture studies on crustacean septate and other gap junctions suggests hexamers (Peracchia, 1973b) or tetramers (Peracchia and Peracchia, 1978) so there is no general concensus yet as to the number of sub-units per particle in arthropod systems. 4.4

DISTRIBUTION OF GAP JUNCTIONS

4.4.1 Homocellular, heterocellular and autocellular junctions in arthropod tissues Gap junctions are commonly homocellular and link cells of the same morphological type, which then can presumably be synchronized physiologically.


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In thin-sections, these homocellular gap junctions have been found between a wide variety of insect cell types and include salivary gland cells (Oschman and Berridge, 1970; Rose, 1971; Burger and Uhrik, 1972; Leslie and Robertson, 1973; Berridge et a[., 1976b), cells of various parts of the gut including midgut, proctodeum and rectum (Berridge and Gupta, 1967; Reger, 1970;NoirotandNoirot-TimothCe, 1971a,b, 1976,1977; Wall and Oschman, 1973; Noirot-TimothCe and Noirot, 1974b; Ito et al., 1975; Dallai 1975; Lane, 1978a, 1979a, c; Noirot-TimothCe et al., 1979; Skaer et al., 1979), haemocyte capsules (Baerwald, 1975), cyst cells of the testis (Szollosi and Marcaillou, 1977; Jones, 1978; Skaer and Jones, 1979), follicular epithelial cells of the ovary (Huebner and Anderson, 1972; Mahowald, 1972; Woodruff, 1979), glial cells of the nervous system (Lane and Treherne, 1972a, 1973; Skaer and Lane, 1974; McLaughlin, 1974a, b; Lane et al., 1977a; Ne’eman and Spira, 1977a; Lane and Swales, 1978a, b, 1979a, b; Lane 1978a) or nerve cells (Osborne, 1966; Landholt and Ris, 1966; Morris and Steel, 1977), sub-cuticular surface epidermal cells (Hagopian, 1970; Caveney and Podgorski, 1975; Lawrence and Green, 1975),cellsoftheeye(EleyandShelton,1976; Ribi, 1978; NickelandSheck, 1978; Shaw, 1979; Carlson and Chi, 1979; Lane, 1979d), fat body cells (Skaer and Lane, unpublished observations), cardiac muscle cells (Fig. 3 l), glandular epithelial cells (Stuart and Satir, 1968), the undifferentiated cellsof haemocytopoietic organs (Monpeyssin and Beaulaton, 1978), imaginal disc cells (Poodry and Schneiderman, 1970) and cells of Malpighian tubules (Wall et al., 1975; Dallai, 1975; Skaer 1979; Green et al., 1979). After Flower’s (1972) initial freeze-cleave study on insect tissue, freezefracture reports of gap junctions in arthropods have been numerous and include, not only tissues from insects (for example, Noirot-TimothCe and Noirot, 1974; Skaer and Lane, 1974; Flower and Filshie, 1975; Skaer ef al., 1975; Baerwald, 1975; Lane et al., 1977a; Flower, 1977; Ne’emen and Spira, 1977a. b; Lane and Swales, 1978a, b, 1979a, b) but also tissues from other arthropods as well, such as from the xiphosauran, Lirnulus (Gilula, 1973; Johnson et al., 1973; Lane and Harrison, 1978; Harrison and Lane, 1980), from ticks (Binnington and Lane, 1980) and from crustacea (Gilula, 1972b; Peracchia, 1973a, b, 1974; Perrachia and Dulhunty, 1976; Graf, 1978b). Shivers and co-workers report on desmosomes and hemi-desmosomes in crayfish tissue (Shivers and Chauvin, 1977; Shivers and Brightman, 1977) that bear all the freeze-fracture attributes of the gap junctions which they probably actually are (see Section 3.4.1). For the most part, these arthropod gap junctions have EF particles. However, there are exceptions. The perineurial/glial gap junctions of crayfish, seen in thin-sections (Lane and Abbott, 1975; Lane et al., 1977b),

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are reported, by freeze-fracture, either to possess conventional EF particles and P F pits (Brightman et al., 1975) or to exhibit particles that are irregularly segregated on both P and E faces (Perracchia and Dulhunty, 1976). In other crustacean tissues, such as caecal epithelium, the gap junctions are found to have mainly P F particles (Graf, 1978b). Gap junctions are sometimes heterocellular; that is, they may link two different cell types within the same organism which are then able to exchange substances and so are functionally coupled. These usually are indistinguishable structurally from homocellular gap junctions (Johnson et al., 1973). For example, the ooplasm of oocytes may be coupled to the encompassing follicle cells in moth ovary (Woodruff, 1979), the principal cells of the insect rectum may be associated by gap junctions to basal cells (Noirot and Noirot-Timothte, 1976, 1977), perineurial cells are linked to underlying glial cells by communicating junctions in the central nervous system of a variety of insects (Lane, 1978a; Lane and Swales, 1979a), neuroblast cells are electrically coupled to epithelial cells in grasshopper embryos (Goodman and Spitzer, 1979) the epithelial cells of the midgut of Limulus are frequently coupled to reserve cells (Johnson et al., 1973), glial and visual cells in Limulus eye are associated by “quintuple-layered junctions” (Lasansky, 1967), and epithelial and underlying interstitial cells are coupled by gap junctions in the midgut of certain fresh water crustacea and terrestrial Arachnids (Reger, 1970). There are a number of examples of heterocellular gap junctions in vertebrate tissues, and the details of these associations are described by Larsen (1977a). It has been established, by co-culturing insect and mammalian cells lines, that low-resistance heterocellular gap junctions cannot be extensively established between two such different cell types (Epstein and Gilula, 1975, 1977). This led these authors to suggest that different gap junctional phenotypes must exist, as is suggested by their differing fracturing characteristics and particle sizes. Although a low incidence of coupling was found between heterologous cell lines derived from different arthropod orders, insect cell lines from the same order will couple extensively (Epstein and Gilula, 1977), suggesting an insect interorder specificity. In this vein, it is interesting that rat granulosa cells may transmit hormonal stimuli through permeable gap junctions formed in culture with mouse myocardial cells: these stimuli were shown to be passed by means of a second messenger, thought to be cyclic AMP (Lawrence et al., 1978) and clearly here functional heterocellular gap junctions have formed between cells from two different genera. No communication specificity appears to exist, therefore, in this instance. Very few examples of such communication-specificity have been as yet documented of which one is the insect interorder specificity (Epstein and Gilula, 1977).


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The functional significance of autocellular gap junctions, referred to as “reflexive” gap junctions (Herr, 1976), which occur between two projections of the same cell (for examples in vertebrates see Larsen, 1977a) is still obscure. Perhaps these have to do with the exchange of ions or trophic molecules which are important locally but which have difficulty in diffusing any great distance. Autocellular gap junctions have been reported between salivary gland cells in the adult blowfly (Oschman, 1976, in Larsen, 1977a), and also occur in tracheal cells where the mestracheon exhibits gap junctions (Lane and Skaer, unpublished observations). They often appear between attenuated cytoplasmic processes of cells in insect tissues [for example, glial cells (Lane et al., 1977a) where diffusion could well be a problem to be overcome. 4.4.2

Other invertebrate tissues

Gap junctions are fairly ubiquitous and are also found in a wide range of other invertebrate groups. In the molluscs (Flower, 1971; Gilula and Satir, 1971), chaetognaths (Duvert et al., 1978) and tunicates (Lorber and Rayns, 1977) they are vertebrate-like in that their component particles fracture onto the P face leaving E F pits, and the particle separation is usually 9-10 nm. Some particles also cleave onto the PF in Hydra (Hand and Gobel, 1972; Filshie and Flower, 1977; Wood, 1977), and in some, but not all, planarian worms (Quick and Johnson, 1977; Flower, 1977). In annelid worms, the situation is complex, but it appears that if the tissue is fixed the particles are mainly PF (Bilbaut, 1979), although some fracture onto the EF (Skaer, 1979b), while in unfixed material, they are primarily EF particles (Flower, 1977). In centipedes, the junctional particles fracture onto the EF in fixed preparations (Juperthie-Jupeau, 1979). A number of particles in the gap junctions of coelenterates adhere to the E face (Wood, 1977; Filshie and Flower, 1977) and here strong intercellular adhesive forces exist too, since fragments of cytoplasm often cover the junctions. This phenomenon can also be observed in some insect tissues (for example see Fig. 36, insert B) and annelid tissues (Skaer, 1979b). There is a good deal of variation in the shape of gap junctions in invertebrates; round or oval macular plaques occur in hydra, planaria, ragworms, centipedes and some arthropod tissues, but other arthropod tissues may have more irregular outlines (Flower, 1977). The centre-to-centre spacings of the invertebrate gap junctional particles is variable because they are rarely very tightly packed (Figs 28 and 30). In those cases where figures have been given the centre-to-centre separation for insects measures from 1 0 to 12 nm (Flower, 1972; Satir and Gilula, 1973; Baerwald, 1975; Dallai, 1975; Caveney and Podgorski, 1975) and

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holds also for crustacea (Hudspeth and Revel, 1971). In planaria and ragworms it appears to be about 12 nm too (Flower, 1977), while in Hydra and other coelenterates it is 9.5-11 nm (Hand and Gobel, 1972; King and Spencer, 1979) and in molluscs it is 10 nm (Flower, 1977). A larger repeat of 18-20 nm has been reported in crayfish nerve fibres (Peracchia, 1973b) but this may reduce to a smaller repeat when the cells are uncoupled (Peracchia and Dulhunty, 1976). 4.5

STRUCTURAL DIFFERENCES BETWEEN ARTHROPOD A N D VERTEBRATE GAP J U N C T I O N S

In thin-sections, the gap junctions of arthropods (for example, Hagopian, 1970; Reger, 1970; Noirot andNoirot-Timothie, 1971b; Laneetal., 1977a) appear virtually identical to those found in vertebrate cells although certain reports indicate that the intercellular gap is slightly larger (3-4 nm) than that of vertebrates(Paytoneial., 1969; Hudspeth and Revel, 1971; Rose, 1971). There is also the exception of the so-called “septate” gap or electrotonic junctions that couple the giant axons of crayfish (Peracchia, 1973a) when fixed in glutaraldehyde-H202solutions; the space between two axonal membranes may be as much as 4-5 nm wide (Zampighi et al., 1978). Electrotonic junctions in vertebrates have been divided into asymmetrical and symmetrical gap junctions, the former exhibiting greater electron opacity in the dendritic rather than the axonal side (Bennet et al., 1967) but this has not been observed in the insect electrotonic junctions thus far studied (Ribi, 1978). The only clear-cut morphological evidence of asymmetry is the presence of 80 nm vesicles in the cytoplasm near the presynaptic membrane of the rectifying electrotonic synapse in crayfish giant axons (Hanna et al., 1978). In freeze-fracture preparations, the insect junctions also have the same basic structure as vertebrate gap junctions, in that they are both composed of macular arrays of intramembranous particles (see McNutt and Weinstein, 1973; Staehelin, 1974; Gilula, 1974,1977; Bennett, 1977) and in that there is a reduction in the intercellular space when fracture faces cleave across the membranes composing a gap junction. Vertebrate gap junctions are, like those of insects, fairly ubiquitous, in their distribution between cells of different tissue types; they appear to be present in most kinds of tissue except for skeletal muscle, red blood cells and spermatozoa (Revel, 1978) and are absent in many differentiating neurons (Gilula, 1978). However, there are some freeze-fracture differences between aphropod and vertebrate gap junctions, in spite of their basic similarity. 1 Arthropod gap junctional particles in general tend to adhere to the E face, leaving complementary P face pits; the opposite fracture face possesses


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the junctional particles in vertebrates. There are, however, some recent reports suggesting that under different circumstances, with respect to fixing or cryoprotection, some of these particles in arthropod tissues may adhere to the PF (Flower, 1977; Wood, 1977; Graf, 1978b), but these are very much in the minority. 2 Arthropod gap junctional particles measured in freeze-fracture replicas are approximately 13 nm in diameter (Flower, 1972; Satir and Fong, 1973; Satir and Gilula, 1973; Flower, 1977; Gilula, 1977; Lane, 1978a) which is much larger than the 6 nm (Revel, 1978) to 7-8 nm (Gilula, 1978) particles that make up vertebrate gap junctions. However, since those of arthropods are larger particles, with different fracturing characteristics, they are readily distinguishable from the tight junctional particles which are smaller, 6-10 nm, and fracture onto the P face. This convenient distinction is not found in vertebrates where PF particles of “ambiguous identity” may occur (Larsen, 1977a). In studying such phenomena as the mode of formation of gap junctions, in vertebrate tissues, where tight and gap junctions co-exist on the same membrane face, no distinction can be drawn between the particles destined to form one of the two junctional types (Revel, 1978) until they have assumed the distribution which is characteristic of their mature state. This is not so in arthropods which is a distinct advantage in following the changes in particle distribution that occur during junction formation (Lane and Swales, 1978a, 1979a, b; Lane, 1978a, b). 3 The gap junctions of arthropods are composed of intramembranous particles which tend to lie in loose formations (Figs 28 and 30) either as disordered arrays or with a 1 2 nm spacing (Flower, 1977), not in the closely-packed hexagonal arrays with a 9-1 0 nm spacing that are characteristic of vertebrate macula communicans (see for example, Gilula, 1974). Differences in the degree of order with which the particles are arrayed may relate to whether or not they are coupled (Peracchia and Dulhunty, 1976; Perrachia, 1978). In this context it is of interest that two types of hexagonal arrays have been reported in vertebrates, one with the usual 9-10 nm spacing (Staehelin, 1974) and the other with a 19-20 nm spacing (Staehelin, 1972). Factors affecting coupling will be considered in greater detail in the later section on the functional significance of gap junctions (Section 4.6.3). Recent studies on mammalian gap junctions with a rapid-freezing technique involving liquid helium-cooled copper block surfaces, has produced freeze-fracture replicas with randomly-distributed junctional particles, rather than hexagonally-packed ones (Raviola et af., 1978). Using this system, the junctions in tissues suffering from anoxia contract into closely packed arrays, suggesting that they are uncoupled and in a high resistance state. This indicates that the hexagonally-packed gap junctional particle arrays normally seen in vertebrate tissue have been uncoupled by the

NANCY J. L A N E AND H E L E N leB. SKAER

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preparative procedures. In coupled cells, low resistance junctions should therefore appear as randomly distributed particles, or connexons, with little mutual affinity (Raviolaet a [ . , 1978). This work supports the hypothesis put forward by Peracchia that states that closely-packed particles represent the uncoupled state. It is interesting to note that insect gap junctions often appear in a relatively loosely packed state; this may mean that conventional fixing does not abolish electrical coupling between insect cells (cf. vertebrate and crayfish cells, Bennett et al., 1972) or that their packing arrangements do not accurately reflect their state of coupling. 4 The size of the gap junctional aggregates in arthropods is highly variable; the plaques in insect tissue may range widely both in diameter and in the number of component particles (Fig. 30), from 5 to 10, up to many 100s. Such small plaques are less frequent in vertebrate tissues, although they have been reported in such situations as embryonic tissues (Argue110 and Martinez Palomo, 1975). Ordinarily, the insect junctional areas are not particularly extensive but, like the vertebrates, larger areas of cell surface may occasionally be junctional, especially in the case of developing junctions (Fig. 34). Although the particles in arthropod tissues are usually in the form of macular arrays, they may sometimes be ring-shaped (Lane, 1978b) or in the case of insect cardiac muscle, linear configurations may occur (Fig. 31). Interestingly, linear arrays have also been reported in cardiac tissue of vertebrates (Larsen, 1977a; Mazet, 1977; Kensler et al., 1977; Shibata and Yamamoto, 1979) as well as in certain other tissues (Raviola and Gilula, 1973; Pricam et al., 1974; Fujisawa et al., 1976; Simionescu et al., 1976). This is highly unusual and more typically such irregular patterns only occur during development; there are also certain distinctions in the mode of formation of gap junctions in arthropods in comparison with those of vertebrate cells which will be considered later (see Section on Developing gap junctions 4.7.1).

4.6

FUNCTIONS O F G A P IUNCTIONS

4.6.1 Adhesion Since gap junctions serve to maintain a very close cell-to-cell association between the cells that are coupled, they inadvertently must serve in an adhesive capacity to some extent. This has been particularly stressed in certain situations, (for example, Lorber and Rayns, 1977; Pannese et al., 1977; Wood, 1977) and in insect tissues, cytoplasm above gap junctions frequently adheres to these as though held by strong adhesive forces (Fig. 36, insert B). In particular, insect interglial gap junctions have been thought


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to be a specific device for cell-to-cell adhesion (Ribi, 1978), while in dipteran midgut, polysaccharides found in the intercellular 2 to 3 nm gap, have been interpreted to function as a “cement” substance for cellular adhesion (Reinhardt and Hecker, 1973). In further support of this, gap junctional complexes are found to be very resistant to disruption by mechanical or physical means, or by treatments such as divalent cation removal or proteolysis (Gilula, 1974; 1977). When cells are separated by these means, one of the dissociated cells retains the intact gap junctional complex (Berry and Friend, 1969). When unfixed coelenterate tissue is exposed to 25% glycerol, commonly used as a cryoprotectant, the septate junctions were lost after 5 min, whereas the gap junctions did not separate, even though considerable distortion of the tissues often took place (Filshie and Flower, 1977); this was taken to suggest that gap junctions may have a “localized bonding function”. Only treatment with hypertonic sucrose effectively splits the gap junctional complex (Goodenough and Gilula, 1974) but such a procedure has not thus far been applied to insect junctions. 4.6.2 Sieve-area effects In some instances, gap junctions have been considered to have a rather different function, in forming an intercellular sieve area for small molecules, that is, acting as a specialized barrier for controlled entry between cells (Forssmannetal., 1975). For example, such regulation of the speed of entry of molecules seems likely to be one function of gap junctions in the crustacean CNS. In the crayfish, unlike the insects, there is no ultimate blood-brain barrier to the entry of substances, but the rate of penetration through the perineurium is significantly slower than in “open” systems (Abbott et al., 1977). This partial restriction seems likely to be due to gap junctions which are found in the perineurium of the CNS (Lane and Abbott, 1975) but which are relatively infrequent or absent in the peripheral nerves which are quite patent to tracers (Lane et al., 1977b). 4.6.3

Cell-to-cell communication and low-resistance path ways

( a ) Historical introduction: gap junctions versus septate junctions as communication channels. Although the cytoplasm of cells has a low specific resistance, that of the plasma membrane is high. In many cases, however, this resistance can be overcome and adjacent cells are found to be electrically coupled, with low resistance pathways between them so that they behave like a synctium. The morphological basis for this coupling appears to be gap junctions. In recent years, in attempts to understand how these are regulated, many experiments have been carried out on the uncoupling of

102


adjacent cells by reducing the permeability of the gap junctions. The most extensively studied systems are both arthropod in nature: the epithelial cells of dipteran salivary glands and the crayfish septate giant axons. These have been studied both electrically with intracellular microelectrodes and by exogenous tracer exchange; substances that uncouple the cells electrically are also found to inhibit the intercellular flow of tracers. However, as will be discussed later, the systems are more complex than this suggests and modulation in the degree of uncoupling is possible. In dipteran salivary glands, much of the early work on coupling was carried out by Loewenstein and his co-workers (Loewenstein and Kanno, 1964; Lowenstein, 1973, 1977). It is now clear that the cells of Chironomous or Drosophila salivary glands have both septate and gap junctions. However, it seemed originally that the former were providing the pathway for intercellular exchange (Bullivant and Lowenstein, 1968; Gilula et al., 1970) since the latter did not occupy a sufficiently large fraction of the lateral cell surface to be responsible for the degree of intercellular coupling observed (Rose, 1971). Doubt, however, began to be expressed as to the role of the septate junction in communication in the developing insect in 1970 (Poodry and Schneiderman). It was then found that when iso-osmotic fixatives were used, which d o not lead to the damaging cell shrinkage that otherwise would occur with a non-isosmotic fixing solution, very extensive regions of gap junctions were observed to co-exist with the septate junctions in dipteran salivary glands (Burger and Uhrik, 1972). In these salivary glands, although electrical coupling is easily disrupted, septate junctions are stable structures, and are unmodified by uncoupling (Bullivant and Loewenstein, 1968; Rose, 1971). Gap junctions are sensitive to osmotic disruption (Burger and Uhrik, 1972) and this suggested that the gap junctions were possible candidates for the sites of intercellular coupling. Poodry and Schneiderman (1970) eliminated the septate junction as the prime communication channel in Drosophila imaginal discs because they occur most frequently afier cell determination and pattern formation. Other evidence with microelectrode measurements and fluorescent labelling indicates that no low resistance pathways are present in the septate junctions of gregarines (Sniginevskaya et al., 1977). On structural grounds, as well as on the basis of morphometric analysis, it was found that in the epidermal cells of the larval beetle Tenebrio, the gap junctional membrane alone can account for the high electrotonic coupling recorded in this epidermal sheet (Caveney and Podgorski, 1975); analysis of the septate junction cast doubt on the possibility that it could serve as the communicating channel between these beetle cells. Although some investigators have not yet rejected the possiblity that the septate junctions may play some role in the intercellular coupling phenome-


