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Effects of temperature upon the electrophysiology of retzius cells in the leech
Camp. Biochem.Physiol.,1974,Vol. 47A, pp. 27 to 38. PergmnonPress. Printed in Great Britain
EFFECTS OF TEMPERATURE UPON THE ELECTROPHYSIOLOGY OF RETZIUS CELLS IN THE LEECH CHARLES Department
H. K. WEST*
and CHARLES
M. LENT?
of Zoology and Microbiology, Ohio University, Athens, Ohio 45701, U.S.A. (Received 6 March 1973)
Abstract-l. The Qia temperature coefficients of several electrophysiological parameters of Retzius cells were examined by microelectrode recording in segmental ganglia from the horse leech, Huemopis marmorata (Say), at temperatures between 5 and 25°C. 2. The action potential characteristics of Retzius cells have temperature coefficients which are suggestive of processes mediated by chemical reactions: duration, 3-l ; rate of rise, 3%; rate of decline, 2-3 ; and amplitude of the afterpotential, 2-5. 3. The membrane and electrotonic characteristics have coefficients which are suggestive of physical or diffusional processes: membrane resistance varies with temperature (Qia, 1.55) while the attenuation factor (QrO, 1.25) and resistance of the electrotonic junction (Qi,,, l-05) vary inversely with temperature. 4. An analysis of the impulses in either Retzius cell and of the electrotonic depolarization of the homologous neuron indicates that the cell pair is functionally asymmetric and their loci of impulse initiation are more distant from their neurosomata than their electrotonic junction.
INTRODUCTION ALTERATIONS
in temperature have significant effects upon neurobiological processes at levels ranging from membrane biophysics to behavioral thermoregulation. A widely used measure of effects induced by temperature changes is the QI,, or temperature coefficient of van’t Hoff which is the ratio of the rate of a process at a given temperature to its rate at a temperature 10°C lower. This coefficient indicates whether the rate-limiting step in a biological function is mediated by physical means or by a chemical reaction. This is because processes such as diffusion which proceed by the physical movements of their molecules have QIO values of l-2 as a result of thermal agitation. However, processes involving chemical reactions usually have co-efficients of 2-35 because as the temperature increases, the relative number of * Present address : Department of Physiology, Michigan State University, East Lansing, Michigan 48823. t Present address: Department of Zoology, University of Oklahoma, Norman, Oklahoma 73069. 27
28
CHARLJZSH. K. WEST ANDCHARLESM. LENT
molecules possessing the critical energy of activation increases exponentially (Prosser & Brown, 1961). Various electrical properties of neurons fall into two different classes of QIO. The membrane resistance and resting potential of many neurons are altered by temperature changes and have coefficients between 1 and 2 (Murray, 1966 ; Chalazonitis et al., 1967; Marchiafava, 1970), while the duration of action potentials usually increases at lower temperatures with coefficients greater than 2 (Kerkut & Ridge, 1961; Willows, 1965 ; Larimer, 1967). Hodgkin& Katz (1949) showed that the rising phase of action potentials in the giant axon of the squid has a QIO of 3.2 while its fall has a value of 2.0, and they concluded that two distinct chemical species with different energies of activation mediate these two phases of the impulse. Payton et al. (1969) studied the electrotonic junctions between the segments composing the lateral giant axons in crayfish and they showed that the resistance of these junctions increases at lower temperatures with a QrO of 3.1 even though the resistance of the non-junctional membrane has a coefficient of only 1.5. They hypothesized that the membranes of these septal junctions might be altered by lower temperatures. The pair of large Retzius cells located within each segmental ganglion of leeches is also coupled by a bidirectional electrotonic synapse (Hagiwara & Morita, 1962; Eckert, 1963). The junction between the Retzius cell pair is unaffected by low Ca2+ concentrations (Gerasimov & Akoev, 1967; Payton & Lowenstein, 1968) and may therefore be qualitatively different from crayfish junctions which are readily uncoupled by low Ca2+ concentrations (Asada et al., 1967). This paper reports on an investigation of the temperature coefficients of many electrical properties of Retzius cells including impulse parameters, membrane resistance and electrotonic coupling. The resistance of Retzius cell electrotonic junctions is essentially independent of temperature, and we conclude, therefore, that these junctions are maintained by a process different from that in crayfish septal junctions. MATERIALS
AND METHODS
Horse leeches, Haemopis marmorata (Say), which had been maintained at 5 & 2°C in a S-10% solution of leech physiological saline (Nicholls & Baylor, 1968) were immobilized for dissection by chilling to about 1°C. The techniques for dissection and the general morphology of the segmental ganglia have been described (Nicholls & Baylor, 1968; Lent, 1972). The isolated ganglion was secured in the recording chamber by its connectives and/or roots in order to minimize movements from spontaneous contractions of the muscles located in the neural sheath. The Retzius cells were viewed with lateral illumination for penetration with microelectrodes. The S-ml recording chamber was situated upon a thermoelectric cold stage by which the bath temperature was controlled. The temperature was monitored by means of a thermistor fully immersed in the saline near the preparation. An inlet to the chamber was connected through branching plastic tubes to Mariotte bottles containing aerated salines. In some experiments designed to block chemical synaptic activity, the saline contained 20 mM Mga+ which presumably interferes with the release of transmitters from the nerve terminals (Stuart, 1970).