103

non (Gilula, 1978), the weight of current evidence may now be seen to support the thesis that the gap junctions represent the communication pathways; the more recent freeze-fracture and lanthanum studies, the evidence from the isolation and biochemical characterization of vertebrate gap junctions (Goodenough and Stoeckenius, 1972; Goodenough, 1974), together with the X-ray diffraction data (Casper et al., 1977; Makowski et al., 1977), all indicate that their structure is uniquely organized so as to permit transport of ions and small molecules through the central channel found in each of their component particles (see Fig. 32). Conversely, the conclusions as to the function of the septate junctions are still indefinite (see Section 2.7). The other arthropod system that has been studied in depth is that of the crayfish septate giant axons. After the original demonstration (Furshpan and Potter, 1957) of electrical transmission in certain synapses of the crayfish nerve cord, other studies established that procion yellow (Payton et al., 1969) and mixtures of fluorescein and microperoxidase (Reese et al., 1971), when injected intra-axonally could move into adjacent axons. Peracchia (1973a, b) demonstrated the existence of two kinds of particle packing within the gap junctions of crayfish giant axon synapsis, the channels of which are thought to be the sites of exchange of these molecules. The gap junctions of arthropod eyes have also been implicated in low resistance pathways, and, for example, are found coupling photoreceptor axons in the optic ganglion of the fly (Chi and Carlson, 1976; Ribi, 1978; Carlson and Chi, 1979); these are found at sites where electrical transmission is observed physiologically (Shaw, 1979). The junctions are symmetrical, and it has been suggested that they are sites of electrotonic synapses so that those retinular cells which share the same visual field are linked together through these low resistance pathways (Ribi, 1978). The functional significance of this coupling of the retinular cells in attaining optimal visual acuity seems clear-cut, since signal enhancement can occur through frequency-selective signal averaging via the gap junctions that connect photoreceptor axons (Shaw, 1979). Electrotonic coupling between adjacent photoreceptor cells in the lateral eye of Lirnulus (Lasansky, 1967) may have a similar physiological basis. There is also some evidence from other invertebrates in support of ionic coupling between excitable epithelial cells having its basis in recognizable gap junctions. In annelid worms (Bilbaut, 1979), the gap junctions in the bioluminescent scales have been analysed by both electro-physiological and freeze-fracture techniques and correlations are drawn between the communicating structures and low-resistance pathways. Moreover, electrophysiological evidence for electrical coupling in Hydru has been reported (Hufnagel and Kass-Simmon, 1976). This is taken to support the contention

104


that the gap junctions are the structural basis of cell-to-cell communication, since gap junctions occur between cell processes extending into the mesoglica, and no other junctional type is present at these particular points, although homocellular septate junctions occur between the ectodermal cells and also between endodermal cells (Hufnagel and Kass-Simon, 1976). However, in the vast majority of cases, the presence of gap junctions in non-excitable tissues has been reported without any accompanying experimental evidence to show that molecules, possibly regulatory, are exchanged between the cells thus coupled. Any specific biological function of such communication is, therefore, as yet unclear (Gilula and Epstein, 1976). It is assumed that transport from cell-to-cell does occur, because in cultured non-excitable cells, a cell type with defective gap junctions has been shown to be communication defective (Gilula et al., 1972) but what the nature of the molecules exchanged may be is still obscure. ( 6 ) Regulation of gap junction permeability: effects of Ca++,hormones and transmission of hormonal stimuli Much of our information about the effects of calcium on gap junctional permeability stems from the work of Loewenstein and his colleagues. Originally, removal of calcium from coupled dipteran salivary glands led to loss of cell coupling (Nakaset al., 1966), while elevated levels of intracellular calcium were also found to induce uncoupling. It now seems that the permeability of the gap junctional membrane channels depends on the cytoplasmic Ca++concentration (Rose and Loewenstein, 1975a). This dependence has been shown by experiments on Chironomus salivary glands in which Ca++is injected into a cell, or the cytoplasmic Ca++concentration is elevated by Ca++ionophores or metabolic inhibitors, while the free cytoplasmic Ca++concentration is monitored in the gap junctional region by the luminescent protein aequorin. This protein is a specific Ca++indicator (Rose and Loewenstein, 1975b; Loewenstein, 1977) since its light emission is approximately proportional to Ca++concentration. At normal concentrations of Ca++,of about 1 0 - 7o~r less, the channels are permeable to a wide range of molecular sizes up to peptides of about 1200 to 1900 daltons (Simpson, Rose and Loewenstein, 1977) or to tracer molecules of up to M.W. 1000 (Caveney and Podgorski, 1975). However, it appears that within the Ca++range from less than ~ O - ’ M and up to 5 x ~O-’M (above which permeability €or all molecular species falls dramatically), there is a graded control by Ca++of junctional permeability (Rose et al., 1977). Although one cannot discount the possibility that a non-selective reduction in channel number takes place, this phenomenon may be due to a binding of Ca++to the junctional membrane in a way that induces a graded change either in the channel’s fixed charge or in its molecular configuration so p to reduce its effective size or induce channel misalignment (Loewenstein, 1977).


105

Support for the effects of Ca++on the junctions include evidence that exists for calcium deposits in those regions of the plasmalemma where the junctional arrays occur (Oschman and Wall, 1972; Larsen, 1975). It is also claimed that ionic lanthanum can be used as a marker of calcium-binding sites (Weihe et al., 1977) and certainly it stains the outer membrane leaflets of gap junctions, as well as the central channel (Fig. 27). In crayfish septate axons, as in insect salivary gland, varying the intercellular calcium concentration also led to the observation that uncoupling occurred with elevated or depressed Ca++levels (Asada and Bennett, 1971). It has subsequently been demonstrated in freeze-fractured preparations of these crayfish giant axons that an increase in the degree of close packing of the component particles of these gap junctions occurs with the elevation of Ca++ (Peracchia and Dulhunty, 1976). This is paralleled by changes in permeability leading to electrical uncoupling; these changes are thought to be due to a conformational change in certain components of the gap junctional particles (Peracchia and Dulhunty, 1976) that effectively close the junctional channels. A decrease in junctional thickness and possibly also particle size occur as well. Recent studies on crayfish gap junctions by Zampighi (personal communication) reveal that, in vivo , ionic lanthanum binds to the intraparticle pore simulating the site of Ca++ binding. This supports the contention that perhaps Ca++acts to change the configuration of the gap junctional particle sub-units during graded permeability by binding directly to them. In this same system (Peracchia and Dulhunty, 1976; Zamphighi et al., 1978; Hanna et al., 1978) vesicles are present in the cytoplasm adjacent to these junctional regions in the presynaptic, but not postsynaptic giant fibre. The function of such vesicles, typical of conventional synapses rather than electrotonic junctions, is not clear, although it has been suggested (Potter, in Peracchia and Dulhunty, 1976) that they may be Ca++-sequestering organelles which would presumably release Ca++in response to appropriate stimuli. Support for the effects of Ca++on uncoupling arthropod cells is also t o be found from experiments with cultured cells. An insect cell line has been found to be rapidly affected by treatment with an ionophore which increases intracellular calcium; within minutes the cell population begins to uncouple, and by the end of one hour's exposure the cells are all rounded and disassociated (Gilula and Epstein, 1976). Such results are compatible with the observations on dipteran salivary gland cells and crustacean giant axons where rises id cytoplasmic calcium lead to uncoupling. Lowering the intracellular pH, independent of Ca++,has also been found to uncouple embryonic cells of vertebrates, such as Xenopus (Turin and Warner, 1977); this may mean that the acidity of the solutions rather than elevated 10-5 6~10-~ 6x lo-'

>1 0 - ~ >10-3 >10-6

' Haim et al. (1979) * Ben-Barak and Dudai (1979)

5~10-~ >loWastek and Yamamura (1978)

ACETYLCHOLINE RECEPTORS OF I N S E C T S

239

choline, in the presence of the anticholinesterase DFP (1 x 1 0 - 5 ~ )also , inhibited [3H]-quinuclidinyl benzilate binding (I5,)= 3 x 1O+M), whereas nicotine which has a high affinity for putative nicotinic receptors in Drosophila (Dudai, 1977; Schmidt-Nielsen, et al., 1977) was ineffective in protecting [3H]-quinuclidinyl benzilate binding sites at concentrations up to 1 x 1 0 - 3 ~(Dudai and Ben-Barak, 1977). The last named authors also showed that a-bungarotoxin at 1 x 1 0 - 5 had ~ no significant effect on [3H]quinuclidinyl benzilate binding. Differential centrifugation has been employed to determine the subcellular distribution of the [3H]-quinuclidinyl benzilate binding sites. Both in isotonic sucrose and Pow ionic strength buffer, the bulk of the activity sedimented between 500xg and 20000xg (Haim e t a l . , 1979). In the same study it was shown by discontinuous sucrose-gradient centrifugation that most of the [3H]-quinuclidinyl benzilate binding activity sedimented between 1.O M and 1.7 M sucrose whereas a large part of the acetylcholinesterase activity sedimented to lower densities. Thus, the sedimentation profile of Drosophila [3H]-quinuclidinyl benzilate binding sites closely corresponds to that for Drosophila ['251]-a-bungarotoxinbinding sites (Dudai, 1978). This indicates that in both cases the binding sites are membrane-located. Several treatments have been applied to Drosophila extracts in attempts to solubilize the muscarinic receptor sites (Table 8). T o date it has not proved possible to solubilize the [3H]-quinuclidinyl benzilate-binding component. TABLE 8 Effect of various treatments on [3H]-quinuclidinyl benzilate binding levels in Drosophila. Aliquots of Drosophila head homogenate were preincubated for 1 h at 25°C with the indicated agent in 0.025 M Tris-C1, p H 7.6. Binding of [3H]quinuclidinyl benzilate was measured. Each value represents mean f S.E.M.for 3-5 experiments. In the case of Triton treatment, ammonium sulfate, precipitation and D E A E adsorption assays were also tried but yielded similar results. From Haimet al. (1979) Treatment Buffer 1 M NaCl 2 M NaCl 0.6% Triton X-100 M dithiothreitol 2 x lo-' M CaClz M EDTA 0.5% (w/w protein) trypsin 1.6% (w/w protein) phospholipase C Boiling 3 min

[3H]- quinuclidinyl benzilate bound (fmol) 12.821.3 14.3f0.3 13.7f0.6 1.3*1.1 11.421.0 14.0f1.0 13.8k0.7 0.620.4 11.820.5 0.1

D A V I D B. SATTELLE

240

A specific [3H]-quinuclidinyl benzilate binding component has recently been reported in extracts of the terminal abdominal ganglion of the cricket Acheta domestica with a dissociation constant calculated from the Hill plot . and Edwards, 1980). In the same study the data of 5.1 x 1 0 - 9 ~(Meyer density of binding sites in this cricket ganglion was estimated to be 480 pmoles per g protein. Thus head extracts of Drosophila contain a [3H]-quinuclidinyl benzilate binding component with the expected properties of a muscarinic acetylcholine receptor. The pharmacological specificity of these binding sites closely resembles that reported for vertebrate muscarinic receptors. However, whereas mammalian brain has about an order of magnitude more muscarinic receptors than nicotinic receptors (Yamamura and Snyder 1974a; Salvaterra et al., 1975; Segal et al., 1978), in Drosophila head extracts the situation is reversed and nicotinic receptors are about an order of magnitude more abundant (Dudai and Ben-Barak, 1977; Haim, et al., 1979).

3 Autoradiographic localization of binding sites

In addition to demonstrating a saturable component of binding with the pharmacological specificity expected of an acetylcholine receptor it is necessary, in order to satis@ the criteria for identification of a specific binding component as a receptor, to show that binding of the radiolabelled ligand is localized to synaptic areas in the central nervous system. Autoradiographic studies of the localization of radiolabelled-ligand binding sites are only available to date in the case of ['251]-a-bungarotoxin and these studies have to date been largely confined to Drosophila melanogaster, Manduca sexta and Periplaneta americana. 3.1

DISTRIBUTION

The distribution of [*Z"I]-a-bungarotoxinbinding in insect tissues was first studied by Hall and Teng (1975) who showed that its distribution in frozen, serial 10 p m sections of the fruit fly Drosophila melanogaster was not uniform. In a subsequent detailed investigation by the same laboratory, it was demonstrated that toxin-binding was confined to neural tissue in the head and thorax of Drosophila. Non-neural tissues exhibited only background levels of binding. Specific binding to neural tissue was reduced to background levels by preincubation of the sections with 1.O x ~O-'M unlabelled a-bungarotoxin (Hall and Teng, 1975; Schmidt-Nielsen et al., 1977). Rudloff (1978) was unable to introduce ['2SI]-a-bungarotoxin into intact


24 1

whole brain tissue and all studies to date of the distribution of toxin-binding have been carried out using frozen sections. The brain of a dipterous fly such as Drosophila bridges the head capsule between the laterally situated compound eyes and consists of: the median protocerebrum and its lateral optic lobes; the anteriorly located deuterocerebrum the neuropile of which contains many glomeruli (synaptic loci for neurones of the antennal nerve); and the tritocerebrum (cf. Miller, 1950). Each optic lobe consists of three readily distinguishable regions - the outer lamina ganglionaris, the medulla and the lobula (Cajal and Sanchez, 1915; Strausfeld, 1976). Toxin-binding to frozen sections of Drosophila heads was shown by three laboratories to be confined to neuropile areas of the brain (Schmidt-Nielsen et al., 1977; Dudai and Amsterdam, 1977; Rudloff, 1978). No binding was detected, for example, over axons in the antennal nerve. Binding in neuropilar (synaptic) regions was not however uniform (Fig. 8). Schmidt-Nielsen et at. (1977) reported that in the medulla of the optic lobes binding was layered in a manner resembling the distribution of neuronal branching found in Calliphora brain by Cajal and Sanchez (1915) and in Musca brain by Strausfeld (1976). By contrast, little or no binding was found over the lamina of the optic lobes. All three laboratories have concluded from autoradiographic studies that toxin binding is confined to synaptic areas of neuropile and is absent on nerve tracts and cell bodies in the CNS of Drosophila. That the pattern of binding appeared to reflect synaptic distribution was consistent with the view that a-bungarotoxin was binding to synaptic nicotinic receptors in Drosophila. Rudloff (1978) provided the first autoradiograms of frozen sections of larvae and pupae of Drosophila. A non-uniform distribution of toxin binding activity was reported in both cases and features of the adult pattern of binding were discernible in the three-day old pupae. Toxin-binding to frozen sections of the brain of Manduca sexta has been studied by Hildebrand and colleagues (1979). The organization of the antennal lobes of the brain of this insect enables a histological distinction to be made between synaptic and non-synaptic regions. Sensory axons from the ipsilateral antenna terminate in condensed areas of neuropile known as the glomeruli which contain the primary afferent synapses. Groups of cell bodies of second order antennal neurones are located peripherally. [12sI]a-bungarotoxin-binding sites were heavily concentrated over the glomeruli (see Fig. 28). Few binding sites were detected over non-synaptic regions such as the non-synaptic neuropile, the cell body regions and the antennal nerves. In recent studies on the localization of [ 1251]-a-bungarotoxinbinding in sections of the sixth abdominal ganglion of Periplaneta arnericana an intriguingpatternof bindingsitesemerged (Sattelleetal. ,1981a). Two distinct areas

DAVID B. SATTELLE

242

Fig. 8 Distribution of [1251]-a-bungarotoxinbinding in the head of Drosophila melanogaster. The small arrow points to the antenna1 nerve running from the antenna into the brain. ( a ) The section is viewed by transmitted light. ( b ) This is the same field shown in ( a ) but viewed by dark-field illumination. From Schmidt-Nielsen et al. (1977)

of densely concentrated toxin-binding sites were located in the central neuropile on either side of the midline of the ganglion. This is the region of the ganglion in which cercal afferent fibres make synaptic contacts with the extensive dendritic brances of the giant interneurones. Densely concentrated toxin-binding sites were also detected in the periphery of the ganglion, a non-synaptic region occupied by glial cells and neuronal cell bodies.

3.2

P H A R M A c o LO G K

Schmidt-Nielsen et al. (1977) showed that binding of [12SI]-cy-bungarotoxin to frozen sections of Drosophila brain was blocked by preincubation with

ACETYLCHOLINE RECEPTORS OF INSECTS

243

1 x ~O-’M unlabelled toxin and 1 x 1 0 - 4 d-tubocurarine. ~ Pre-incubation ~ reduced binding but did not completely eliminate it. with 1 x 1 0 - 4 atropine Rudloff (1978) also showed that 1 x ~O-’M unlabelled a-bungarotoxin prevented toxin binding to sections. Dudai and Amsterdam (1977) noted that ~ prevented binding. pretreatment with 1x l W 3 nicotine In pharmacological studies designed to test the specificity of toxin binding to sections of the brain of Manduca sexta, Hildebrand and colleagues (1979) showed that 1 x 1 0 - 3 acetylcholine ~ in the presence of 1 X 1 0 - 3 neostig~ mine blocked toxin binding. d-Tubocurarine (1 X 1 0 - 3 ~ )completely ) blocked toxin binding whereas quinuclidinyl benzilate (1 x 10 - 3 ~ only slightly reduced toxin binding. Thus autoradiographic studies of the [1251]a-bungarotoxin binding sites in Drosophila melanogaster, Manduca sexta and Periplaneta americana reveal a distribution and pharmacological specificity consistent with the notion that they are acetylcholine receptors.

4

4.1

Efectrophysiological responses of neurones to cholinergic ligands EXPERIMENTAL APPROACHES

In order to ascribe a functional role to any of the putative acetylcholine receptors characterized by radiolabelled ligand binding techniques it is necessary (1) to show a comparable pharmacological specificity in vivo to that demonstrated in the binding studies and (2) in cases where a radiolabelled receptor-specific antagonist is used for receptor characterization (see Sections 2.3 and 2.4), to demonstrate a synaptic blocking action of the receptor probe. A variety of electrophysiological recording techniques have been utilized to monitor the actions of cholinergic ligands on insect central neurones. A brief consideration of experimental methods is an essential preliminary to a survey of the results of pharmacological experiments of this kind. External hook-electrode recordings (see for example Roeder, etal., 1947; Twarog and Roeder, 1957; Yamasaki and Narahashi, 1958,1960; Shanklandet al., 1971) and suction electrode recordings (for example Kerkut etal., 1969b) have been used to monitor synaptic transmission in insect ganglia. In all cases a large number of neurones contribute to the observed recording. Using such techniques it is difficult to obtain more than an estimate of the threshold concentration of a ligand that will produce a response and the response cannot be attributed to specific neurones. However, using sucrosegap recordings (Fig. 9) of excitatory postsynaptic potentials (EPSPs) and average postsynaptic polarization derived from a population of neurones,

244

DAVID B. SATTELLE

dose-response curves can be constructed for the actions of Iigands on cercal-afferent, giant-interneurone synaptic transmission in the cockroach (Callec and Sattelle, 1973). Although quantitative comparisons of the synaptic actions of ligands can be achieved by this technique, the responses can be ascribed only to a group of neurones. Experiments on multifibre preparations in conjunction with bath-application of drugs can therefore lead only to rather general conclusions concerning the pharmacological properties of receptors in such tissues.

Evoked Monosynaptic

EPSP

I2mv

-

toms

Fig. 9 Schematic representation of the oil-gap and sucrose-gap recordingtechniques. Excitatory postsynaptic potentials (EPSPs) evoked by the application of depolarizing pulses to nerve 11 (nXI) and recorded by these two methods are illustrated. Mechanical stimulation (MS) can be applied to single mechanoreceptor hairs. Electrical stimulation (ES) of many cercal mechanoreceptor afferents can be achieved by hook electrodes applied to nerve 1 1 . From Callec et al. (1980)

Studies on single invertebrate neurones, and in particular identified cells, have proved fruitful in the study of a variety of neurotransmitter-receptors ( c f . Gerschenfeld, 1973; Ascher and Kehoe, 1976). Two complementary experimental approaches to the pharmacology of single insect neurones were developed in the late 1960s. Boistel, Callec, and collaborators pioneered the microdissection of axons of giant interneurones (Fig. 10) and the oil-gap, single-fibre recording technique (cf. Boistel, 1968; Callec, 1972) for monitoring synaptic phenomena in single giant interneurones of the sixth abdominal ganglion of the cockroach Periplaneta americana. A t about the same time, Kerkut and collaborators (cf. Pitman 1971) made a detailed microelectrode study of the cholinergic sensitivity of certain nerve cell bodies (somata) of cockroach ganglia. The particular advantage of micro-

ACETYLCHOLINE RECEPTORS OF INSECTS a

-

245

b

300pm

Fig. 10 Stages in the microdissection of the axon of giant interneurone 2 of the cockroach Peripfaneta americana. ( a ) Ventral view of the isolated sixth abdominal ganglion. (b) From one of the paired connectives linking the fifth and sixth abdominal ganglia the nerve sheath is split and the cut ends retracted towards the ganglia. (c) The axon is isolated from adjacent fibres using fine stainless-steel needles. (d) The axon is dissected to within about 150 p m of the ganglion so that in the experimental chamber it is located close to the barrier between the oil and saline compartments. From Callec (1972)

electrode recording is that the precise site of recording can be defined. The method is widely applicable and has been extensively employed for investigating soma membrane chemosensitivity. The non-electrolyte gap techniques (oil-gap and sucrose-gap) enable prolonged, stable recordings of synaptic events, and in the case of the oil-gap technique (Fig. 9) these can be ascribed to a single identifiable postsynaptic neurone. Also, pre- and postsynaptic drug actions can be simultaneously assessed (Callec et al., 1980). However, these non-electrolyte gap techniques are less widely applicable,

DAVID 6.SATTELLE

246

TABLE 9 Comparison of nonelectrolyte-gap and microelectrode techniques for recording synaptic transmissionin the terminal abdominal ganglion of the cockroach (Peripfaneta americana). From Callec et al. (1980)

Properties Unitary EPSPs Unitary IPSPs Evoked - EPSP Evoked - IPSP Postsynaptic action potential

Manni tol-gap (whole or part Microelectrode Oil-gap of the (in giant fibre) (single giant fibre) connective) 0.7-3.2 mV 0.5-2 mV 15-20 mV 5 mV Up to 75 mV

0.5-2 mV 0.04-0.32 mV 3-8 mV up to 4 mV up to 115 mV

0.2-0.5 mV 0.15 mV 2-5 mV up to 1 mV up to 35 mV (compound response) 1.5-3 mV (giant axon)

although they are particularly well suited to investigations of the cercalafferent, giant-interneurone synapses of the cockroach. Table 9 compares some of the synaptic phenomena that can be recorded by different electrophysiological techniques from the sixth abdominal ganglion of the cockroach Periplaneta americana. Although most pharmacological studies have employed bath-application of cholinergic ligands to the cell or tissue under investigation, more recently localized application of ligands to individual neurones has been achieved using microsyringe application (Callec and Boistel, 1967) and microiontophoresis (Kerkut et a/..,l968,1969a, b; Pitman and Kerkut, 1970; Callec and Boistel, 1971; Callec 1972; David and Pitman, 1979; David, 1979; Goodman and Spitzer, 1979a, b; Sattelle et al., 1980). By these means, the substance under investigation is delivered to the cell from a micropipette. In the first case ejection is achieved by the application of a pressure pulse and in the second case ejection is induced by the application to the micropipette of rectangular current pulses of appropriate polarity. Before discussing in detail the results of electrophysiological experiments some consideration will be given to the limitations and advantage of the methods of ligand application. When pharmacological agents are bath applied to a ganglionic preparation, although the final concentration in the bath is known, this may depart from the true concentration at the cell surface. Also, all cells sensitive to the ligand will respond. Furthermore, appreciable time is required for equilibrium of the agonist concentration at the cholinoceptive membrane. This is


247

undoubtedly the cause of considerable desensitization which precludes repeated applications of test compounds (Fig. 11). The limitations imposed by this method of application to insect ganglia have been discussed in detail (Sattelle el al., 1976). A further possible problem resulting from bath application stems from the observations on electroplax (Karlin 1967) and 30

-20

a

I

I

I

-

> E

v

C

210 m

-

-

N

m 0

I

Concentration

(M)

Fig. 11 Dose-response data for carbamylcholine-induced depolarization recorded from the sixth abdominal ganglion of the cockroach Periplaneta americana. In ( a ) results of experiments on 18 ganglia are summarized. The mean depolarization for a particular dose is determined. Vertical bars show twice the standard error. In ( b ) , the upper curve ( 0 )is constructed by plotting results obtained from 13 ganglia which were tested once or twice only; the lower curve (0)is derived from an experiment in which a single ganglion was challenged successively by increasing concentrations of carbamylcholine. Between each test pulse repolarization was observed in normal Ringer. From Sattelle et al. (1976)

muscle (Jenkinson and Terrar, 1973) tissue that prolonged exposure to acetylcholine can cause ionic changes inside the cell. Nevertheless, when care is taken to eliminate physical and metabolic barriers that may restrict access of the ligand to the neuronal surface, it remains a valuable method for comparative studies since under these conditions dose-response data can be accumulated with the ligand dose expressed in terms of molarity. Localized application of a ligand to the cell body region of an individual neurone can be achieved via micropipettes. In the same way, ligands can be delivered to localized regions within the neuropile although clearly in this

DAVID

248

B. SATTELLE

case it is not possible to confine the actions to a single cell. By varying the duration of the pressure pulse (in the case of pressure ejection) or by changing the iontophoretic current amplitude (in the case of microiontophoresis) the amound of ligand discharged can be varied and a doseresponse curve constructed (see Fig. 12 for an example). By means of localized, brief applications of ligands many of the problems of desensitization that limit bath-application experiments can be avoided. Although only charged ligands can be applied by microiontophoresis, this restriction does not apply to pressure ejection via micropipettes. In the remainder of this section consideration is given to the results of experiments in which acetylcholine and related compounds are applied to insect neurones.

4.2

MULTIFIBRE FREPARATIONS

4.2.1 Actions of acetylcholine The relative insensitivity of ganglionic synaptic transmission in insects to bath-applied acetylcholine was consistently reported in the earlier pharmacological investigations using extracellular recording electrodes. Transmission across the cercal-afferent, giant-interneurone synapses of the sixth abdominal ganglion of Periplaneta americana, first described by Pumphrey and Rawdon-Smith (1937), was unaffected by acetylcholine at concentra~ ganglia with the nerve sheath removed (Roeder tions below 1.0 x 1 0 - 3 for et al., 1947; Roeder, 1948; Tawrog and Roeder, 1956, 1957). The same authors showed that the anticholinesterase agent eserine (1.0 x 1 0 - 6 ~ ) ~ desheathed ganglia. reduced the threshold concentration to 1.0 x 1 0 - 4for Yamasaki and Narahashi (1958, 1960) showed that the threshold concentration for the same preparation but with the nerve sheath intact was ~ pretreatment with eserine reduced from 1.0 x 1 0 - ’ ~to 1.Ox 1 0 - 3following (1.0 x 10%). Also, application of acetylcholine by perfusion or by direct addition to the saline surrounding the metathoracic ganglion of Periplaneta increased the spike activity recorded from the fifth thoracic nerve (Kerkut et al., 1969b). The threshold level for this action of acetylcholine was esti. studies on the grasshopper Crampsockis mated to be about 5.5 X 1 0 - 5 ~In buergeri (Suga and Katsuki, 1961), 5.5 x 1 0 - * ~acetylcholine produced excitatory effects on the auditory synapses of the prothoracic ganglion. It has been suggested that the nerve sheath surrounding the CNS presents a diffusion barrier to the penetration of acetylcholine (Twarog and Roeder, 1956; O’Brien, 1957; O’Brien and Fisher, 1958). Nevertheless Treherne and Smith (1965a) noted that radiolabelled acetylcholine rapidly penetrated nerve cords of the cockroach Periplaneta americana. The most likely explanation of the insensitivity of ganglia to bath-applied acetylcholine, an


IONTOPHORETIC

CURRENT

249

( nA

I

Fig. 12 Dose-response curve obtained by iontophoretic application of acetylcholine to a neuronal cell body in the sixth abdominal ganglion of the cockroach Periplanefa americana. The relationship between the amount of acetylcholine applied to the nerve cell (iontophoretic current) and the depolarization of the cell. Retaining current = 60 nA. Acetylcholine-filled electrode had a resistance of 12 M a . From Kerkut et al. (1969b)

insensitivity that persists even following removal of the nerve sheath, has been advanced by Smith and Treherne (1965) and Treherne and Smith (1965a, b). These authors proposed that the high concentration of cholinesterase in the CNS is responsible for the hydrolysis of most of the applied acetylcholine so that the final concentration in the extracellular spaces adjacent to cholinoceptive membranes in the CNS is much lower than the applied concentration. For example, Treherne and Smith (1965a) calculated that when the abdominal nerve cord was bathed in 1.0 X ~ O - * Macetylcholine, the extracellular concentration was approximately 8.1 x 1O+M. This notion of a biochemical barrier to the penetration of bath-applied acetylcholine is supported by the report from several laboratories that carbamylcholine, a cholinergic agonist that is not subject to hydrolysis by cholinesterase is more potent than acetylcholine when bath applied to intact ganglia (see Fig. 13). The first study that attempted to examine the detailed cholinergic pharmacology of the cercal-afferent, giant-interneurone synapses, giving due

30

I

I

I

I

I

I

I

1

ACE T Y LCHOI-INE j ACh) 0 ACh + ESERItdE 0 ACh . CURAFE 0

20 -

ACh 4 ATROPINE

mv 10 -

0

'

I 0

NICOTINE

a CARBACHOL Q

PILOCARPINE

20 -

M

Fig. 13 Actions of acetylcholine and various cholinergic agonists (bath applied) on synaptic transmission between cercal afferent fibres and giant interneurones in the sixth abdominal ganglion of the cockroach Periplanefa amerzcana. Ganglionic (postsynaptic) depolarization (in mV) recorded by the sucrose-gap technique is plotted against ligand concentration (M). Vertical bars represent twice the standard error of the mean. From Sattelle (1978)

consideration to the t (me required for ligand diffusion in the ganglion and the enzymic hydrolysis of bath-applied choline esters by endogenous cholinesterase, was that of Shanklandet al. (1971). These authors reasoned that the response of the synapses to bath applied acetylcholine and cholinergic drugs would be subject to interference by (1) endogenous transmitter, and (2) the degrading actions of endogenous cholinesterase in cases where the drug was susceptible to hydrolysis by this enzyme. Pretreatment of the desheathed sixth abdominal ganglion with hemicholinium-3 (which competitively inhibits choline transport in membranes - Birks and MacIntosh, 1961 - thereby blocking acetylcholine synthesis), and either dichlorvos or paraoxon (which are potent cholinesterase inhibitors O'Brien, 1960), followed by washing, resulted in preparations highly sensitive to bath-applied acetylcholine. Three quarters of these pretreated ganglia responded to 1.0 x 1 0 - 6 ~acetylcholine and Shankland er al. (1971)

ACETYLCHOLINE RECEPTORS

OF I N S E C T S

251

estimated the average minimal effective concentration to be between 1.0 X 1 0 - 7and ~ 1.O x 1 0 - 6 ~In. subsequent studiesusing the sucrose-gap recording technique, dose-response data were obtained for the actions of acetylcholine on cercal-afferent, giant-interneurone synaptic transmission in the sixth abdominal ganglion of Periplaneta americana (Callec and Sattelle, 1973; Sattelle etal., 1976).It was shown, for example, that pretreatment of the ganglion with 1.O x 1O-% eserine considerably shifted the dose-response curve for the post-synaptic depolarizing action of acetylcholine so that the preparation was consistently sensitive to 1.0 x 1 0 - 6 acetylcholine ~ (Fig. 13).

4.2.2 Actions of cholinergic ligands A variety of cholinergic agonists have been applied to insect central nervous tissue and their effects monitored using external-electrode recordings from multifibre preparations. Twarog and Roeder (1975) demonstrated that muscarine and pilocarpine when bath applied to the sixth abdominal gang~ 1.0 X 1 0 - 4 ~ lion of Periplaneta at concentrations in the range 1.0 x 1 0 - 3 evoked asynchronous bursts of spikes in giant interneurones. By contrast nicotine and acetyl-P-methylcholine (see Table 10) excited the same postsynaptic giant fibres at concentrations as low as 1.0 X ~ O - ' M (Flattum and Sternberg, 1970a, b; Shankland, et al., 1971; Flattum and Shankland, 1971). Urocanylcholine induced bursts of large and small spikes followed by a synaptic block in the same preparation (Twarog and Roeder, 1957). The metathoracic ganglion-fifth nerve preaparation of Periplanefa was also shown to be most sensitive to nicotinic agonists, the order of effectiveness being nicotine>carbamylcholine>pilocarpine>acetylcholine(Kerkut et al., 1969). The auditory synapses of Gampsocleis were stimulated by the perfusion of butyrylcholine (5.5 x ~O-'M) over the prothoracic ganglion (Suga and Katsuki, 1961). Using sucrose-gap techniques the actions of a variety of cholinergic agonists and antagonists were studies at cercal-afferent, giant-interneurone synapses of Periplaneta (Sattelle et al., 1976; Sattelle, 1978; Sattelle et al., 1980). Nicotine was the most potent of the ligands used (Fig. 13) producing postsynaptic depolarization at concentrations of 1.O x 1 0 - * ~ and above. Acetylcholine (in the presence of 1.O x 1 0 - 6 .eserine) ~ at 1.O x and higher concentrations also produced a postsynaptic depolarizing action (see Section 4.2.1). Carbamylcholine and pilocarpine were three to four orders of magnitude less effective than nicotine (Fig. 13). Several cholinergic antagonists have been shown to block transmission at the synapses in the sixth abdominal ganglion of Periplaneta americana. d-Tubocurarine a nicotinic antagonist which in earlier pharmacological

DAVID B. SATTELLE

252

TABLE 10 Actions of cholinergic ligands on synaptic transmission in the insect central nervous system using multifibre preparations Ligand

Nicotine Acetylcholine d-Tubocurarine Hexamethonium Acetyl-fl-methylcholine Carbamylcholine Decamethonium Atropine Pilocarpine

Threshold concentration (M) for minimal detectable response at cercal-nerve giant fibre synapses in the sixth abdominal ganglion of the cockroach Periplaneta americana Hook-electrodel recordings

Sucrose-gap2 recordings

10-8

10-8

10-7-

10-6

10-7 - I O - ~

10-6 10-8 10-~ 10-7 -

10-6 10-7 10-5 -

1o

-

-~

-

10-5

Shanklandetal. (1971) Sattelle (1978); Sattelle (1980)

studies had been reported to have no effect at 1.0 x ~ O - * M(Roeder, 1948), was more recently shown to suppress transmission at cercal-afferent, giantinterneurone synapses at concentrations as low as 1.0 X ~O-’M.(see Table 10). This same antagonist inhibited the excitatory effects of acetylcholine in the auditory synapses of Gampsocleis (Suga and Katsuki, 1961). Antagonism of the actions of applied acetylcholine by d-tubocurarine was noted at cercal-afferent, giant-interneurone synapses in the cockroach in both hook-electrode recording tests (Shankland et al., 1971) and sucrose-gap experiments (Sattelle, 1978). At high concentrations d-tubocurarine blocked conduction in cockroach axons (Friedman and Carlson, 1970). Atropine, an antagonist at vertebrate muscarinic receptors was reported to inhibit transmission at cercal-afferent, giant-interneurone synapses ~ al., 1971) and the at a threshold concentration of 1.0 x 1 0 - 7(Shanklandet same authors demonstrated its ability to suppress the excitatory effects of acetylcholine on the same preparation. Sucrose-gap experiments have also shown that atropine can completely block transmission at cercal-afferent, giant-interneurone synapses but only at much higher concentrations (1 .O X 1 0 - 4 ~ than ) required for d-tubocurarine block. Using the same method it was also shown that atropine was less effective than d-tubocurarine in displacing the dose-response curve for acetylcholine (Fig. 13). Recent sucrose-gap studies demonstrated that benzoquinonium


2 53

(1.0 x ~O-’M) and hexamethonium (1.0 x 1 0 - 3 ~will ) block cercal-afferent, giant-interneurone synaptic transmission (cf Sattelle, 1980). Decamethonium also blocked synaptic transmission across the terminal abdominal ganglion of the cockroach (Twarog and Roeder, 1957). Shankland et al. (1971) estimated that the concentrations required to block were 1.0 x 1 0 - 6in ~ the ~ the case of decamethonium. case of hexamethonium and 1.0 x 1 0 - 3 in Twarog and Roeder (1957) reported that tetramethylammonium (1.O X 1 0 - 3 ~ )hydroxyphenyltrimethylammonium , (1 .O x 10-*h1) and benzoquinonium (1.0 x 1 0 - 2 ~produced ) synaptic block in this preparation. The reasons for the discrepancies between the effective concentrations of hexamethonium and benzoquinonium reported by different laboratories has yet to be resolved. Only in the case of the sucrose-gap experiments, however, have dose-response curves for a series of cholinergic antagonists been prepared following bath-application for a fixed period of time (Fig. 14). The rank order of effectiveness of antagonists revealed in this way was as follows: a-bungarotoxin > d-tubocurarine > benzoquinonium > hexamethonium and atropine.

Fig. 14 Actions of cholincrgic antagonists on synaptic transmission between cercal-afferents and giant interneurones. Antagonists were bath applied and their effects on transmission were monitored by the sucrose-gap recording technique. Abbreviations: a-Bgt, a-bungarotoxin; d-TC, d-tubocurarine; Benzo, benzoquinonium; Hexa, hexamethonium; Atr, atropine. From Sattelle. (1980)

4.3

SINGLE NEURONES

This section is confined to a consideration of pharmacological data obtained in studies on single neurones which were not subsequently identified anatomically using single-cell marking methods.

DAVID B. SATTELLE

254

4.3.1 Actions of acetylcholine Depolarization accompanied by a decrease in membrane resistance (Rm) has been observed to follow bath-application of acetylcholine (3.0 X 3.0 x ~ O - ’ M ) to the desheathed sixth abdominal ganglion of Periplaneta, using an oil-gap, single-fibre technique to record from single giant interneurones (Fig. 15). In an attempt both to circumvent the peripheral

OlO

mV

mV

Time (min)

Fig. 15 Actions of bath applied acetylcholine on a giant interneurone in a desheathed sixth abdominal ganglion of the cockroach Periplaneta americana recorded using the oil-gap technique. The cell depolarizes in response to 3.0 x 1 0 - 3acetylcholine ~ (ACh), ultimately giving rise to a volley of action potentials. Acetylcholine-induced depolarization is accompanied by a progressive reduction in membrane resistance (Rm) and the amplitude of the monosynaptic EPSP evoked by contralateral electrical stimulation of ipsilateral nerve 11. Repolarization and recovery of the EPSP and Rm follow re-exposure to normal saline. Em = membrane potential. From Callec (1972)

cholinesterase barrier and to apply acetylcholine close to the synapses, Callec used microiontophoresis of acetylcholine (Callec and Boistel, 1971; Callec, 1972). With an acetylcholine-filled electrode located in the dendritic field of an interneurone, the injection of acetylcholine induced a transitory depolarization of the giant interneurone (Fig. 16). The response obtained increased as the applied current, and hence the quantity of acetylcholine discharged, was increased. This acetylcholine-induced depolarization was accompanied by a decrease in the membrane resistance of the giant interneurone. During the acetylcholine potential, the EPSPs obtained by stimulation of cercal nerve 11 decreased in amplitude, with a corresponding decrease in Rm (Fig. 16). Changes in amplitude of both the EPSP and the acetylcholine-induced potential as a function of the polarizing current were also investigated (Fig. 17). With hyperpolarizing current the acetylcholine potential increased in amplitude, but when depolarizing currents were applied it decreased (Callec, 1974). These changes followed closely the changes in the EPSP. Both responses have similar reversal potentials and are therefore likely to be supported by the same ionic currents. Reversal potentials for the EPSP and the acetylcholine potential of around -35mV were


255

A mV 7-

6-

-z

.-

5 -

C

+

2

4 -

1

0 Q

3-

't!

-

AC h

500 m s

I

100

200

I

300

I

I

400

500

~~~

600

nA

B 1

Fig. 16 Effects on a giant interneurone of iontophoretic injection of acetylcholine in the region of neuropile in the sixth abdominal ganglion of Periplaneta containing part of the cell's dendritic tree. Oil-gap, single-fibre recordings are used to show: (A), the relation between the amplitude of the acetylcholine-induced potential response (see insert) and the injection current (nA); (B,), that during the application of acetylcholine the monosynaptic EPSP evoked by electrical stimulation of nerve 11, which was initially subthreshold, is now able t o generate an action potential; (BJ, the reduction in membrane resistance (Rm) during the acetylcholineinduced potential. From Callec (1972)

estimated by extrapolation (Callec, 1974). Evidence available to date therefore has established that acetylcholine has a depolarizing action which is accompanied by an increase in the conductance of the postsynaptic membrane. It also appears that the acetylcholine potential and the EPSP originate from the same ionic currents. The ionic basis of these synaptic currents remains to be explored.

D A V I D B. SATTELLE

256 mV 14, 0

0

12 ' 0

--

0 .

oo2\

ACh

-

I2OnA

300ms

O

Em.

Fig. 17 Relation between the amplitude of the monosynaptic EPSP (evoked by ipsilateral electrical stimulation of cercal nerve 11) and the amplitude of the iontophoretic acetylcholine response. Data were obtained from the same giant interneurone in the sixth abdominal ganglion of the cockroach (Periplunetu umericuna) at a variety of different membrane potentials Em. The amplitude of the response to iontophoretically applied acetylcholine was initially adjusted to be equal to the EPSP amplitude. From Callec (1972)

Excitatory postsynaptic potentials have also been recorded from insect neuronal somata (Hagiwara and Watanabe, 1956, Kerkut ef al., 1969a, b, Crossman ef al., 1971). The cell bodies of insect neurones are not in synaptic contact with presynaptic fibres. Interpreting the data obtained on nerve cell bodies therefore requires caution in that the effects of any given cholinergic ligand on the neuronal cell body membrane may differ from its actions on synaptic membranes. In 1967 Callec and Boistel impaled cell bodies located near the dorsal midline of the cockroach sixth abdominal ganglion. These authors showed that previously silent and autoactive cell bodies responded to the localized application of acetylcholine at concentrations above 5 x 1 0 - 6by ~ membrane depolarization and an associated increase in action potential activity. Later it was shown that iontophoretic ejection of acetylcholine from a microelectrode located close to a recording microelectrode inserted into a nerve cell body of the same region of the sixth abdominal ganglion (Kerkut et al., 1968; 1969a, b; Pitman and Kerkut, 1970) resulted in a transient

2 57


depolarization (Fig. 18). The acetylcholine response and the EPSP evoked by stimulating the right anterior connective both increased in amplitude when the membrane potential was increased. Based on these observations, Pitman and Kerkut (1970) estimated, by extrapolation of plots of the amplitude of transient responses versus membrane potential, reversal potentials of -45.3 t 3 . 1 mV for the EPSP and -40.3 -+ 1.6 mV for the acetylcholine depolarization, These findings suggested that the same ionic currents were involved in both responses. Kerkutetal. (1969b) also showed that acetylcholine-induced depolarization of the soma membrane was reduced in the absence of external sodium ions (Fig. 19), pointing to a role for this cation in the generation of the EPSP. The threshold concentration of acetylcholine required to produce a response was estimated by extrapolating the depolarization amplitude-iontophoretic dose curve (Kerkut et al., 1969a, b). A value of 1.31 x 1 0 - 1 3 was ~ obtained. This resembles the value obtained for snail neurones and is not far removed from the value of 1 0 - l ' ~ reported for the vertebrate neuromuscular junction (del Castillo and Katz, 1955). b

a

-

lJ

66

-761

--93 831

,

20s

-86] -96

\ \

c

i

41

-

030

O - k -

M.P. mV

-50

-70

-90

-110

M.P. mV

Fig. 18 The effects on (u) the EPSP driven through the right anterior connective and (b) the iontophoretic acetylcholine response of hyperpolarizing the membrane of a neuronal cell body of the cockroach sixth abdominal ganglion in 10 mV steps (c) shows a plot of the amplitude of the EPSP against the membrane potential of the cell. The EPSP reversal potential is -44 m V ( d ) shows a graph of the amplitude of the iontophoretic acetylcholine (ACh) response against the membrane potential of the cell. The acetylcholine reversal potential is -38 mV. From Pitman and Kerkut (1970)

2 58

DAVID B . SATTELl F

a

I 5OOpg

ACh.

b

10 min

Na+ FREE

C

/ I

,

"I

1 I

1

10 rnin WASH

Fig. 19 Effects of reducing the external sodium concentration on the response of a neuronal cell body to acetylcholine. Removal of external sodium reduced the effect of acetylcholine. From Kerkut et al. (1969b)

4.3.2 Actions of cholinergic ligands The application of nicotine to the desheathed sixth abdominal ganglion of PeripEaneta resulted in a rapid depolarization of a single giant interneurone at concentrations of 1.0 x 1 0 - 6 to ~ 4.0 x 1 O w 6 (Callec ~ 1972). In the same study it was noted that acetyl-8-methylcholine depolarized a single giant interneurone at concentrations of 5.0 x 1 0 - 3 ~to 3.0 x ~ O - ' M . d-Tubocurarine (1.0 x ~O-'M to 8.0 x 1 0 - 4 ~induced ) a progressive decline

A C E T Y L C H O L I N E RECEPTORS OF I N S E C T S

259

in the unitary and evoked EPSP at cercal-afferent, giant-interneurone synapses. The blocking action of d-tubocurarine was reversible and did not result in a significant change in either the membrane potential or the resistance of the postsynaptic cell. If acetylcholine is the neurotransmitter released by the excitatory cercal nerve terminals then it would be predicted that antagonists would have similar effects on the potentials caused by the natural transmitter and those induced by artificially injected acetylcholine. ) observed to progressively Bath applied d-tubocurarine (7.0 x 1 0 - 4 ~was block both the monosynaptic EPSP recorded from a giant interneurone in response to the stimulation of cercal nerve 11 and the acetylcholine potential resulting from the microapplication of acetylcholine into the neuropile at a depth of 210 p m below the ganglion surface (Fig. 20). Both potentials

RI

5

4 2 nin

6

53mn

7

I

9 2 rnn

Fig. 20 Actions of d-tubocurarine (d.TC) on a giant interneurone in a desheathed sixth abdominal ganglion of Periplanetu arnericunu. Using the oil-gap technique changes in the monosynaptic EPSP evoked by stimulation of ipsilateral nerve 11 and the depolarization resulting from iontophoretic application of acetylcholine into the dendritic tree region are followed in a single giant interneurone during the bath application of d-tubocurarine (7.0 x 1 0 - 4 ~ )The . two responses decline at approximately the same rates. Times in minutes after exposure to test solution are shown. Traces 1 and 8 were obtained with the preparation bathed in normal saline (Ri). Traces 2-7 show progressive blocking actions of d-tubocararine. From Callec (1972)

were largely reversible when the ganglion was rebathed in normal saline. The uniformity of action of d-tubocurarine on the EPSP and the acetylcholine potential indicated that the same postsynaptic receptors were involved in both cases. Atropine, a classic muscarinic antagonist, also blocked cercal-afferent, giant-interneurone synaptic transmission in Periplaneta (Callec and Boistel 1971c; Callec, 1972). Effective concentrations

DAVID B. SATTELLE

260

required for complete block (2.0 x - 1.0 x 1 0 - 3 ~were ) higher than those required for d-tubocurarine. The acetylcholine potential was also blocked by these rather high concentrations of atropine. A comparative study of the effects of various cholinergic ligands has been made on cell bodies of Peripfaneta central neurones. The following sequence of effectiveness was detected: nicotine > carbamylcholine > pilocarpine > acetylcholine (Kerkut et ~ l . 1969b). , In dissociated neuronal cell bodies isolated from the thoracic ganglia of adult locusts (Schistocerca gregaria), membrane depolarization resulted from bath application of acetylcholine at concentrations above 1.0 x 1 0 - 6 ~ (Holden et al., 1977). Iontophoretic application of acetylcholine also depolarized the isolated neurones and this response was antagonized by d-tubocurarine and atropine. Although there appear to be broad similarities in their sensitivity to cholinergic ligands between cell body (extrasynaptic) membranes and synaptic membranes, to date a direct comparison of the properties of synaptic and extrasynaptic receptors of a particular insect neurone has not been performed. 4.4

S I N G L E IDENTIFIEI) NEURONES

A limited number of studies have recently been initiated aimed at characterizing the pharmacological properties of single identified neurones in the insect central nervous system. Giant interneurones 2 and 3 of the sixth abdominal ganglion of Peripfaneta americana and the fast (Df) and slow (D,) coxal depressor motoneurones of the third thoracic ganglion of the same insect, have been investigated. 4.4.1

Giant interneurones

Using a novel, single-axon backfill technique, selective cobalt staining of individual giant interneurones in the sixth abdominal ganglion of the cockroachPeripfanetaamericanahas beenachieved (Harrowetaf., 1979; 1980a,b). Each of the three giant interneurones (GI 1-3) investigated has unique morphological features (Fig. 21). Cell body position, neurite shape, dendritic branching pattern, the presence or absence of an axon collateral and the characteristic position of the axon in sections of the fifth abdominal ganglion provide anatomical criteria for identifying giant interneurones (Harris and Smyth, 1971; Camhi, 1976; Harrow et al., 1980a). Since it is possible to select by dissection one of these three giant interneurones it is possible to complement neuroanatornical studies with pharmacological studies of synaptic transmission in selected, identified interneurones using the oil-gap, single-fibre recording technique. In this way it has been shown, for giant interneurone 3, that a-bungarotoxin (1 .O x ~O-'M)when bath applied to the

261


b

Y I

,

\

Fig. 21 Morphology of giant interneurones of the cockroach (Periplaneta americana, L.). ( a ) Camera lucida drawing of a section through the fifth abdominal ganglion showing the relative positions of giant axons. (b-d) Camera lucida representations of cobalt-backfilled, silverintensified, single, giant interneurones in the sixth abdominal ganglion: ( b ) GI 1; (c) GI 2; ( d ) GI 3. ( e ) Photograph of the sixth abdominal ganglion containing the cobalt-filled giant interneurone (GI 3) prior to intensification. Scale bar represents 100 pm.From Harrowetal. (1980a)

DAVID 6.SATTELLE

262

desheathed sixth abdominal ganglion, completely and irreversibly blocks unitary excitatory post-synaptic potentials (EPSPs) recorded in response to the deflection of a single cercal mechanoreceptor (Fig. 22). Cobaltbackfilling (Harrowetal., 1979) confirmed that giant interneurone 3 was the SALINE 0 rnin

W-Bgt

30 rnin

SALINE after 210 min wash

Fig. 22 ( a ) The unitary EPSP recorded from giant interneurone 3 (GI 3) in response to mechanical stimulation of a single cercal mechanoreceptor is irreversibly blocked by a-bungarotoxin (I x ~ O - * M ) . ( b ) The EPSP evoked by electrical stimulation of nerve 11 and recorded from GI 3 is irreversibly blocked by 1 X lo-% a-bungarotoxin. ( a ) from Harrower a l . (1979); ( b ) from Harrow efal. (1980b)

postsynaptic cell under investigation (Fig. 23). Using the same combination of techniques, it was shown for giant interneurone 2 that both the unitary EPSP and the EPSP evoked by electrical stimulation of cercal nerve 11were completely blocked by 1.O x 1 0 - * a-bungarotoxin ~ (Sattelleetal., 1981). This neurotoxin is a specific, irreversible antagonist of vertebrate peripheral cholinergic receptors and some vertebrate central cholinergic receptors (Heidmann and Changeux, 1978). The blocking action of a-bungarotoxin at cockroach synapses at which nicotinic agonists and antagonists are particularly active, and at concentrations close to the KDs(see Table 11) estimated for binding to various insect extracts, provides strong evidence for a functional role in synaptic transmission of the membrane component that binds a-bungarotoxin. 4.4.2

Fast coxal depressor motoneurone

The fast coxal depressor motoneurone of the third thoracic ganglion of the cockroach Periplaneta americana, numbered cell 28 by Cohen and Jacklett (1967), was designated D, by Pearson and lles (1970). Located on the ventral surface of the ganglion, the cell body of this motoneurone is 8 0 p m in diameter and can readily be located visually in the whole ganglion. This cell

-


263

innervates the coxal depressor muscles 177d, 177e, 178 and 179 through the fifth ganglionic nerve trunk (Carbonell, 1947, Pearson and Iles, 1970). Recently, David and Pitman (1979) studied the sensitivity of the cell body membrane to both bath-applied and iontophoretically-applied acetyl-

lo&m

Fig. 23 Camera-lucida drawing of a cobalt-stained, silver-intensified, giant interneurone of the sixth abdominal ganglion of Peripfaneta americana following an experiment in which all cercal afferent input to the cell via nerve 11 was blocked by 1.0 X 1 0 - ' ~a-bungarotoxin. The criteria of cell body position, neurite shape, major dendritic branching pattern and the position of the axon in sections of the fifth abdominal ganglion confirmed that the cell under test was GI 3. From Harrow et al. (1 979)

choline. Depolarization of the cell membrane followed the application of acetylcholine and repeated applications produced desensitization of the response. The sensitivity of D, cell bodies to acetylcholine (David, 1979) appeared to be similar to that of the dorsal unpaired median (DUM) cells of the metathoracic and sixth abdominal ganglia reported earlier (Kerkut er al., 1969a, b; Pitman and Kerkut, 1970). Carbamylcholine also depolarized the cell body membrane when locally applied to D, using iontophoretic techniques (David, 1979). Axotomy did not result in a change in sensitivity to carbomylcholine (David and Pitman, 1979). These authors showed that the anticholinesterases (physoconcentrations produced up stigmine and neostigmine) at 1.0 x 1 0 - 7 ~ to a 1000-fold potentiation of the normal acetylcholine response indicating the presence of large amounts of cholinesterase near the soma membrane. It was also concluded that the increased acetylcholine sensitivity of axotomized Df motoneurones may in part be attributable to a fall in the activity of acetylcholinesterase at the cell surface. Recent experiments on D r (Sattelle et al., 1980) demonstrated that a-bungarotoxin (5.0 x ~O-'M)irreversibly reduced the sensitivity of this cell to iontophoretically applied acetylcholine without any appreciable change in the membrane resistance and resting potential (Fig. 24). Thus it appears that in Periplaneta there are

DAVID

264

B. SATTELLE

16

-> E

v

r e 0 .c

m

N ._ I

m 0

a a,

n

C 1

10

102

I 103 1 o4 Charge ( n C )

Fig. 24 a-Bungarotoxin suppresses the response to acetylcholine of the cell body membrane of the right coxal depressor motoneurone (Df) of the metathoracic ganglion of Periplaneta americana. As shown in the upper traces a-bungarotoxin (5.0 x lo-%) substantially reduced the depolarizing response of the cell body membrane to iontophoretically applied acetylcholine. Changes in the dose-response curve are shown following a 2 h exposure to a-bungarotoxin. From Sattelle et al. (1980)

acetylcholine receptors on the soma membrane of Dfwhich in their sensitivity to a-bungarotoxin resemble the receptors mediating cercal-afferent, giantinternpiirnne cvnantir- trancmiccinn in t h e ciyth ahclnminal onnolinn

4.4.3

Trochanteral hairplate-to-rnotoneurone D,reflex

The pharmacological properties of the monosynaptic connection between the trochanteral hair plate afferents and motoneurone D, of the metathoracic ganglion of Periplaneta americana (Fourtner et al., 1978) were investigated by Carr and Fourtner (1978). Electrical stimulation of the

ACETYLCHOLINE RECEPTORS

OF I N S E C T S

265

hairplate produced a 1:1 reflex activation of D,. Changes in the D, reflexresponse to stimulation and changes in the level of activity in D, were monitored during perfusion of cholinergic ligands. A variety of antagonists including a-bungarotoxin, nicotine, hexamethonium and atropine blocked synaptic transmission at this central synapse. 4.4.4

Dorsal unpaired median (DUM) neurones

Goodman and Spitzer (1979a) have examined the sensitivity of the cell bodies of identified embryonic DUM neurones in the grasshopper Schistocerca nitens to bath application and iontophoresis of various cholinergic ligands. The iontophoretic application of acetylcholine resulted in a transitory depolarization of the soma membrane. For the acetylcholine response, a reversal potential of +20 mV was estimated by extrapolation. The acetylcholine-induced depolarizations were blocked following the substitution of sodium in the physiological saline by choline and were reduced by the bath application of the nicotinic antagonists d-tubocurarine (1.O X 1 0 - 4 ~ ) , ) decamethonium (1.0 x lo-’~).Nicotine hexamethonium (1.0 x 1 0 - 3 ~and was found to be an agonist, but a-bungarotoxin (1.0 x 1 0 - 6 ~failed ) to block the acetylcholine response. The muscarinic antagonists quinuclidinyl benzi) atropine ( 1 x lo-%) were also without effect. Thus, late (1.0 x 1 0 - 7 ~and although the cholinergic receptors on embryonic DUM cell somata of Schistocerca nitens appear to be nicotinic in nature, they differ from the receptors on D r motoneurone cell bodies of adult Periplaneta americana in their sinsitivity to a-bungarotoxin (see also Section 4.4.2). In recent studies on DUM cells of the metathoracic ganglion of adult Periplaneta, a relative insensitivity to a-bungarotoxin has been noted (Sattelle et af., 1980) similar to that reported for the embryonic DUM cells of Schistocerca (Goodman and Spitzer, 1979a). However, whereas the responses to acetylcholine of the embryonic DUM neurone cell bodies (Schistocerca) did not desensitize (Goodman and Spitzer, 1979a), desenstization was always noted in the case of the acetylcholine-induced responses of adult DUM neurone cell bodies (Periplaneta) (Kerkut et al., 1969a, b). Clearly a more detailed comparison of the acetylcholine sensitivity of embryonic and postembryonic DUM neurones of a single species would be desirable.

5 Comparative pharmacology of CNS acetylcholine receptors Having surveyed the results of biochemical, localization and electrophysiological studies, it is now possible to ask whether or not a physiological role can be ascribed to any of the three putative acetylcholine receptors

DAVID B. SATTELLE

266

characterized by radiolabelled ligand-binding techniques. Having attempted this, it will then be appropriate to compare the pharmacological properties of insect acetylcholine receptors with acetylcholine receptors of other invertebrates and vertebrates. 5.1

INSECTS

Three putative acetylcholine receptors have been reported in insect central nervous tissue (see Section 2). As a first step in considering a possible physiological role €or one or more of these receptors, we have compared in Table 11 the sedimentation properties of these putative receptors and the density of binding sites. Clearly the bulk of the [1251]-a-bungarotoxin binding sites and many of the [3H]-quinuclidinyl benzilate binding sites sediment in membrane or particulate fractions (pelleted at 20 00040 000 xg). Also, the difficulties noted in solubilizing these binding components indicate that they are tightly bound (intrinsic) to the membrane as would be expected for receptors. The density of binding sites defined by these two receptor probes is within the range reported for other tissues such as vertebrate CNS (Snyder and Bennett, 1976) and electroplax tissues (Changeux et al., 1970). The main difference is that whereas in vertebrate brain [3H]-quinuclidinyl benzilate (muscarinic) binding sites are more abundant by one or two orders of magnitude than the ['251]-abungarotoxin-binding sites (Yamamura et al., 1974; Segal et al., 1978), the reverse appears to be the case in the one insect preparation (Drosophila rnelanogaster) for which comparative data is available (Dudai, 1978; Haimet al., 1979). The extremely high density of binding sites defined by various reversible radiolabelled ligands in Musca heads (Eldefrawi et al., 1971; Jewess et al., 1975) and the solubilization of the cholinergic binding molecules by homogenization in water distinguishes this putative receptor from all other putative cholinergic receptors. Of the three putative acetylcholine receptors reported in insect central nervous tissues (see Section 2) only in the case of the abungarotoxin-sensitive receptor is there substantial evidence for a physiological role in synaptic transmission. Although the [ '2'I]-a-bungarotoxin-binding component has been most fully characterized in Drosophila rnelanogaster, the evidence that it is a constituent of a synaptic acetylcholine receptor stems largely from experiments on Periplanetu arnericana, the central nervous system of which has proved amenable to both radiolabelled ligand binding studies and single-cell electrophysiology. The evidence can be summarized as follows: (a) Extracts from abdominal nerve cords of Periplaneta contain a compowith many of the expected nent of specific [ 1251]-a-~bungarotoxin-binding

TABLE 11 Comparison of putative CNS acetylcholine receptors from different insects

z

a-Bungarotoxin binding parameters

rn

n Insect preparation Periplaneta americana (abdominal nerve cord extracts) Drosophila rnelanogasfer (whole fly extracts)

Receptor probe

ki k-, K D = k - , / k , pmoles (lo-'%) binding sites (lO5W1s-') (lo-%')

[~2511-~-

5.7

Irreversible binding

112

bungarotoxin

76% pelleted after 40000xg for 30 min

0 rn W

+

Gepner er al. (1978)

V)

Sattelle (1980)

51 z

[ q ~ - ~ 7.7 -

[w-

-

6.2

0.81

10

-

0.21

88

a-Bungarotoxin does not bind

6.5

a-Bungarotoxin does not bind

3000

decame ttronium (head extracts)

Reference

0 Y

bungarotoxin (head extracts treated with [1251]-aTriton X-100) bungarotoxin (head extracts) ['HIquinuclidinyl benzilate Musca domestica (head extracts)

Sedimentation properties

[12511-~-

bungarotoxin

89% pelleted after 40000xg for 30 min not done 48% pelleted after 20 000% for 20 min

In supernatant after 100 OOOxg for 60 min

0.3

2.07

6.8

-

Schmidt-Nielsen ef a l . (1977), Gepner (1979) Schmidt-Nielsen ef al. (1977) Haimetal. (1979)

Mansour et al. (1977). Eldefrawi et al. ( 1 971 b) Harriser al. (1979)

V)

rn

v)

268

D A V I D 6.SATTELLE

properties of an acetylcholine receptor. For example, toxin-binding is saturable and sediments in a membrane fraction. Nicotine and d-tubocurarine are highly effective at displacing a-bungarotoxin from its binding site, whereas atropine and pilocarpine are much less effective. ( b ) Nicotinic ligands are similarly more effective in modifying synaptic transmission between cercal afferent neurones and giant-interneurones in the sixth abdominal ganglion of Periplunetu. In fact, a close correspondence has been reported for the pharmacological specificity of the [1251]-abungarotoxin-binding component and the pharmacological specificity of cercal-afferent, giant-interneurone synapses. This is illustrated in Table 12 in which the ligand concentrations required for inhibition by 50% of the binding [1251]-a-bungarotoxinare compared to concentrations estimated to produce half-maximal physiological actions at cercal-afferent, giantinterneurone synapses. ( c ) The most effective blocking agent of all those tested to date for their ability to block cercal-afferent, giant-interneurone synapses has proved to be a-bungarotoxin, which completely blocks transmission at nanomolar concentrations. This is close to values for the K D estimated from toxinbinding studies on Drosophilu (Schmidt-Nielsen et ul., 1977; Gepner, 1979). No K Dvalues are available for Periplunetu since toxin bound essentially irreversibly but from saturability experiments it was noted that half~ (see Fig. 6). maximal binding was achieved at 1.1 x 1 0 - 9 a-bungarotoxin ( d ) Autoradiographic studies have shown specific binding of [1751]-abungarotoxin in the neuropile of the sixth abdominal ganglion, the region known to contain the cercal-afferent, giant-interneurone synapses. Thus there is strong evidence for a functional role in synaptic transmission at Peripfunetu cercal-afferent, giant-interneurone synapses of an acetylcholine receptor for which a-bungarotoxin is a specific irreversible probe. Recent microelectrode studies on the cell body membrane of the coxal depressor motoneurone (Df) of the metathoracic ganglion of Periplunetu have shown that the depolarizing response to iontophoretically applied acetylcholine can be largely suppressed by the bath application of 5.0 x ~ O - * M a-bungarotoxin (Sattelle et ul., 1980). This provides further evidence for the existence of a neuronal a-bungarotoxin-sensitive acetylcholine receptor. Dudai and collaborators (1980) have shown that cholinergic drugs administered to Drosophilu by injection could result in the death of the insect. When LD5{,values were compared, it was clear that nicotinic ligands were far more active than muscarinic drugs, but the results of such whole-animal responses to injected ligands are difficult to interpret in terms of possible receptor actions of the ligands. A physiological role for the other two putative acetylcholine receptors of insects has yet to be demonstrated. It seems unlikely that these putative

,

D 0 rn -I

s 0

I

-

TABLE 12 Comparative pharmacology of insect cholinergic receptors as determined electrophysiologically and by binding studies

z

Concentration (M)for 50%of maximum effect

0

m

P Ligand

Penpianera amncana '

Peripianera amencana '

Electrophysiology

["'I]-a-Bungarotoxin hindlng

~

~

0.8 x 0.3 X '1.3 x 5.0 x 15.0 x 6.6 X 5.4 x

lo-* lo-'' lo-"

lo-'

['i~I]-a-Bungarotox~n binding (15")

(150)

Nicotine d-Tubocurarine Acetylcholine Carbamylcholine Decamethonium Pilocarpine Atropine PrBCIvf'

Drosophila mclanogrrrter'

2.7 x lo-" 2.4 x 10-7 '8.3 x lo-" 1.0 x 10-4 1.7~10-~ 2.1 x 10-5 7.2 x 10-5

4.5 x lo-' '2.8 x lo-'' 1.2 x 10-4 2.5 x 7.9 x low 5.7 x lo-'

rn

Musca domesrrca'

Musca domesrxa'

Drosophila meianogasfer'

Labelled ligand

['HI-Decamethonium

[1H]-Qumuclidmyl

binding

binding

bemilate binding

( K Oor K,)

(15")

3.0 - 4.4 X 1.6 X lo-' -d5.0

X

6.5 x lo-" 8.5 x lo-" 3.5 X lo-' 6.0 x lo-'

1 . 9 - 4 . 8 ~lo-' 5.4 x 10-5 2.1 - 5.2 X lod6 2.2 x lo-'

~

~

" + eserine ( 10-6M); + neOStigmine (10-'M); '+ neostigmine (lo-'#); + paraoxon (1W'M); ' + diethylfluorophosphate lo-'^); 'PrBCM = propylbenzilylcholinemustard 'Sattelle (1978; 1980) *Schmidt-Nielsenet al. (1977) 'Eldefrawi et al. (1971b), Aziz and Eldefrawi (1973); Jewess et al. (1975) Tattell et al. (1979) 'Dudai and Ben-Barak (1977); Haim et al. (1979)

(ED,,

01

KJ

110-3

m D

0 n

z v)

' 5 x lo-" 3 x 10-5 >lo-) 3 x lo-" 4 x 10-9

rn 0 -I

v)

270

DAVID 6 . SATTELLE

receptors contribute substantially to acetylcholine-mediated transmission at cercal-afferent, giant-interneurone synapses since both the muscarinic receptor (characterized by [3H]-quinuclidinyl benzilate binding) and the mixed receptor (characterized by reversible ligand binding) are unaffected by micromolar a-bungarotoxin (Mansouretal., 1977; Haimet al., 1979). By contrast, cercal-afferent, giant interneurone synaptic transmission is completely blocked by nanomolar toxin concentrations (Harrow et al., 1980b). Nevertheless, some support for the existence of a-bungarotoxin-insensitive acetylcholine receptors on certain insect neurones has emerged from recent electrophysiological experiments. For example. DUM neurone cell bodies of the grasshopper (Schistocerca nitens) and cockroach (Periplaneta arnericana) metathoracic ganglia are sensitive to acetylcholine applied by microiontophoresis, but the resulting depolarization is not blocked by 10 - 6 ~ a-bungarotoxin (Goodman and Spitzer 1979a; Sattelleetal., 1980). Further studies are needed to define the detailed pharmacological specificity of these acetylcholine responses of insect neurones which are relatively insensitive to a-bungarotoxin. In this context, it is of interest to note that Greenspan et al. (cited in Dudai, 1980) have shown that Drosophila mosaics with acetylcholinesterase-less mutant tissue in the lamina lack a synaptic component of the electroretinogram - the “off” transient. Dudai (1980) obtained the same modification of the electroretinogram by feeding flies with the acetylcholinesterase inhibitor neostigmine. Since the lamina contains very few [L251]-a-bungarotoxinbinding sites, it may be that a-bungarotoxin-insensitive receptors mediate the “off” response recorded from the retina. Confirmation of the identity of putative insect acetylcholine receptors by immunological methods would be highly desirable and work in this area has recently been initiated (see Eldefrawi and Eldefrawi, 1980). Several antibodies form against the nicotinic acetylcholine receptor purified from Torpedo (Lindstrom et al., 1977; Eldefrawi, 1978; Gomez, et al., 1979) all of which react with the receptor antigen to precipitate it from solution. In double-diffusion tests Mansour et al. (1977) compared the electric-organ receptor with the housefly putative receptor purified from high-speed supernatants (see Section 2.2). No line appeared between the purified Torpedo receptor and the purified housefly cholinergic binding proteins (Fig. 25a). Sharp lines did appear between the well containing a 1:1mixture of antisera against the pure Torpedo and housefly proteins and the two wells containing their respective antigens (Fig. 25b). However, these lines did not connect smoothly indicating that the antigenic determinants were distinct. The same laboratory employed a radioimmune assay to test for crossreactivity between Torpedo acetylcholine receptor and the Triton-extracted [‘251]-a-bungarotoxin-binding component from houseflies (Eldefrawi and


27 1

Eldefrawi, 1980). No such cross-reaction was detachable. On the other hand, the sheep antiserum against Torpedo nicotinic receptor did precipitate the [1251]-a-bungarotoxinlabelled Torpedo receptor. These authors concluded that the toxin-binding proteins of Torpedo electroplax and housefly heads differ at least in respect of the antigenic site(s) against which these antibodies are produced.

Fig. 25 Double diffusion assay of ( a ) rabbit antisera for the isolated housefly proteins (bottom well) against the two isolated housefly proteins (top left well) and the high-speed (100 000 x g) supernatant extract S , (top right well) and ( b ) 1: 1 mixture of rabbit antisera for the purified housefly and Torpedo ACh receptor (bottom well) against the purified Torpedo ACh receptor (top left well) and the two isolated housefly proteins (top right well). From Mansour et al. (1977)

Thus biochemical and electrophysiological experiments have shown that several insect species (Drosophila melanogaster, Musca domestica and Periplaneta arnericana) possess more than one type of CNS putative acetylcholine receptor. Evidence has been presented for a physiological role in synaptic transmission of an acetylcholine receptor for which nicotine and acetylcholine are potent agonists and d-tubocurarine and a-bungarotoxin are potent antagonists.

5.2

INVERTEBRATES OTHER THAN INSECTS

In this section the properties of acetylcholine receptors of the central nervous tissues of invertebrate organisms other than insects are considered. Emphasis will be given where possible to those groups of organisms for which both biochemical and electrophysiological data are available. Few data are available for the Annelida although it has been established that acetylcholine (Kerkut etal., 1970; Kerkut and Walker, 1967); and nicotinic agonists (Woodruff et al., 1971) depolarize and excite the cell body membrane of Retzius cells in the central nervous system of the leech Hirudo

272

D A V I D 6.SATTELLE

medicinalis. The excitatory action of acetylcholine was blocked by benzoquinonium. Muscarinic agonists at low concentrations were able to produce inhibition but at higher concentrations their application resulted in excitation (Woodruff, et af., 1971). a-Bungarotoxin was ineffective in blocking the acetylcholine-induced depolarization of Retzius cells (Magazanik, 1976). Sargent et al. (1977) showed that whereas cell body membranes of pressure-sensitive (P) and nociceptive (N) sensory neurones of the CNS of Hirudo were depolarized by acetylcholine, the touch sensitive (T) cells exhibited a biphasic response (depolarization followed by hyperpolarization). Of the motoneurones investigated, the A E cells which control the formation of skin ridges were hyperpolarized by acetylcholine, but in the L neurones which control shortening of the animal a biphasic response was elicited. Nicotine and d-tubocurarine were highly effective ligands at the cholinergic receptors of N cells. Signhcant differences were noted between extrasynaptic (cell body) and synaptic receptors in respect of their sensitivity to d-tubocurarine - the extrasynaptic receptors being much more sensitive. No radiolabelled ligand binding studies have been performed to date on annelid central nervous tissues. The nerve cell soma membranes of various species of Mollusca have been the subject of the most detailed pharmacological analysis of CNS acetylcholine receptors in invertebrates (Ascher and Kehoe, 1976; Walker, 1980). Acetylcholine, applied iontophoretically to soma of identifiable Aplysia neurones or released by synaptic activation of cholinergic cells, can give a variety of responses which result from the activation of three different acetylcholine receptors each with a distinct pharmacological specificity (Kehoe, 1972 a, b, c; Kehoe et al., 1976; Shain et a f . , 1974; Yarowsky and Carpenter, 1978). For example, a receptor mediating an increased membrane permeability to sodium ions generated an EPSP that was sensitive to hexamethonium and d-tubocurarine, but relatively insensitive to a-bungarotoxin.This receptor is, therefore, comparable to the acetylcholine receptor giving rise to the rapid EPSP in vertebrate sympathetic ganglion cells ( c t Barlow, 1964: Volle and Koelle, 1975). In addition, a receptor mediating an increase in permeability to chloride ions generated a rapid inhibitory postsynaptic potential, that was sensitive to d-tubocurarine and a-bungarotoxin but insenstive to hexamethonium. This receptor therefore exhibits a pharmacological profile resembling that of the acetylcholine receptor of vertebrate skeletal muscle (cf. Barlow, 1964; Koelle, 1975). Finally a receptor mediating an increased permeability to potassium ions generated a slow IPSP. This inhibitory postsynaptic potential was insensitive to d-tubocurarine, atropine and a-bungarotoxin but was blocked by tetrethylammonium ions. There is no vertebrate or invertebrate counterpart to this receptor which is not readily incorporated into traditional schemes of


273

receptor classification. Three similar acetylcholine-induced responses were reported in Helix neurones (Gerschenfeld and Tauc, 1961; Kerkut et al., 1973; Chadetal., 1979; Yavarietal., 1979) and the sodium-dependent and chloride-dependent responses were also observed in neurones of Limnaea, Planorbarius (Zeimal and Vulfius, 1967) and Navanax (Levitan and Tauc, 1972). A biphasic response to acetylcholine was recorded from neurones of the pedal ganglion of Planorbarius (Ger and Zeimal, 1977). The initial (depolarizing) phase was mimicked by nicotinic ligands, whereas the slower (hyperpolarizing) phase was mimicked by muscarinic agonists such as 2-methyl-4-trimethylammonium-1, 3 dioxolane (F-2268). Recently, Walker and colleagues (Walker and Kerkut, 1977; Chadetal., 1977; Yavari et al., 1979) obtained evidence for the existence of an excitatory muscarinic receptor on cell E4 of Helix aspersa. The effects of a-bungarotoxin on the three types of acetylcholine-induced responses in Aplysia neurones have been investigated. Shain et al. (1974) were able to block all three acetylcholine responses at toxin concentrations as low as 2.0 x ~O-'M. However, Kehoe et al. (1976) found a-bungarotoxin (from Bungarus multicinctus) completely ineffective on the sodium and potassium mediated responses but able to produce reversible blockade of the response resulting from an increase in chloride permeability when . al. (1976) also showed applied at high concentrations (1 x 1 0 - 5 ~ )Kehoeet that a-bungarotoxin from B. caeruleus was ineffective on all three cholinergic responses. They suggested that the results of Shain et al. (1974) might have been due to contamination of their toxin samples by acetylcholinesterase. Other a-toxins, the Dendroaspis toxins from the green mamba Dendroaspis aspis, were also only effective on the chloride dependent response (Szczepaniak, 1974). Thus it appears that the so-called long toxins (e.g. B. multicinctus and D. aspis) are effective on the chloridemediated response but the short toxins (e.g. B. caeruleus toxin) are ineffective. Differences have been observed between the chloride-mediated response of Aplysia neurones and vertebrate skeletal muscle acetylcholine receptors. For example, whereas only the toxin of B. multicinctus was effective on Aplysia neurones, both B. multicinctus and B. caeruleus toxins were effective on vertebrate neuromuscular receptors (Kehoe et al., 1976). Also, the a-bungarotoxin block reported for Aplysia neurones was achieved only at high (1.O x ~O-'M)concentrations and was reversible, whereas neuromuscular junction receptors of vertebrates were irreversibly antagonized by ~ and Potter, 1971). Using [1251]-aa-bungarotoxin at 8.0 x l W 9 (Miledi bungarotoxin, Shain et al. (1974) demonstrated the existence of a saturable component of binding with some of the expected properties of a nicotinic acetylcholine receptor (Table 6). It is premature to consider this binding

274

DAVID

B. SATTELLE

component as a constituent of the receptor mediating chloride permeability changes, in view of the lack of correlation between the physiological and biochemical findings. Also, a number of ligands formerly considered to act at the receptor recognition site (including d-tubocurarine) are now known to exert their actions a t the receptor ionophore (Ascher et al., 1978). A high concentration of a-bungarotoxin-binding sites was reported in squid optic ganglia (Kato and Tattrie, 1976) and a-bungarotoxin-sensitive receptors were found on the Schwann cells enveloping the squid giant axon (Villegas, 1975). Of the arthropods other than insects that have been investigated both ligand-binding studies and electrophysiological results are available but in only one case to date (the Xiphosuran, Limulus polyphemus) have these complementary approaches been applied to the same tissue. Walker and James (1 978) found that acetylcholine depolarized Limulus neurones and this response was blocked by hexamethonium. Although nicotine was the most effective agonist, d-tubocurarine was a weak antagonist. An ['251]-a-bungarotoxin-binding component with the properties of a nicotinic acetylcholine receptor was characterized in homogenates prepared from the CNS of Limulus polyphemus (Thomas et al., 1978). As shown in Table 6, nicotine was a more effective inhibitor of toxin-binding than carbamylcholine and of the antagonists tested d-tubocurarine was more effective than atropine. Another acetylcholine binding component was demonstrated in axons of Limuluspolyphemus with a low affinity for both acetylcholine and a-bungarotoxin (Jonesetal., 1973). The sensitivity of Crustacean neurones to acetylcholine has been described by several laboratories (see Wiersma etal., 1953; McLennan and York, 1966; Barker eta/., 1972). For example, Barkerelal. (1972) showed that the depolarizing response to acetylcholine, iontophoretically-applied to cell bodies in lobster (Homarus americanus) CNS was blocked by 7.0 x 10 - 4 ~ d-tubocurarine. Atropine and hexamethonium at similar concentrations were less effective. The same workers noted that when bath applied to the abdominal ganglion cell. M15, d-tubocurarine and atropine (7.0 x 1 0 - 4 ~ ) blocked the depolarization recorded from the soma membrane in response to electrical stimulation of the slowly-adapting muscle receptor neurone. Hexamethonium, mecamylamine, choline and succinylcholine were either less effective or induced depolarization of the soma membrane (Barker, et al., 1972). The amine containing neurones of the second roots of lobster thoracic ganglia were depolarized by iontophoretically applied acetylcholine (Konishi and Kravitz, 1978). Both the response to iontophoretically applied acetylcholine and synaptic potentials evoked by nerve stimulation were blocked by 7.0 x ~ O - ' M hexamethonium. Nicotine and d-tubocurarine were the most effective of the ligands tested for their ability to suppress


275

the synaptic response and a-bungarotoxin (1.0 x 10-6-1.0 x 1 0 - 5 ~were ) ineffective even after perfusion for 30 min. Neurones of the stomatogastric ganglion of the crab Cancer pagurus showed several responses to iontophoretically applied acetylcholine, the predominant one being a depolarizing response (Marder, 1977; Marder and Paupardin-Tritsch, 1978) which was mimicked by agonists of vertebrate nicotinic ganglionic acetylcholine receptors such as TMA and DMPP ( c t Volle and Koelle, 1975). These responses were blocked by antagonists of vertebrate nicotinic ganglionic transmission such as mecamylamine and hexamethonium. Decamethonium, a potent ligand at vertebrate skeletal muscle acetylcholine receptors, had no effect on the stomatogastric ganglion cell depolarizing response to acetylcholine. Evidence was also obtained for the existence on stomatogastric ganglion neurones of acetylcholine receptors comparable to the muscarinic receptors of vertebrates (Marder, 1977; Marder and Paupardin-Tritsch, 1978). Thus studies on a variety of Crustacean neurones have revealed more than one kind of response to acetylcholine, but common to several species is an acetylcholine receptor resembling somewhat the properties of the vertebrate ganglionic nicotinic receptor. Few radiolabelled ligand binding studies have been performed on tissues of Crustacea. However. an acetylcholine-binding component has been demonstrated in axons of the lobster (Homarus americanus) (Denburget al., 1972; Denburg, 1973; Denburg and O’Brien, 1973; Joneset al., 1977) but not in axons of crabs such as Cancer pagurus (Balerna et al., 1975), Maia squinado (Balerna et al., 1975) and Callinectes sapidus (Jonesetal., 1977). Nevertheless this component has a low affinity for acetylcholine and binds a-bungarotoxin with low affinity and reversibly. This binding component to which no function has yet been ascribed does not appear to be either an axonal or glial acetylcholine receptor (Jones et al., 1977). Thus work on insects and other invertebrates has revealed a diversity of neuronal acetylcholine receptor types. Although there is some evidence for the existence of acetylcholine receptors resembling vertebrate muscarinic receptors, most invertebrate cholinergic receptors are characterized by a high affinity for nicotine and nicotinic ligands. Several invertebrate species possess more than one kind of CNS acetylcholine receptor and identified molluscan and annelid neurones with more than one kind of cholinergic receptor have been described. Extrasynaptic (cell body) receptors and synaptic receptors of Aplysia neurones share the same pharmacological profile, whereas differences have been detected in the case of Hirudo neurones.

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276

5.3

VERTEBRATES

Acetylcholine receptors in the central nervous systems of vertebrates have been investigated using both electrophysiological and radiolabelled ligand binding techniques. Several distinct responses to acetylcholine have been recorded from vertebrate neurones. Fast excitatory responses resulting from an increased cation permeability have been well documented in autonomic ganglion cells (Blackman et al., 1963; Dennis et al., 1971) Renshaw cells (Curtis and Ryall, 1966a, b) and a variety of other regions of the CNS (cf. Krnjevic 1974). These responses are nicotinic in nature but the most effective blocking agents are not d-tubocurarine and its derivatives, but hexamethonium and tetraethylammonium (cfi Volle and Koelle, 1975). In studies on a cholinergic pathway in the frog spinal cord, Miledi and Szcepaniak (1975) showed that, although four neurotoxins from Dendroaspis venom appeared to block neuronal acetylcholine receptors, abungarotoxin (8.0 x LO-'M) was ineffective. This pathway in amphibia corresponds to the motoneurone-Renshaw cell pathway of higher vertebrates. Hunt and Schmidt (1978) have shown that Renshaw-like cells in the anterior horn of the rat spinal cord possess binding sites for ['*51]- a-bungarotoxin. Recently a saturable component of [ '251]-cr-bungarotoxin binding with a nanomolar dissociation constant ( K , ) and a pharmacological specificity corresponding to that of a nicotinic acetylcholine receptor was reported in the following nervous tissues of vertebrates: mammalian brain (Moore and Loy, 1972; Salvaterra and Moore, 1973; Eterovic and Bennett, 1974; Moore and Brady, 1976; McQuarrieetal., 1976; Schmidt, 1977; McQuarrie et al., 1977); mammalian autonomic (sympathetic) ganglia (Fumagallietal., 1976; Greene, 1976); cultured sympathetic neurones (Patrick and Stallcup, 1977a; Carbonetto et al., 1978); mammalian adrenal medulla (Wilson and Kirschner, 1977); avian retina (Yazulla and Schmidt, 1977); avian autonomic (parasympathetic) ganglia (Chiappinelli and Giacobini, 1978); fish brain (Oswald and Freeman, 1979) and amphibian brain (Oswald and Freeman 1977). The receptor-like properties of these sites was established and a good correlation demonstrated between the distribution of binding sites and sites of known central cholinergic pathways (cf. Schmidt et al., 1979). Also, in autonomic (sympathetic) ganglion preparations, toxin-binding sites appeared to be confined to neurones rather than to non-neuronal cells (Greene et al., 1973; Fumagalli et al., 1976). Synaptic localization of toxin binding sites has been confirmed by electronmicroscopy (Daniels and Vogel, 1975; Lentz and Chesher, 1977; Hunt and Schmidt, 1978). However,


277

attempts to block nicotinic receptors with a-bungarotoxin and related a-neurotoxins have met with variable results. Several laboratories have failed to block transmission in vertebrate sympathetic ganglia (Chou and Lee, 1969; Magazanik et al., 1974; Brown and Fumagalli, 1977). Also the responses to nicotinic ligands of cultured vertebrate sympathetic neurones were not blocked by a-bungarotoxin (Obata, 1974;Nurse and O’Lague, 1975; Giller et al., 1975; KOet al., 1976; Carbonetto et af., 1978). a-Bungarotoxin was also unable to block the nicotine-induced release of catecholamines in the adrenal medulla (Wilson and Kirschner, 1977). Patrick and Stallcup (1977b) working on clone PCR, a cell line from a rat pheochromocytoma showed that receptor activation (measured as carbamylcholine-induced sodium ion fluxes) was unaffected by a-bungarotoxin. The same authors also showed that antibodies raised against Torpedo acetylcholine receptors inhibited receptor function but did not precipitate the toxin-binding macromolecules. Thus it appears that a-bungarotoxin binds to a nicotiniclike binding site on sympathetic neurones but that occupation of this site does not inhibit receptor activation. The visceral (parasympathetic) ganglia of rabbit, guinea pig, and chick were unaffected by a-bungarotoxin and no binding of radiolabelled toxin was detected (Bursztajn and Gershon, 1977), whereas in chick ciliary (parasympathetic) ganglion specific toxin binding sites were detected (Chiappinelli and Giacobini, 1978) and some, but not all, batches bf a-bungarotoxin proved effective at blocking transmission though at rather ) (Chiappinelli and Zigmond, 1978). A high (1.O x 1 0 - 6 ~ concentrations recent investigation of the inhibition of neuronal acetylcholine sensitivity by a-toxins from B. multicinctus venom has been performed using ciliary ganglion neurones in cell culture (Ravdin and Berg, 1979). These authors have identified six separate a-toxins. The widely used a-bungarotoxin (aBgt 2.2) is one of these fractions. Although a high-affinity specific toxin-binding site was detected on these cells, this particular a-toxin did not affect the response of the cultured cells to iontophoretically applied acetylcholine at 1.O X 1O-’M (a concentration which should saturate the high-affinity binding site). Higher concentrations of a-bungarotoxin (1.O x 1 0 - 5 ~ ) produced a partial inhibition of acetylcholine sensitivity. Whether this indicates that the cultured neurones possess two classes of binding sites or that other a-toxins present as minor components in the aBgt 2.2 were responsible for the blockade remains to be determined. In contrast to the results on spinal cord neurones and autonomic ganglia, other laboratories have provided physiological evidence that abungarotoxin acts at a site that is a constituent of a functional acetylcholine receptor. For example, excitatory postsynaptic potentials recorded from neurones in toad tectum (Freeman, 1977) are irreversibly blocked by

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a-bungarotoxin at a concentration of 1.0 x ~O-’M.This tissue contains a specific toxin-binding component. a-Bungarotoxin also blocks acetylcholineinduced dopamine release from rat brain synaptosomes (de Belleroche and Bradford, 1978) and cholinergic transmission in mammalian retinal ganglion cells (Masland anti Ames, 1976). It is possible that the complex results that have emerged from studies on vertebrate neuronal nicotinic acetylcholine receptors reflect an underlying heterogeneity of nicotinic receptor types, in particular those sensitive to, and those insensitive to a-bungarotoxin. In this context it might be recalled that certain neuromuscular junction nicotinic receptors of vertebrates are also insensitive to a-bungarotoxin (Burden et al., 1975). Alternatively, it is possible that all the a-bungarotoxin binding sites are constituents of the receptor complex. The different actions of a-bungarotoxin could then be explained if the binding sites for small ligands (including acetylcholine) and a-bungarotoxin vary in the degree to which they overlap (cf. Schmidt et a1., 1979). The techniques of electrophysiology and radiolabelled ligand binding have also been applied to the study of muscarinic acetylcholine receptors in the CNS of vertebrates. A slow prolonged excitation in response to acetylcholine and muscarinic agonists that was blocked by atropine and hyoscine, was recorded from ganglion cells and from a variety of central neurones (cf. Krnjevic, 1974). This type of response proved to be the most common excitatory response of acetylcholine in the vertebrate CNS and was recorded from neurones that, in addition, exhibited nicotinic responses (Phillis, 1970). Inhibitory actions of acetylcholine were reported in many areas of the CNS, even in cells predominantly excited by acetylcholine (cf. Krnjevic, 1974). These responses appeared to be due solely to the activation of muscarinic receptors. In recent years, the binding of a variety of reversible tritiated muscarinic antagonists to vertebrate CNS tissues, and to subcellular fractions derived from CNS tissues has been investigated. These include atropine (Paton and Rang, 1965; Schleifer and Eldefrawi, 1974; Birdsall et al., 1975; Hulme et al., 1976) quinuclidinyl benzilate (Yamamura and Snyder, 1974a, b, c Yamamura et al., 1974a, b; Snyder et al., 1975) and propylbenzilylcholine (Birdsall et al., 1976; Hulme et al., 1976). The tritiated form of the irreversible ligand propylbenzilylcholine mustard has also been used as a muscarinic receptor probe (Hiley et al., 1972; Burgen et al., 1974b; Hiley and Burgen, 1974; Hiley and Bird, 1974; Birdsall and Hulme, 1976; Birdsall et al., 1978). By these methods, a specific, saturable binding component with the expected pharmacological properties of a muscarinic receptor has been characterized. Also, the distribution of [3H]-antagonist binding sites in the central and peripheral nervous system detected by autnradinpranhv rlncelv n n m l l e l s the dictrihlltinn eynepted frnm eler-


279

trophysiological studies (Kuhar and Yamamura, 1975; Yamamura and Snyder, 1974b). The cerebral cortex is particularly rich in muscarinic binding sites (Table 7). Thus several general conclusions emerge from a comparative study of CNS acetylcholine receptors of insects, other invertebrates and vertebrates. For example, several different kinds of receptor can be recognized with reference to both pharmacological specificity and receptor mediated cellular responses. That several acetylcholine receptors appear to be present in vertebrate, insect and molluscan central nervous tissues indicates that this diversity may have been established early in animal evolution. It is also clear that the traditional classification based on experiments on vertebrates becomes an inadequate framework when both invertebrate and vertebrate receptors are considered. The existence of more than one type of acetylcholine receptor on a single neurone has been demonstrated for both vertebrate and invertebrate receptors, so the ability of a single neurotransmitter to exert different effects on a particular postsynaptic cell also appears to have been established early in evolution. Finally, the density of acetylcholine receptors is comparable in both vertebrate and invertebrate organisms, though it appears that whereas muscarinic receptors predominate over nicotinic receptors in the mammalian brain the reverse is true in the insect brain. Comparative studies of this kind may point to distinct functional roles for the various acetylcholine receptors.

6 Genetic and developmental studies

6.1

G E N E T I C A P P R O A C H E S TO RECEPTOR S T R U C T U R E A N D F U N C T I O N

The wealth of genetic information that has accumulated on the fruit fly Drosophila melanogaster provides scope for a detailed genetic analysis of receptor structure, function and role in behaviour and development. As a first step, it is necessary to isolate mutants with altered receptors that can be used for subsequent genetic analysis. Although the existence of a nicotineresistant mutant of Drosophila was briefly reported over ten years ago (Lindsley and Grell 1968), the most detailed study was recently performed by Hall and collaborators (1979). These authors exposed Canton-S wildtype Drosophila to 3 mM nicotine in the culture medium. This concentration of nicotine killed 96% of the Canton-S strain and was routinely used to screen for nicotine-resistant strains in wild-type populations. One of the eight resistant strains that the authors identified was designated H R (isolated from Hikone-R wild type stock) and 50% of this resistant strain survived when exposed to 3 m M nicotine. To identify resistant strains with modified receptors, Hall and co-workers

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subjected a solubilized receptor-[ 1251]-cy-bungarotoxincomplex to isoelectric focusing. As shown in Fig. 26 whereas the receptor-toxin complex in extracts from the Canton-S strain focused as a single peak with an isoelectric point of 6.60, the receptor-toxin complex from the nicotine-resistant H R strain focused as a single peak at pH 6.69. By running a mixture of extracts

N tc ot i ne - res i st ant H - R slroin

150-

C

%

7

@bacD, %No

1 100 E

M l x t u r e of

Canton-S and H-R

50.-

20

40

60

Fraction

Fig. 26 Isoelectric focusing of t h e acetylcholine receptor-a-bungarotoxin complex from wildtype Canton4 and nicotine-resistant H R strains of Drosophila. Extracts were prepared and run on isoelectric focusing gels: ( a ) Canton-S strain alone; (b) Nicotine-resistant H R strain alone; ( c ) Mixture of extracts of Canton-S and H R strains. From Hall ef al. (1978)

from the two strains on the same gel, Hall et al. (1979) demonstrated that this difference in isoelectric point was due to an alteration in receptor properties and not due to variations between gels. In this way, a hereditary alteration in acetylcholine receptor structure has been identified. Not all nicotine-resistant strains would be expected to affect the structure of the nicotinic acetylcholine receptor. Alterations in components involved in


28 1

nicotine metabolism permeability and membrane environment of the receptor could also conceivably produce nicotine resistance. Of the eight nicotine-resistant strains identified by Hall et al. (1979), three of the stocks (including the resistant HR strain) show shifts in isoelectric point relative to Canton-S. These authors concluded that the iso-electric focusing variants represented either mutations in structural genes coding for the polypeptide subunits of the receptor or mutations in genes coding for enzymes which modified the receptor complex. It has been noted that the mutations identified so far have resulted in changes in the receptor which do not appear to greatly modify its function, since normal locomotor behaviour is observed in the nicotine-resistant strains. Recently using reciprocal crosses to test for X-chromosome linkage the same laboratory has shown that the gene causing the PI shift detected by isoelectric focusing is X-linked (Hall, 1980). When this PI variant has been mapped, the location of a gene affecting receptor structure will be identified. The major nicotine-resistance factor also segregated with the X-chromosome. Genetic mapping experiments could determine whether or not these two phenotypes are the result of a change in the same gene. Hybrid females carrying genetic information for two different forms of the variant polypeptide always revealed material which migrated to an isoelectric point between that of the parental types. The HR locus therefore codes for a structural polypeptide in the a-bungarotoxin-binding complex. Also at least two copies of this polypeptide must be present in the receptor complex. As Hall (1980) has pointed out, mapping the isoelectric focusing point variants will enable the determination of both the number and gene location of loci affecting receptor structure and could also provide information on receptor subunit composition. The structural gene for acetylcholinesterase has been identified on the third chromosome of Drosophita by Hall and Kankel(l976). Dudai (1978) tested acetylcholinesterase-less mutants (both point mutations and deletions covering the locus) for their ['2'I]-a-bungarotoxin-binding activity. This author showed that flies which were heterozygous for mutations in that gene and contained only half the normal acetylcholinesterase activity exhibited normal toxin-binding activity. In addition, pharmacological studies established that a-bungarotoxin did not bind to the active site of acetylcholinesterase and subellular fractionation revealed that the toxin-binding and enzyme activities did not co-purify (Dudai 1978). Furthermore, the fact that deletions covering the acetylcholinesterase locus from both sides did not abolish receptor activity was a strong indication that the genes coding for the toxin-binding receptor of Drosophila were not situated adjacent to the gene for acetylcholinesterase (Dudai, 1978). In a parallel study from the same laboratory Haim et al. (1979) using similar genetic techniques demonstrated

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that the [3H]-quinuclidinyl benzilate binding component could be distinguished from acetylcholinesterase and genes coding for the muscarinic receptor were not located adjacent to the acetylcholinesterase gene. These findings for the muscarinic receptor (Table 13) are of interest in the light of earlier reports that muscarinic ligands bind to peripheral sites on acetylcholinesterase molecules (Kato et al., 1972). In their studies on the [3H]-decamethonium binding component in head extracts of Musca, Tripathi et al. (1979) have compared a wild type with a mutant strain that shows a remarkable insensitivity to poisoning by organophosphates and carbamates. The mutant showed a four fold greater affinity for [3H]-decamethonium binding but the total amount of binding was reduced by seven fold. Thus the initial genetic studies on insect acetylcholine receptors have proved fruitful and the prospects are optimistic for an increased understanding of receptor structure, function and role in behaviour and development.

6.2

RECEPTORS I N DEVELOPMENT

Insects provided experimental material well suited to the analysis of pre- and postembryonic neuronal development. Identifiable neurones are a particularly attractive source of material since it is possible to follow individual cells from birth to maturity. It is therefore possible to ask at what stage in development functional acetylcholine receptors first appear. Also, the ease with which experimental manipulations can be performed early in development enables analysis of neuronal development in the absence of normal synaptic input. The embryonic development of the dorsal unpaired median (DUM) neurones of the grasshopper (Schistocerca nitens) was recently investigated (Goodman et al., 1979; Goodman and Spitzer, 1979). On the seventh day of embryonic development neither the DUM neuroblast nor its progeny showed responses to bath application of acetylcholine. Beginning on day 8, the cell bodies of the older DUM neurones exhibited sensitivity to bath application and iontophoretic application of acetylcholine (Fig. 27). Simultaneously, processes of the older DUM neurones became sensitive to acetylcholine. It appears therefore that soon after the first appearance in the cell membrane of functional acetylcholine receptors, they are distributed over the whole surface of the cell (Goodman and Spitzer, 1979; 1980). The same authors showed that sensitivity to acetylcholine develops whilst the cells are still electrically coupled. The reversal potential (+ 20 mV) and the ionic dependence of the acetylcholine-induced depolarization of the cell body membrane appeared to be unchanged between days 8-1 8 of embryonic development.

D

0

rn

-I

< TABLE 13 [3H]-QNB binding in acetylcholinesterase-deficient mutants. Heads (400/ml) were homogenized in 0.32 M-sucrose and assayed for specific [3H]-QNB binding and acetylcholinesterase activity. Mutants were kept in heterozygous state (homozygous is lethal) over the chromosomal balancer MRS. As seen in the table, the balancer itself had no significant effect on esterase activity or QNB-binding. Values are percentages - mean one standard error with number of experiments in parentheses. From Haim et nl, (1979)

*

Acetylcholinesterase activity Strain

Description

c-s

Wild type

z

rn

['HIQNB binding

Absolute activity (%) Specific activity (%) Absolute activity (%) Specific activity (%)

-I

0 SJ

Heterozyygous for a point mutation in the acetylcholinesterase gene Heterozygous for a point mutation 1(3)m38/MRS in the acetylcholinesterase gene Df(3R)126d/MRS Heterozygous for a small deletion covering the acetylcholinesterase gene Df(3R)ry615/+ Heterozygous for a larger deletion covering the acetylcholinesterase gene + /MRS Control for the balancer chromosome employed in part of the above experiments

100 (22 ? 1 nmol/min/ 10 PI) (1 1)

100 (0.52 ? 0.02 pmol/ min/ mg protein) (11)

100 (2.7 2 0.2 fmol/ 10 p1) (6)

100 (62 2 2 fmol/mg protein) (6)

cn

: z

cn

1(3)m15/MRS

rn

52

*3 (6)

5 3 k 2 (8)

* 2 (8)

87 k 10 (2)

101 ? 11 (4)

97 2 7 (2)

80 2 9 (4)

55?2(6)

43

56 ? 2 ( 6 )

51?6(8)

loo? 12 (2)

105 "20 (4)

61 k 3 (3)

54? 5 (3)

86 5 2 (4)

9 6 ? 3 (3)

103 k 4 (7)

82?4(7)

92?7(7)

77?9(7)

0

-I

cn

N

m W

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284

1

4

10

rnvL 2s

b

13 D A Y I

ACh

GABA

Fig. 27 Chemosensitivity of identified DUMneurones in the grasshopper (Schistocera nitens). ( a ) Response to iontophoretic application of acetylcholine in a day 8 embryo. The diagram

shows the embryonic neuropile of a single segmental ganglion (T3), as viewed from the dorsal surface(anteri0rat top). The DUMneuroblast(MNB)isshown withitspacketofprogeny; those most anterior (oldest) send their axons anteriorly in a median bundle of processes which cross the posterior commissure (PC) and bifurcate near the anterior commissure (AC). The extent of branching of an individual neurone was determined by intracellular injection of Lucifer Yellow. The longitudinal connectives extend into the ventral nerve cords (VNC) and are seen on either side. The lateral neuropile extends (beyond the margin of the figure) into three fibre tracts which become peripheral nerves 3,4 and 5. The responses to applicationof acetylcholine at the points indicated by the arrows were recorded by an intracellular electrode in the cell body. Processes are sensitive to ACh over their whole length. Small vertical or lateral displacements of the iontophoretic electrode abolished the response. (b) Responses to sequential iontophoretic application of acetylcholine and y-aminobutyric acid from a double-barrelled micropipette, recorded with an intracellular electrode in a day 13 embryo. At a resting potential of -55 mV, acetylcholine depolarizes the cell to threshold, eliciting overshooting action potentials, while y-aminobutyric acid hyperpolarizes the cell. When the cell is hyperpolarized by injected current, the response to acetylcholine is larger, but remains subthreshold; the cell is then depolarized by y-aminobutyric acid. From Goodman and Spitzer (1979a)


285

In the same study the first spontaneous synaptic activity (origin of input unknown) was reported in DUM neurones at 15 days, one week after the onset of chemosensitivity. Although causal relations in the sequence of phenotypes in embryonic neurones have yet to be established, Goodman and Spitzer (1979) have pointed out that several events appear to be closely linked. For example, outgrowth of processes and sensitivity to acetylcholine occur at about the same time. A possible regulatory function of acetylcholine receptors in the maintenance of synaptic connections has been proposed based on experiments on amphibian retino-tectal synapses (Freeman 1977). Insect neurones which can be identified during development could provide a direct test of this hypothesis. The role of innervation in the normal development of excitable tissues has been most fully documented in the case of the regulation of the distribution of acetylcholine receptors in vertebrate skeletal muscles developing in vitro andin vivo (cf. Fambrough, 1979). It is established that in response to motor innervation clusters of acetylcholine receptors are formed in the subsynaptic membrane. In the first comparable study of the development of receptors during de novo synapse formation in the central nervous system, Hildebrand and colleagues (1979) have investigated the antennal lobes of the brain of the moth, Manduca sexta. The antennal lobes are well suited to such a study as they appear to have a high concentration of cholinergic synapses, a simple anatomical organization and their development is amenable to experimental manipulation (Sanes and Hildebrand, 1976a, Sanes and Hildebrand 1976b, Sanes et al., 1977, Hildebrand, 1980). The antennal lobes are the first synaptic stations for processing most of the antennal sensory inputs to the central nervous system (Strausfeld, 1976). Both the antennae and the antennal lobes of Manduca develop de novo during adult development, commencing at about the time of the metamorphic moult of the larva to the pupa (Sanes and Hildebrand 1976, Saneset al., 1977; Prescott, et al., 1977). Sensory neurones with cell bodies in the antennae send fibres into the lobes. Antennae and antennal nerves contain acetylcholine. The antennae synthesize and store [14C]acetylcholine whereas several other putative neurotransmitters do not accumulate. Also, the presence of choline acetyltransferase is further evidence that the sensory axons under investigation are cholinergic (Sanes and Hildebrand, 1976b). Levels of acetylcholine, choline acetyltransferase and acetylcholinesterase rise dramatically in the antennal lobes as these sensory (probably cholinergic) axons grow into the lobes through the antennal nerves (Sanes et al., 1977). An ['251]-cu-bungarotoxinbinding activity was shown to develop in the antennal lobes with a time-course different from that of the other cholinergic components, rising steadily throughout metamorphosis (Sanes et al., 1972). This toxin-binding activity was specific

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to nervous tissue and was blocked by a range of cholinergic ligands (Saneset a f . ,1977). The pharmacological properties of this toxin-binding component indicated that it closely resembled the nicotinic acetylcholine receptor characterized by [1251]-a-bungarotoxinbinding in several other insect species (see Section 2 . 3 ) . Antenna1 lobes were deprived of their normal antennal sensory inputs throughout adult development by deafferentation. (Hildebrand et a f., 1979). This was routinely achieved by removal of an antenna from one side of the head, the remaining antenna serving as a control. Following deafferentation, levels of acetylcholine, choline acetyltransferase and acetylcholinesterase were greatly reduced in the antennal lobe but the toxinbinding activity of the lobe was not significantly lowered (Sanesetaf., 1977). Furthermore, the deafferented lobe, although somewhat stunted and ectopic, displayed histological features similar to those of the normal antennal lobe (Hildebrand et a f . , 1979). These same authors showed that deafferentation did not lead to the appearance of toxin- binding sites over other regions of the neuropile or over the cell bodies of neurones in the antennal lobe (Fig. 28). To check that localized toxin-binding activity was not due to the influence of the unoperated side on the contralateral side of the brain bilaterally deafferented animals were investigated. Both deafferented lobes showed the same pattern of toxin-binding noted in the deafferented lobe of a unilaterally deantennated animal. In the complete absence of antennal sensory input to the brain, therefore, the developing antennal lobes nevertheless develop toxin-binding activity in the neuropile region normally destined to receive afferent synaptic inputs (Hildebrand et a f . , 1979). Finally, these authors were unable to detect any affects of deafferentation on the histology or toxin-binding activity of higher-order neuropile regions. Thus, antennal sensory neurones of Manduca appear to undergo normal development following deafferentation as judged by morphological, neurochemical and electrophysiological criteria. The deafferented antennal lobes produce a characteristically organized neuropile and elaborate acetylcholine receptors in the appropriate region of the neuropile. This finding for the innervation of central neurones is in contrast with the situation in vertebrate muscle ( c j Fambrough, 1979). These initial autoradiographic studies reveal a pattern of development in excitable cells that is distinct from that of vertebrate neuromuscular junctions. In the antennal system of the moth Manduca sexta, therefore, there is strong evidence that neurones complete much or all of their development autonomously, independent of their normal synaptic input.


Fig. 28

287

Effects of deaffererltation on [li5]-a-Bgt binding in the antennal lobes of Manduca on the left were taken with bright-field illumination and those on the right, with dark-field illumination. ( a ) Frontal section of antennal lobes from a unilaterally deantennated animal showing normal distribution of toxin-binding sites on the unoperated, control side and dense toxin binding confined to protoglomeruli (pgl) on the deafferented side. ( b ) Same section as that shown in ( a ) . Note the absence of binding over cell bodies (cb). (c) Nearly frontal section through the antennal lobes and subesophageal ganglion (SEG) of a bilaterally deantennated animal showing a-Bgt binding to the condensed protoglomeruli. (d) Same section as that shown in (c). ( e ) Horizontal section of a unilaterally deantennated animal showing a-Bgt binding to the mushroom bodies (mb) in the protocerebrum. The unoperated, control antennal lobe is marked with the AL, but the deafferented lobe is not visible in this plane of section. cf) Dark-field view of same section as that shown in ( e ) . Scale bar = 100 pm. From Hildebrand er a l . (1979)

sexta. Micrographs of toluidine blue stained sections

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288

7 Receptor actions of toxins and insecticides 7.1 7.1.1

RECEPTOR-ACTIVE TOXINS

a-Neurotoxins

Several of the potent snake a-neurotoxins have been tested for receptor activity in insects using both electrophysiological and radiolabelled ligandbinding techniques. Irreversible blockade of cercal-afferent, giantinterneurone synaptic transmission in Periplaneta americana at nanomolar concentrations was noted (Harrow et a f . , 1979; 1980b). Suppression of the depolarization induced by iontophoretic application of acetylcholine to the cell body membrane of the Df motoneurone of Peripfaneta at 5.0 x 1 0 - 8 ~ was also demonstrated (Sattelle et al., 1980). By contrast, DUM neurone cell bodies from adults of Peripfaneta (Sattelleet al., 1980) and embryos of Schistocerca nitens (Goodman and Spitzer, 1979; 1980) were unaffected by (see also Section 4.4.2). In these experia-bungarotoxin at 1.0 X 1 0 - 6 ~ ments, the a-bungarotoxin used corresponded in all cases to the fraction aBgt 2.2 from the venom of Bungarus multicintus (nomenclature of Ravdin and Berg, 1979). Other a-toxins purified from the venom of Bungarus multicintus were ineffective on the acetylcholine-induced depolarization of embryonic DUM neurone cell bodies (Goodman and Spitzer, 1979a, b). These included: aBgt 3.1; aBgt 3.2; aBgt 3.3; aBgt 3.4 (Goodman and Spitzer, 1980). The a-neurotoxin from the Siamese cobra (Naja naja siamensis) was ineffective in blocking the response of DUM neurone cell bodies to iontophoretic application of acetylcholine (Goodman and Spitzer, 1980). An a-toxin from the green mamba (Dendroas viridis) which blocked a vertebrate central cholinergic synapse (Szcepaniak and Miledi, 1975) was also ineffective in blocking the acetylcholine response of DUM neurone cell bodies at 1.0 x ~O-’M (Goodman and Spitzer, 1980). The highly selective nature of the receptor blocking actions of the snake a-toxins could be further exploited in order to characterize in more detail the differences between neuronal acetylcholine receptors of different organisms. 7.1.2 Nereistoxin Nereistoxin (NTX) isolated from the marine annelid Lumbriconereis heteropoda is a potent insect neurotoxin (Nitta, 1934, Sakai, 1964; Narahashi, 1972). Of the variety of synthesized derivatives of NTX, those toxic to insects are the 4-alkylamino-1, 2-dithiolanes and the 2-dimethylamino-l , 3-propane dithiols (Sakai, 1966). Nereistoxin blocks cholinergic synaptic transmission but its mechanism of action is complex.


289

Deguchi et a f . (1971) showed that the toxin reduced the amount of acetylcholine released from presynaptic nerve terminals of neuromuscular junctions. The same authors found that NTX reduced the sensitivity of the postsynaptic nicotinic receptors to applied acetylcholine. In addition to exhibiting antagonistic actions at nicotinic synapses, NTX acted as an agonist at certain vertebrate muscarinic synapses. For example, the toxin both decreased mammalia heart rate and increased salivary gland secretion, both effects being antagonized by atropine (Nitta, 1941). The actions of NTX at both nicotinic and muscarinic receptors of mammals led to the suggestion (Eldefrawi, 1976) that this compound did not act at the ligand binding site of these acetylcholine receptors. Some confirmation of this notion comes from the actions of NTX on the insect central nervous system. Nereistoxin blocked cercal-afferent, giant-interneurone synapses in the sixth abdominal ganglion of Periplaneta (Sakai, 1967; Bettini e f al., 1973; Sattelle and Callec, 1977). Concentrations as low as 5 X ~ O - ' M NTX partly suppressed excitatory postsynaptic potentials in Periplaneta giant interneurones (Sattelle and Callec, 1977; Sattelle, 1977). Nevertheless, a discrepancy was noted between the ability of NTX to suppress transmission at synapses at which nicotinic cholinergic receptors appear to mediate transmission (see Fig. 29) and its ability to inhibit the binding of [1251]-abungarotoxin to Periplaneta extracts. The concentration of NTX that produced an inhibition of 50%of the toxin binding was 5 x 1 0 - 4 (Sattelleetal., ~ 1981b). One possible explanation is that NTX acts on the nicotinic receptor not at the ligand binding site but on the ionophore. The amphibian toxin histrionicotoxin is considered to act in this way at vertebrate neuromuscular junctions (Kato et al., 1975; Eldefrawi et a f . , 1977). However, NTX did not inhibit the binding of ['HI-perhydrohistrionicotoxin to Torpedo membranes rich in nicotinic receptors (Eldefrawi et a f . , 1980). Although its detailed receptor interactions are not resolved, NTX primarily acts as an antagonist at insect and vertebrate nicotinic receptors.

7.2

C H O L I N E R G I C RECEPTORS AS SITES OF INSECTICIDE ACTION

For a limited number of insecticidally active molecules acetylcholine receptors have been proposed as a possible site of action (see Narahashi, 1973; Corbett 1974; Eldefrawi, 1976). The present discussion will consider the potential of specific cholinergic receptors as target sites for insecticides in the light of recent advances in the characterization of insect acetylcholine receptors. Clearly, the demonstration of receptor activity of an insecticide or related compound does not necessarily indicate that the molecule acts primarily at this site. Also, metabolism, permeability barriers and indirect effects of the compound may conspire to produce only a weak correlation

DAVID B. SATTELLE

290

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( I ~ ~ -xI ~. ~6 5 ~ )

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Fig. 29 ( a ) Concentration dependence of inhibition by Nereistoxin of [1Z51]-~-bungarotoxin binding to extracts of both whole flies (Drosophila melnnogaster) and cockroach abdominal nerve cords (Periplaneta americana). Results are expressed as a percentage of toxin bound to extract in the absence of Nereistoxin. Each point is the average of three replicates and vertical bars denote one standard deviation. Concentrations for 50% inhibition of toxin binding are estimated to be 6.6 x lo-% (Drosophila; 0 )and 1.8 X 1 0 - 4 (Periplaneta; ~ 0). ( b )Effects of a range of nereistoxin concentrations on the amplitude of the evoked EPSP recorded from the cercal-nerve, giant-interneurone synapses in the sixth abdominal ganglion of the cockroach Periplaneta americana by means of a sucrose-gap recording technique. Clearly the inhibition by nereistoxin of an ['Z51]-a-bungarotoxin-sensitivereceptor does not fully account for its synaptic blocking action. Modified from Sattelle er al. (1981b)

between insect toxicity and receptor activity even in those cases where there are strong indications of a primary site of action on an acetylcholine receptor. Nevertheless, in approaching a rational design of environmentally more acceptable insecticides it is instructive to test the actions of both currently used and future potential insecticides at specific receptors. Nicotine, one of the earliest commercial insecticides (Corbett, 1974), is a potent cholinergic agonist when applied to insect neurones (see Section 3), and binds both to the nicotinic receptor and the mixed receptor characterized by radiolabelled ligand binding to insect extracts (see Section 2). For example, studies on the mixed receptor of Musca have shown that nicotine binding is inhibited by both optical isomers of nicotine and by toxic nicotinoids but not by non-toxic nicotinods (Eldefrawietal., 1970). The common


291

requirement for toxicity was 3-pyridylmethylamine with a basic amino nitrogen (Yamamoto et al., 1962, 1968; Kamimura et al., 1963). The correlation between toxicity measured as LDSousing houseflies and the blockade of binding of [3H]-muscarone to the mixed receptor was poor possibly as a result of metabolism and barriers to penetration. Furthermore, in studies on Periplanetu it was shown that the receptor actions of nicotine can largely account for its observed effects on cercal-afferent, giant-interneurone synaptic transmission in the sixth abdominal ganglion (Gepner et ul., 1978). So, there is strong evidence that nicotine acts at two cholinergic receptors in insects, one of which appears to have a functional role in synaptic transmission (see Section 2.3). As an insecticide, however, nicotine is largely of historical interest. Cartap (4-N,N-dimethylamino-1, 2-dithiolane) was the first synthetic insecticide based on the structure of a natural toxin and in vivo appears to be metabolized to Nereistoxin (NTX) (Sakai and Sato, 1971). Although most studies have applied NTX directly to insect nerve preparations (see Section 7.1.2), some studies have been performed with cartap. Similar results to those reported for NTX were obtained (Bettini et al., 1973). The bulk of the present generation of insecticides including the organophosphates and carbamates are anticholinesterase agents (cf. Corbett, 1974). They are considered to exert their primary action by retarding the hydrolysis of acetylcholine and thereby prolonging the actions of the neurotransmitter at cholinergic synapses. Nevertheless, in electrophysiological experiments on Electrophorus electroplax, evidence has accumulated that the organophosphate anticholinesterases DFP, paraoxon and phos) pholine can act as receptor antagonists at high (1-8 X 1 0 - 3 ~concentrations (Bartels and Nachmanson, 1969). Eldefrawietal. (1971d) obtained an inhibition of acetylcholine binding to electroplax tissue by similar concentrations ( 1 0 - 4 ~of) the anticholinesterases DFP, Guthoxon, Tetram and 2- (0, S-dimethylthiophosporylimino) 3-ethyl-5-methyl-l , 3-oxazolindine (known as R-16661). The carbamates neostigmine and pyridostigmine also inhibited the binding of acetylcholine to electroplax tissue (Eldefrawi et a1 ., 1972). Since this competitive effect was only noted at high concentrations, Eldefrawi (1976) has suggested that it may result from an electrostatic attraction between the anionic site of the receptor and the positively charged carbamates. It has also been shown that the anticholinesterase edrophonium has a high affinity for peripheral nicotinic receptors (Seifert and Eldefrawi, 1974) which is consistent with earlier electrophysiological evidence of its agonistic actions (Riker, 1953). Thus, in vertebrates, actions as cholinergic receptor agonists and antagonists have been reported for concentrations of anticholineresterases in excess of the concentrations required for enzyme inhibition.

292

DAVID 6.SATTELLE

A number of anticholinesterase insecticides inhibited [3H]-decamethonium binding to housefly brain (Eldefrawiet al., 1971d). These were in order of effectiveness Tetram > Guthoxon > R-16661> DFP. Cholinesterase inhibitors eserine and neostigmine also inhibited the binding of [ '251]-a-bungarotoxin to extracts of Drosophila. In these studies I,,, values of 1.0 x 1 0 - 5 ~were reported for eserine (Schmidt-Nielsen et al., 1977) and values of 2.0 x 1 O W s were ~ reported for neostigmine (Schmidt-Nielsen et a!., 1977). It may be, therefore, that certain anticholinesterase insecticides owe some of their toxicity to interactions with acetylcholine receptors (Eldefrawi et al., 1971d; Jones et al., 1979). Recent studies on isothiocyanates as potential insecticides by Baillie and collaborators (1975) have shown their considerable potency as choline acetyltransferase inhibitors. When applied to the isolated abdominal nerve cord of Periplaneta at concentrations higher than those required to inhibit the enzyme in vitro, postsynaptic actions consistent with those of a cholinergic agonist were recorded (Sattelle and Callec, 1977). A good correspondence was noted between the postsynaptic blocking actions of 2-isothiocyanato-ethyltrimethylammoniumiodide and its ability to inhibit [1251]-a-bungaratoxinto extracts of Periplaneta nerve cords (Fig. 30). Although this particular molecule of the series of isothiocyanates synthesized is not a likely candidate insecticide because of its charge and water solubility, it is possible that related compounds in the series may owe part of their toxic action to a cholinergic receptor action in addition to an inhibitory action on the enzyme choline acetyltransferase. In 1976, Bigg and Purvis reported that a range of muscarinic agonists showed acaricidal activity against both organophosphate resistant and susceptible strains of the tick Boophilus microplus. In the case of oxotremorine the compounds were and 1-(4-Dimethylaminobut-2-ynyl)pyrrolid-2-one, more effective on the susceptible strain than nicotine. Several muscarinic agonists were active against mites but were completely inactive against the following insects: Musca dornestica (adults), Aedes aegyptii (larvae), and Lucilia pericata (larvae). Oxotremorine at 9 x 10% inhibited 50% of the binding of [3H]-quinuclidinylbenzilate to Drosophila extracts (Haim et a1., 1979). It is of interest to note that a much greater abundance of nicotinic receptors compared to muscarinic receptors has been reported in extracts of insect CNS (Dudai and Ben Barak, 1977; Haimetal., 1979). The potency of muscarinic ligands on ticks and mites may point to fundamental biochemical differences between these two groups of arthropods. Thus some differences between acetylcholine receptors of insects and other organisms have emerged (Section 5 ) . Also, evidence presented above shows that acetylcholine receptor ligands show differential toxicity to differ-


-

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Fig. 30 ( a ) Concentration dependence of inhibition by 2-isothiocyanatoethyltrimethylammonium iodide of [1251]-a-bungarotoxinbinding to extracts of both whole flies (Drosophila melarrogaster) and cockroach abdominal nerve cords (Periplaneta americana). Each point is the average of three replicates and vertical bars denote one standard deviation. Concentrations for 50% inhibition of toxin binding are estimated to be 6.9 x 1 0 - 6 ~ (Drosophila; 0) and 1.6 x 1 0 - 5(Periplunetu; ~ 0 ) .(b) Effects of various isothiocyanate concentrations on the amplitude of the evoked EPSP recorded from the cercal-nerve, giant interneurone synapses in the terminal abdominal ganglion of P. americana using a sucrose-gap recording technique. Concentration for 50% suppression of the EPSP is estimated to be 2.6 X 1 0 - 5 ~Inset . shows the EPSP recorded in normal saline (100%). From Gepner et al. (1978)

ent groups of arthropods. Such differences might conceivably be exploited in the future design of pesticides.

8 Conclusions

Insects have provided material well suited to the investigation of CNS acetylcholine receptors. The central nervous tissues of these organisms have proved amenable to both radiolabelled ligand-binding studies and singlecell electrophysiology of identifiable neurones. Binding studies have resulted in the characterization in vitro of three putative acetylcholine recognition sites each with a distinct pharmacological specificity. Of these

294

D A V I D B . SATTELLE

three putative acetylcholine receptors defined biochemically, the a-bungarotoxin-sensitive receptor has been most fully investigated. The ['251]-a-bungarotoxin-binding components characterized from Drosophila melanogaster (heads), Musca domestica (heads), Periplaneta americana (abdominal nerve cords) and Manduca sexta (brain) are indistinguishable in their pharmacological properties. A highly purified form of the solubilized receptor has been prepared from Drosophila melanogaster which maintains the pharmacological profile of the membrane-bound receptor. A physiological role has been established for this receptor at synapses between cercal mechanoreceptor afferent neurones and giant interneurones (GI 2 and GI 3) in the sixth abdominal ganglion of the cockroach Periplaneta americana. The cell body membrane of the coxal depressor motoneurone (Df) of Periplaneta americana also contains a-bungarotoxin-sensitive acetylcholine receptors. Pharmacological differences have emerged between this insect receptor and the nicotinic receptor of vertebrate muscle and electroplax tissue. The insect receptor is, for example, much less sensitive to decamethonium and carbamylcholine. Also the receptors mediating cercal-afferent, giantinterneurone synaptic transmission are much more sensitive to a-bungarotoxin and less sensitive to hexamethonium than the nicotinic receptors of vertebrate autonomic ganglia. Two other putative insect acetylcholine receptors both of which are insensitive to a-bungarotoxin have been investigated by radiolabelledligand binding methods. A putative receptor in Drosophila heads characterized by its high affinity for [3H]-quinuclidinyl benzilate closely resembles vertebrate muscarinic receptors in its pharmacological properties. A putative receptor exhibiting a pharmacological profile quite different from any acetylcholine receptor so far described has been characterized in heads of Musca dornestica. This putative receptor which has been highly purified is present at an unusually high density and is very readily solubilized when compared to most other membrane receptors studied. Caution is needed in interpreting these two binding components as true receptors until physiological evidence for a functional role is available. Nevertheless abungarotoxin-insensitive acetylcholine responses have been reported for the cell body membranes of dorsal unpaired median (DUM) neurones of Schistocera nitens and Periplaneta americana . Further studies are needed to determine the detailed pharmacological specificity of these toxin-insensitive acetylcholine responses. Insect material is particularly well suited for developmental and genetic approaches to receptor biology. Using ['251]-a-bungarotoxin as a receptor probe, work on Manduca sexta has provided the first analysis of the development of acetylcholine receptors duringde novo synapse formation in


295

the central nervous system. A pattern of development has emerged quite different from that of the vertebrate neuromuscular junction. Acetylcholine receptors on insect neurones appear to cluster and develop normally in the absence of presynaptic inputs. Also, studies on acetylcholine receptors of embryonic DUM neurones of Schistocera nitens have shown that the oldest progeny of the DUM neuroblast become sensitive to acetylcholine at day 8 of embryonic life. On this day, both the cell body and processes acquire sensitivity to acetylcholine indicating that functional receptors are distributed over the whole surface of the cell soon after they first appear. Using these embryonic neurones it may be possible to determine the distribution and properties of acetylcholine receptors on an identified neurone during synapse formation. Genetic studies offer another approach to the study of receptor biology for which insect material is supremely well suited. Hereditary changes in acetylcholine receptor structure (detected as isoelectric point (PI) variants) have been reported in nicotine-resistant strains of Drosophila melanogaster. The gene responsible for this structural change is on the X-chromosome. Gene mapping techniques could also provide information on receptor subunit composition. In addition temperature-sensitive mutants in which the receptor is active, or not, depending upon the ambient temperature should be useful for determining the role of receptors in behaviour and in development. Arising from genetic studies on Drosophila is the finding that the gene causing the PI shift and the gene controlling a major resistance factor are both located on the X-chromosome. These two phenotypes may result from a change in the same gene. If the mechanism of nicotine-resistance does involve a change in receptor structure this would be of considerable interest in view of the recent demonstrations that several insecticidally active molecules are receptor-active. The molecular basis of one type of insecticide resistance would then be directly accessible to experimental analysis.

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Aziz, S. A. and Eldefrawi, M. E. (1973). Cholinergic receptors of the central system of insects. Pestic. Biochem. Physiol. 3, 168-174 Bacon, J. P. and Altman, J. S. (1977). A silver intensification method for cobalt-filled neurones in wholemount preparations. Brain Res. 138, 359-363 Baillie, A. C., Corbett, J. R., Dowsett, J. R., Sattelle, D. B. and Callec, J.-J. (1975). Inhibitors of choline acetyltransferase as potential insecticides. Pestic. Sci. 6, 645-653 Baillie, A. C., Corbett, J. R. and Sharpe, T. M. (1978). The synthesis of potential insecticides designed to bind to the acetylcholine receptor. Pestic. Sci. 9, 1-6 Balerna, M., Fossel. M., Chicheportiche, R., Romey, G. and Lazdunski, M. (1975). Constitution and properties of axonal membranes of Crustacean nerves. Biochemistry 14, 5500-551 1 Barker, D. L., Herbert, E., Hildebrand, J. G. and Kravitz, E. A. (1972). Acetylcholine and lobster sensory neurones. J. Physiol. 226, 205-229 Barlow, H. B. (1964). In “Introduction to Chemical Pharmacology”, 2nd edn, p. 134. Methuen, London Barrantes, F. J., Changeux, J.-P., Lunt, G. G. and Sobel, A. (1975). Differences between detergent-extracted acetylcholine receptor and “cholinergic proteolipid”. Nature, Lond. 256, 325-327 Bartels, E. and Nachmanson, D. (1969). Organophosphate inhibitors of acetylcholine receptor and -esterase tested on the electroplax. Arch. Biochem. Biophys. 133, 1-30 Ben-Barak, J. and Dudai, Y. (1979). Cholinergic binding sites in rat hippocampal formation: properties and ontogenesis. Brain Res. 166, 245-257 Bettini, S., D’Ajello, V. and Maroli, M. (1973). Cartap activity on the cockroach nervous and neuromuscular transmission. Pestic. Biochem. Physiol. 3, 199-205 Biesecker, G. (1973). Molecular properties of the cholinergic receptor purified from Electophorus electricus. Biochemistry 12, 4403-4409 Bigg, D. C. H. and Purvis, S. R. (1976). Muscarinic agonists provide a new class of acaricides. Nature, Lond. 262, 220-222 Birdsall, N. J. M., Burgen, A. S. V., Hiley, C. R. and Hulme, E. C. (1976). Binding of agonists and antagonists to muscarinic receptors. J. Supramol. Struct. 4, 367-371 Birdsall, N. J. M., Burgen, A. S. V. and Hulme, E. C. (1978). The binding of agonists to brain muscarinic receptors. Mol. Pharmacol. 14, 723-736 Birdsall, N. J. M. and Hulme, E. C. (1976). Biochemical studies on muscarinic acetylcholine receptors. J. Neurochem. 27, 7-16 Birks, R. and MacIntosh, F. C. (1961). Acetylcholine metabolism of a sympathetic ganglion. Can. J. Biochem. Physiol. 39, 787-827 Blackman, J. G., Gauldie, R. W. and Milne, R. J. (1975). Interaction of competitive antagonists: the anti-curare action of hexamethonium and other antagonists at the skeletal neuromuscular junction. Br. J. Pharmacol. 54, 91-100 Boistel, J. (1968). The synaptic transmission and related phenomena in insects. In “Advances in Insect Physiology” (Eds J. W. L. Beament, J. E. Treherne and V. B. Wigglesworth) 5, 1-64. Academic Press, London and New York Boistel, J. and Coraboeuf, E. (1954). Potentiels de membrane et potentiels d’action de nerf d’insecte receuillis ?I I’aide de microelectrodes intracellulaires. C.R . Acad. Sci. Paris 238, 21 16-2118 Briley, M. S. and Changeux, J.-P. (1978). Recovery of some functional properties of the detergent extracted cholinergic receptor protein from Torpedo marmorata after reintegration into a membrane environment. Eur. J. Biochem. 84,429-439


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Brown, D. A. and Fumagalli, L. (1977). Dissociation of a-bungarotoxin binding and receptor block in the rat superior cervical ganglion. Brain Res. 129, 165-168 Burden, S. J., Hartzell, H. C. and Yoshikami, D. (1975). Acetylcholine receptors at neuromuscular synapses: phylogenetic differences detected by snake a-bungarotoxins. Proc. natn. Acad. Sci. USA 72. 3245-3249 Burgen, A. S. V. and Hiley, C. R. (1975). The use of an alkylating antagonist in investigating the properties of muscarinic receptors. In “Cholinergic Mechanisms” (Ed. P. G. Waser) pp. 381-385. Raven Press, New York Burgen, A. S. V., Hiley, C. R. and Young, J. M. (1974a). The binding of [3H]propylbenzilylcholine mustard by longitudinal muscle strips from guinea pig small intestine. Br. J. Pharmacol. 50, 145-151 Burgen, A. S. V., Hiley, C. R. and Young, J. M. (1974b). The properties of muscarinic receptors in mammalian cerebral cortex. Br. J. Pharmacol. 51, 279-285 Bursztajn, S. and Gershon, M. D. (1977). Discrimination between nicotinic receptors in vertebrate ganglia and skeletal muscle by alpha-bungarotoxin and cobra venoms. J. Physiol. Lond. 269, 17-3 1 Cajal, S. R. and Sanchez y Sanchez, D. (1915). Contribuctional conocimiento de 10s centros nerviosos de 10s insectos. Parte I. Retina y centros opticos. Trab. Lab. Invest. Biol. Univ. Madr. 13, 1-168 Callec, J.-J. (1972). fitude de la transmission synaptique dans le systkme nerveux central d’un insecte (Periplaneta americana ). Th2se d‘Etat Rennes, 323 pp. CNRS No. A 0 7165 Callec, J.-J. (1974). Synaptic transmission in the central nervous system of insects. In “Insect Neurobiology” (Ed. J. E. Treherne) pp. 119-178. North-Holland, Amsterdam and New York Callec, J.-J. and Boistel, J. (1967). Les effets de I’acetylcholine aux niveaux synaptique et somatique dans le cas du dernier ganglion abdominal de la Blatte, Periplaneta americana. C. R . Se‘ances SOC.Biol. Paris 161, 442-446 Callec, J.-J. and Boistel, J. (1971). Further evidence for ACh transmission in the cockroach central nervous system studied at the unitary level. Proc. XXV. Int. Congr. IUPS. Munich 9 , 9 5 Callec, J.-J., Guillet, J. C . , Pichon, Y. and Boistel, J. (1971). Further studies on synaptic transmission in insects. 11. Relations between sensory information and its synaptic integration at the level of a single giant axon in the cockroach.J. exp. Biol. 55, 123-149 Callec, J.-J. and Sattelle, D. B. (1973). A simple technique for monitoring the synaptic actions of pharmacological agents. J. exp. Biol. 59, 725-738 Callec, J.-J., Sattelle, D. B., Hue, B. and Pelhate, M. (1980). Central synaptic actions of pharmacological agents in insects: oil-gap and mannitol-gap studies. In “Insect neurobiology and pesticide action” pp. 93-100. Society of Chemical Industry, London Camhi, J. M. (1976). Non-rhythmic sensory inputs: influence on locomotory outputs in Arthropods. In “Neural Control of Locomotion” (Eds R. M. Herman, S. Grillner, P. S. G. Stein and D. G. Stuart) pp. 561-589. Plenum Press, New York Carbonell, C. S. (1948). The thoracic muscles of the cockroach, Peripluneta americana. Smithson. misc. Colls. 107, 1-23 Carbonetto, S. T., Fambrough, D. M. B. and Muller, K. J. (1978). Nonequivalenceof a-bungarotoxin receptors and acetylcholine receptors in chick sympathetic neurons. Proc. Natn. Acad. S h . USA 75, 1016-1020

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Smallman, B. N. (1975). Synthesis of acetylcholine in the blowfly (Calliphora erythrocephala). Pestic. Biochem. Physiol. 5, 170-183 Smith, D. S. and Treherne, J. E. (1965). The electron microscopic localization of cholinesterase activity in the central nervous system of an insect Periplaneta americana. J . Cell Biol. 26, 445 -465 Snyder, S. H. and Bennett, J. P. Jr (1976). Neurotransmitter receptors in the brain: biochemical identification. Ann. Rev. Physiol. 38, 153-175 Snyder,S. H.,Chang, K. J.,Kuhar,M.J. and Yamamura, H. I. (1975). Biochemicalidentificationof the mammalianmuscariniccho1inergicreceptor.Fedn.Proc. 34,191 5 -1 92 1 Sobel, A., Heidmann, T., Hoffler, J. and Changeux, J.-P. (1977). Distinct protein components from Torpedo marmorata membranes carry the acetylcholine receptor site and the binding site for local anaesthetics and histrionicotoxin. Proc. Natn. Acad. Sci. USA 75, 510-514 Strausfeld, N. J. (1976). “Atlas of an Insect Brain”. Springer-Verlag, Berlin Suga, N. and Katsuki, Y. (1961) Pharmacological studies on the auditory synapse in a grasshopper. J . exp. Biol. 38, 759-770 Szczepaniak, A. C. (1974). Effect of a-bungarotoxin and Dendroaspis neurotoxins on acetylcholine responses of snail neurones. J . Physiol. Lond. 241, 55-56P Thomas, W. E,., Brady, R. N. and Townsel, J. G . (1978). A characterization of a-bungarotoxin-binding in the brain of the horseshoe crab, Limulus polyphemus. Arch. Biochem. Biophys. 187,53-60 Tobias, J. M., Kollross, J. J. and Savit, J. (1946). Acetylcholine and related substances in the cockroach fly and crayfish and the effect of DDT. J . Cell Comp. Physiol. 28, 159-182 Treherne, J . E. and Smith, D. S. (1965a). The penetration of acetylcholine into the central nervous tissue of an insect Periplaneta americana. J . exp. Biol 43, 13-21 Treherne, J. E. and Smith, D. S. (1965b). The metabolism of acetylcholine in the intact central nervous system of an insect Periplaneta americana. J . exp. Biol. 43, 441-454 Triggle, D. J. and Triggle, C. R. (1976). “Chemical Pharmacology of the Synapse”. Academic Press, London Tripathi, R. K., Tripathi, H. L. and O’Brien (1979). Properties and affinity purification of the mixed-type putative acetylcholine receptor from wild and a mutant strain of house flies. Biochim. Biophys. Acta 586, 624-631 Twarog, B. M. and Roeder, K. D. (1956). Properties of the connective tissue sheath of the cockroach abdominal nerve cord. Bzol. Bull. 111, 278-286 Twarog, B. M. and Roeder, K. D. (1957). Pharmacological observations on the desheathed last abdominal ganglion of the cockroach. Ann. Ent. SOC. Amer. 50, 231 -236 Tyrer, N. M. and Bell, E. M. (1974). The intensification of cobalt-filled neuron profiles using a modification of the Timm’s silver sulphide method. Brain Res. 73, 151-154 Villegas, J. (1975) Characterization of acetylcholine receptors in the Schwann cell membrane of the squid nerve fibre. J . Physiol. Lond. 249, 679-689 Volle, R. L. and Koelle, G. B. (1975) Ganglionic stimulating and blocking agents. In “The Pharmacological Basis of Therapeutics” (Eds L. S. Goodman and A. Gilman) 5th edn, pp. 565-574. Macmillan, London Walker, R. J. and Hedges, A. (1968). The effect of cholinergic antagonists on the response to acetylcholine, acetyl-P-methylcholine and nicotine of neurones of Helix aspersn. Comp. Biochem. Physiol. 23, 979-989

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Walker, R. J. and James, V. A. (1978). The action of putative transmitters and related compounds on neurones in the abdominal ganglion of the horseshoe crab Limulus polyphemus Neuropharmac. 17, 765-769 Walker, R. J. and Kerkut, G. A. (1977). The actions of nicotinic and muscarinic cholinomimetics and a series of choline esters on two identified neurones in the brain of Helix aspersa. Comp. Biochem. Physiol. 56C, 179-187 Walker, R. J., Hedges, A. and Woodruff, G. N. (1968). The pharmacology of the neurones of Helix aspersa. Symp. Zool. SOC.lond. 22, 33-74 Walker, R. J., James, V. A., Roberts, C. J. and Kerkut, G. A. (1980). Neurotransmitter receptors in invertebrates. In “Receptors for Neurotransmitters, Hormones and Pheromones of Insects” (Eds D. B. Sattelle, L. M. Hall and J. G. Hildebrand) pp. 41 -52. Elsevier/North-Holland, Amsterdam Waser, P. G. (1960). The Cholinergic Receptor. 1. Pharm. Pharmac. 12, 577-594 Wastek, G. J. and Yamamura, H. I. (1978). Binding of [3H]-quinuclidinyl benzilate to human cerebral cortex. Mol. Pharmacol. 14, 768-780 Weber, M. and Changeux, J.-P. (1974). Binding of Naja nigricollis [3H]-a-toxin to membrane fragments from Electrophorus and Torpedo electric organs. Mol. Pharmacol. 10, 15-34 Wiersma, C. A. G., Furshpan, E. and Florey, E. (1953). Physiological and pharmacological observations on muscle receptor organs of the crayfish Cambarus clarkii Girard. J . Exp. Biol. 30, 136-150 Wilson, S. P. and Kirschner, N. (1977). The acetylcholine receptor of the adrenal medulla. J . Neurochem. 28, 687-695 Witzemann, V. and Raftery, M. (1978). Ligand-binding sites and subunit interactions of Torpedo californica acetylcholine receptor. Biochemistry 17,3598-3603 Woodruff, G. N., Walker, R. J. and Newton, L. C . (1971). The action of some muscarinic and nicotinic agonists on the Retzius cells of the leech. Gen. comp. Pharrnacol. 2, 106-1 17 Yamamoto, I., Kamirnura, H., Yamamoto, R., Sakai, S. and Goda, M. (1962). Studies on nicotinoids as an insecticide. I. Relation of structure to toxicity. Agr. Biol. Chem. 26, 709--716 Yamamoto, I., Soeda, Y., Kamimura, H. and Yamamoto, R. (1968). Studies on nicotinoids as an insecticide. VII. Cholinesterase inhibition by nicotinoids and pyridylalkylamines - its significance to mode of action. Agr. Biol. Chem. 32, 1341 Yamamura, H. I., and Snyder, S. H. (1974a). Muscarinic cholinergic binding in rat brain. Proc. Natn. Acad. Sci. USA 71, 1725-1729 Yamamura, H. I. and Snyder, S. H. (1974b). Muscarinic cholinergic receptor binding in the longitudinal muscle of the guinea pig ileum with [3H]-quinuclidinyl benzilate. Mol. Pharmacol. 10, 861 -867 Yamamura, H. I. and Snyder, S. H. ( 1 9 7 4 ~ )Postsynaptic . localization of muscarinic cholinergic receptor binding in the rat. Brain Res. 78, 320-326 Yamamura, H. I., Kuhar, M. J., Greenberg, D. and Snyder, S. H. (1974a). Muscarinic cholinergic receptor binding: regional distribution in monkey brain. Brain Res. 66,541 -546 Yamamura, H. I., Kuhar, M. J. and Snyder, S. H. (1974b). In vivo identification of muscarinic cholinergic receptor binding in rat brain. Brain Res. 80, 170-176 Yamamura, H. I., Enna, S. J. and Kuhar, M. J. (Eds) (1978). “Neurotransmitter Receptor Binding”. Raven Press, New York Yamasaki, T. and Narahashi, T. (1958). Synaptic transmission in the cockroach. Nature, Lond. 182, 1805-1806


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Yamasaki, T. and Narahashi, T. (1960). Synaptic transmission in the last abdominal ganglion of the cockroach. J. Insect. Physiol. 4, 1-13 Yarowsky, P. J. and Carpenter, D. 0. (1978). A comparison of similar ionic responses to y-Aminobutyric acid and Acetylcholine. J. Neurophysiol. 41, 531-541 Yavari, P., Walker, R. J. and Kerkut, G. A. (1979). The pA2 values of cholinergic antagonists on identified neurons of the snail Helix aspersa. Comp. Biochem. Physiol. 63C, 39-52 Yazulla, S. and Schmidt, J. (1977). Two types of receptors for a-bungarotoxin in the synaptic layers of pigeon retina. Brain Res. 138, 45-57 Zeimal, E. V. and Vulfius, E. A. (1968). The action of cholinomimetics and cholinolytics on Gastropod neurons. In “Neurobiology of Invertebrates” (Ed. J. Salanki) pp. 255-265. Acad. Kiado, Budapest


Biogenic Arnines in the Insect Nervous System Peter D. Evans A. R. C. Unit of Invertebrate Chemistly and Physiology, Department of Zoology, Cambridge, UK

I 2

3 4

5

6

7

Introduction 318 The distribution of biogenic amines in the insect nervous system 320 2.1 Quantitative studies on biogenic amine distribution - an historical perspective 321 2.2 Cellular localization of biogenic amines 330 2.3 Subcellular location of biogenic amines 346 Metabolic studies on biogenic amines 349 3.1 Synthesis of biogenic arnines 350 3.2 Inactivation of biogenic amines 356 Octopamine and the dorsal midline neurones 365 4.1 Are the individual cells of the dorsal midline group uniquely identifiable? 367 4.2 The octopaminergic nature of D U M cells 373 4.3 DUMETi and the modulation of a myogenic rhythm 376 4.4 DUMETi and the potentiation of neuromuscular transmission 381 4.5 Studies on the terminal abdominal ganglion 387 4.6 Functions of D U M neurones and parallels with other systems modulated byamines 389 Biogenic amines and firefly light organs 394 5.1 Innervation of light organs 394 5.2 Pharmacology of light responses 397 5.3 Mode of action of neurotransmitter 400 5.4 Future studies on firefly light organs 402 Dopamine and insect salivary glands 402 6.1 Catecholamine distribution and innervation pattern 403 6.2 Effects of nerve stimulation and application of biogenic amines 406 6.3 Further studies on dopaminergic transmission in insect salivary glands 412 Biogenic amines and the insect heart 414 7.1 Innervation pattern 414 7.2 Biogenic amine distribution 417 7.3 Pharmacology of responses to biogenic amines 418 317

P E T E R D. EVANS

318

8 Biogenic amines in the control of gut muscle 420 8.1 Innervation of gut muscle 421 8.2 Pharmacological studies on gut muscle 422 9 Amines and neurohaemal organs 426 9.1 Corpora cardiaca 427

9.2 Median neurohaemal organs 429 9.3 Function of aniines in neurohaemal organs 433 10 Amine-stimulated adenylate cyclase activity 436 10.1 Studies on insect preparations 437 10.2 Comparisons with other invertebrate and vertebrate preparations 442 10.3 Cellular location of responses 444 10.4 Functional role in insects 444 11 Conclusions 445 Acknowledgements 449 References 449

1 Introduction

In the insect nervous system the presence of the catecholamines, dopamine and noradrenaline, and the indolalkylamine, 5-hydroxytryptamine (5-HT), has been known for some time and the presence of the phenolamine, octopamine, has recently been demonstrated (see Fig. 1 for structures). The

5 -HYDROXY TRYPTAMINE

H

w

ADRENALINE

Fig. 1 Structures of some biogenic amines

functional roles played by these amines in the insect nervous system have remained obscure, however, due to a lack of correlation between biochemical and histochemical data on the one hand, and physiological and pharmacological information from amine-containing neurones on the other. Several previous reviews have critically examined the evidence for the role of biogenic amines as potential neurotransmitters in the insect nervous system (Pitman, 1971; Murdock, 1971; Klemm, 1976). Relevant information is also collated in certain sections of other reviews (Davey, 1964;

B l O G E N l C AMINES I N THE INSECT NERVOUS SYSTEM

319

Kerkut, 1973; Gerschenfeld, 1973; Pichon, 1974; Axelrod and Saavedra, 1977; Hicks, 1977; Robertson and Juorio, 1976; Lafon-Cazal, 1978). The aim of the present review is to take a wider view of biogenic amines in the insect nervous system and to assess critically the evidence for their roles as neurotransmitters, neuromodulators and neurohormones. The term neurotransmitter will be used to define a chemical messenger that is released at a specialized synaptic structure and then diffuses across a narrow synaptic cleft to act on a specialized region of membrane on a post-synaptic cell. This cell may be another neurone, a muscle cell or a specialized gland cell. The specificity in this form of communication is dependent on the anatomical distribution of the synapses made by the pre-synaptic cell. The term neurohormone will be used to refer to chemical messengers released from the nervous system into the circulatory system of the insect, but will be modified to “local neurohormone” to describe the release of a messenger within a localized region of the nervous system or a particular end organ. In the case of neurohormones, both general and local, the chemical message is not necessarily confined to a single anatomically apposed post-synaptic cell, but can affect many post-synaptic cells, the specificity of the system relying on the distribution of cells with appropriate receptors. The currently favoured term “neuromodulator” will be used to designate the special case of a neurohormone that either changes the quality of the information being passed through a conventional synapse, or changes the spontaneous activil. of a receptive neurone or muscle cell. In the case of neurohormones, lo,d neurohormones, and neuromodulators, no specific synaptic structures are present and release often occurs from “blindly-ending neurosecretory terminals”. The above definitions obviously represent the extremes of a continuum, making it difficult to define absolutley the point where one category finishes and another starts. The terms, however, appear to convey useful distinctions. This review will attempt to answer the following questions. Where are biogenic amines located in the insect nervous system? How are they synthesized and inactivated? What physiological roles d o they perform and how do they bring about their effects? Particular emphasis will be placed on the correlation of biochemical, physiological, pharmacological and anatomical information from systems containing identified aminergic neurones. Attempts will be made to point out instances where data from one or more of the above approaches is either missing o r conflicts with that from another and experiments will be suggested to resolve these points. When appropriate, the function of biogenic amines in the insect nervous system will be compared with that in the vertebrate nervous system. In this context it is appropriate to summarize here what is known of the role of biogenic amines as chemical messengers in the vertebrate nervous system.

320

PETER

D. E V A N S

In vertebrates, biogenic amines serve as neurotransmitters, and neuromodulators, and also as true hormones, which are released into t h e circulatory system. Dopamine, noradrenaline and 5-HT act as neurotransmitters in the central nervous system (KrnjeviC, 1974) whilst recent evidence suggests that noradrenaline and 5-HT may also serve as central neuromodulators (Dismukes, 1977a). In addition, noradrenaline is released from the terminals of the peripheral sympathetic nervous system where it acts as a transmitter or local neurohormone (Smith, 1973). The third well known member of the catecholamine family, adrenaline, is a circulatory hormone released from the cells of the adrenal medulla and has widespread actions throughout the body (Douglas and Rubin, 1963; see also Berridge, 1975). Evidence is also accumulating for an adrenaline based neural pathway in the vertebrate brain, but the extent of this pathway appears small compared to the corresponding dopamine and noradrenaline pathways, and its physiology and function have yet to be investigated (Moore and Bloom, 1979). Recently attention has also been focussed on the possible role of the phenolamines, octopamine and tyramine, as neurotransmitters or neuromodulators in the vertebrate nervous system, but little positive evidence has been presented to date (see Boulton, 1976; Hicks, 1977; Evans, 1978a; Hicks and McLennan, 1978a, b). Finally, it is worthwhile to re-emphasize one of the major themes of the present review. In contrast to the vertebrate nervous system, the insect nervous system, along with those of many other invertebrates, often presents the researcher with the advantage of working with single, physiologically identified aminergic neurones. This advantage arises from the large size of many of the neuronal somata, which can be isolated for biochemical studies, and also from the relative ease with which the same individual neurone can be identified from one preparation to the next (e.g. for 5-HT see McAdoo, 1978; Qsborne, 1978; for dopamine see Berry and Pentreath, 1978; for octopamine see Evans, 1978b; for histamine see Weinreich, 1978). But, for the most part these attributes have not yet been fully exploited in studies of biogenic amines in the insect nervous system. It is hoped that this review will serve to focus attention on potentially rewarding areas of future research.

2

The distribution of biogenic amines in the insect nervous system

Studies on the distribution of biogenic amines in the insect nervous system have attempted to answer a number of questions of increasing complexity. Initially the questions asked were, which amines are present, in which ganglia are they contained, and how much of each is present? Subsequently,

BlOGENlC A M l N E S I N THE INSECT NERVOUS SYSTEM

321

questions were raised about which cells in the ganglia contained the amines and where they were localized at a subcellular level. More recently attention has centered on the functional roles fulfilled by the various amines present in identified aminergic neurones. In the present section, information on the cellular and subcellular distribution of biogenic amines in the insect nervous system will be reviewed from an historical viewpoint. Throughout the section attempts will be made to examine the extent to which information on the distribution of biogenic amines has contributed to an understanding of their different functional roles. 2.1

QUANTITATIVE STUDIES O N BIOGENIC AMINE DISTRIBUTION - A N HISTORICAL PERSPECTIVE

2.1.1 Fluorescence-based assays The use of fluorescence-based assays has provided evidence for the presence of dopamine, noradrenaline and 5-HT in the insect nervous system. Early studies on biogenic amines in insects were made on homogenates of whole insects using relatively insensitive fluorescent techniques, e.g. Wense (1939); Ostlund(1953); Dresseetal. (1960). These studiessuggested that a wide variety of insect species contained dopamine, noradrenaline and adrenaline, but conflicting results were obtained by Gregerman and Wald (1952) and von Euler (1961) who were unable to detect any adrenaline. The results of the above studies on whole insects tell us very little about the aminergic content of the insect nervous system for several reasons. First, dopamine and its derivatives are found in insect cuticle, where they are thought to be involved in the tanning process (Sekeris and Karlson, 1966; Andersen, 1979). Second, catecholamines occur in the venom glands of some Hymenoptera (Owen, 1971; Ishay et al., 1974) and third, insect haemocytes contain many enzymes capable of synthesising and metabolizing biogenic amines (see section 3 present review). One interesting result, that has emerged from studies on catecholamine levels in homogenates of whole insects, comes from the genetic selection experiments of Tunnicliff et al. (1969). These authors selected two behakioural strains of Drosophila for locomotor activity. They found that noradrenaline levels were highest in the active strain and lowest in the inactive, whilst dopamine levels were highest in the inactive strain m d lowest in the active strain. Unselected control strains had intermediate levels of both amines. These results are obviously open to all the criticisms outlined above and tell us nothing about changes in amine levels within the nervous system, but they suggest an interesting area for research on the role of catecholamines in the modulation of behaviour, especially if more precise techniques for the localization of catecholamines in the nervous system are employed.

PETER D. EVANS

322

The advent of the Falck-Hillarp histochemical technique for the localization of catecholamines and indolalkylamines (see Falck and Owman, 1965) led to the demonstration of the presence of catecholamines in the insect nervous system. Frontali and Norberg (1966) and Frontali (1968) were able to show the presence of a primary catecholamine (either dopamine or noradrenaline -it was not resolved which), but not adrenaline, in the central body and p-lobes of the corpora pedunculata (mushroom bodies) of the cockroach, Periplaneta americana (see Fig. 2 for details of anatomy). The GC

1

MC

LC

I

I

Fig. 2 Diagram of the brain (cerebral ganglia) of the adult cockroach, viewed from the front, to show position of regions of highly structured neuropile. The basic arrangement of a typical globuli cell is illustrated schematically within each corpus pedunculatum. The various regions of the corpus pedunculatum are labelled as follows: A, a-lobe; B, P-lobe; P, pedunculus; MC, medial calyx; LC, lateral calyx; GC, globuli cells. Other labelling: PROTO, protocerebrum; DEUTO, deutocerebrum; TRITO, tritocerebrum; PI, pars intercerebralis: PB, protocerebral bridge; CB, central body; OL, optic lobe; ASN, antennal sensory nerve; and GA, glomeruli within antennal sensory lobe. (Adapted from Weiss, 1974)

Falck-Hillarp technique is able to localize catecholamines to cell bodies and nerve processes but the fluorophore reaction is not quantitative (Lindvall et al., 1974). The amounts of dopamine and noradrenaline present in cockroach brain were measured using a trihydroxyindole based fluorescence assay (Frontali and Haggendal, 1969). The results from this study, together with those of other fluorescence based determinations of catecholamines in insect nervous tissue (Hiripi and S.-Rozsa, 1973; Klemm and Axelsson, 1973; Kusch, 1975) are summarized in Table 1 . In all cases much more dopamine was found than noradrenaline and no adrenaline could be detected in the majority of the studies. The weight of evidence is thus against the presence of adrenaline in the insect nervous system. Only two studies have presented evidence against this conclusion. In the first, adrenaline is reported to be present in the brain of

323

B l O G E N l C A M l N E S IN T H E I N S E C T NERVOUS S Y S T E M

TABLE 1 Biogenic amine content of insect nervous tissue expressed as nglg wet weight Species

Tissue

Periplaneta americana

Cerebral ganglion Suboesophageal ganglion Thoracic nerve cord Prothoracic ganglion Mesothoracic ganglion Metathoracic ganglion Abdominal ganglia 1-5 6th Abdominal ganglion

Schirrocerca Cerebral ganglion gregariu Optic lobes Brain minus optic lobes Whole C.N.S. Leg muscle Salivary gland Fat body Locusta migratoria

Cerebral ganglion Suboesophageal ganglion

Melamplus CNS sanguinipes Acheta domesticus

Octopamine

1 3450,425w 9 4 ~

Dopamine )2500c,

107@

Noradrenaline 5-HT

) 370c

21 50'

20504 1460' 1580' ll5od 27md

490' 350d 1804 130d 310d

} lolw

8706, 800' 66@ 7706

24306 39106 860 18206 9w

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