TEMPERATURE
AND RETZIUS
CELLS
29
Glass microelectrodes with tip resistances of 15-40 MS2 were pulled from stock tubing containing three to four glass fibers that serve as a wick to draw the 3 M KC1 conducting solution into the pipette tip. The Retzius cells in a ganglion were each impaled with a pipette and their intracellular potentials were displayed upon either a dual beam or a storage oscilloscope. Hyperpolarizing currents were passed into one of the neurons by a third microelectrode. A single microelectrode for simultaneous recording and stimulation by passing current through a balanced bridge circuit is an unsatisfactory procedure for measuring d.c. attenuation when the temperature is changed. The resistance of a 25 MR electrode in 20°C saline increases by about 1 Mn/‘C of cooling. The electrode resistance constitutes one of the four legs in the bridge circuit and temperature alterations produced imbalances for which we found it difficult to compensate with consistency. Hyperpolarizing currents were used to measure d.c. attenuation and the resistances of the neurons, as depolarizations generate impulses which confound the measurement of membrane potential. The Qr,, coefficients were calculated by the van’t Hoff equation which is valid over all temperature ranges :
Q,, = (Kl/K&lo’t,-tr, where K, is the measure of the process at the higher temperature, tI (in “C) and K,, the measure at the lower temperature, tr (Prosser & Brown, 1961). For those processes which varied inversely with temperature, their inverse values were used in order to have all temperature coefficients with values greater than one.
RESULTS
The effects of temperature upon the characteristics of the non-overshooting action potentials recorded in the neurosomata of Retzius cells are summarized in Fig. 1. It can be seen in the top panel that the duration of these impulses is Panel B shows that impulse duration varies increased at lower temperatures. The Qr,, for the impulse duration is 3.1 (7-S25°C) inversely with temperature. and this coefficient is the same regardless of whether the temperature is changed by heating or cooling. In other words, the impulse duration is related to the temperature per se and not of the direction of temperature changes; therefore, impulse duration shows no hysteresis, as is true for all the electrophysiological characteristics measured in this study. The increase in the impulse duration at lower temperatures can be accounted for by the decreases in the rates of the rising and falling phases of the impulse, dV/dt. The effects of temperature upon these two phases are illustrated in Fig. 1C and D which show that the rising slope (Qr,,, 3.8) is more affected by temperature than is the falling slope (QiO, 2.3). The hyperpolarizing after-potential is an additional impulse characteristic which is affected by temperature (Fig. 1D). As the figure indicates, magnitude of the hyperpolarization varies inversely with temperature and it has a QIO of 2.5. When we examine the electrotonic and membrane characteristics of the Retzius cell pair, we see that injecting a current into either of the cells produces a potential, Y1, and a proportionally smaller potential, V,, in its bilaterally homologous cell. The ratio of these potentials, VJV,, is the attenuation factor, A,. It can be seen in Fig. 2 that Vz is a linear function of V, for hyperpolarizing currents of up to
CHARLES H. K, WEST AND CHARLESM. LENT
30
FIG, 1. The characteristics of action potentials recorded in Retzius cells from as functions of temperature. A. Traces of individual action potenB. Impulse duration as a function of tials at six experimental temperatures. temperature showing an inverse relationship (data from five experiments). C. The rising phase of Retzius cell impulses as a function of temperature. D. The falling phase of Retzius cell impulses as a function of temperature (data in C and D were obtained from the same two cells in a single ganglion). E. Magnitude of the hyperpolarizing after-potential of Retzius cell impulses as a function of temperature (for C, D and E: -V-, cooling; - - -A- - -, heating).
H. mamorata
The A, is l-55 at WC, increases of 2-27 at 55 mV at each of five temperatures. 7GY’C, has a QIO of 1.25 and is shown as a function of temperature in Fig. 4. The high Mga+ had no apparent effects upon the d.c. attenuation. However, the A, is not an adequate measure of the resistance of the electrotonic junction, I?,, since it is also affected by the membrane resistance, R,, of the Retzius cell pair. A simple relationship between I$ and the experimentally measured parameters can be derived from an equivalent circuit for coupled cells
TEMPERATURE
0
0
15
AND
RRTZIUS
30 VI,
31
CELLS
45
60
mV
FIG. 2. V, as a linear function of V, at five different temperatures. The attenuation of these hyperpolarizations is represented by the slope of the line at each temperature (data from a single experiment, similar results were obtained in four other experiments).
R9 R9
1 ti FIG. 3. A schematic diagram of the equivalent circuit for electronically coupled cells is shown. I is the current source to cell 1 (modified after Bennett, 1966). in Fig. 3. The injected current, I, is divided between the two arms of the parallel circuit and we assume that all elements are linear and that R,. = Rm,. Therefore, which is illustrated
I=
VI/%
+ WR,
+ %I
(1)
CHARLES H. K. WEST AND CHARLIB M. LENT
32
and since &II = (VI f ~~)~(~~+ &z),
(2)
we can substitute (2) into (1) to eliminate Rm, solve for R, and simplify to this equation for calculating junctional resistance : R, = (VT - &?)/(I
* V,).
(3)
Since Y, = VI- vs, Ij = I, and Ij = VJR,, we can also calculate Rm by setting it equal to Ys/Ij. The calculated values for Rj and R, are shown as functions of temperature in Fig. 4 where it can be seen that I?, rises from 8.7 MQ at 75°C to 18*6yvZsZat 25°C in a roughly linear fashion. Thus, the membrane resistance of 2.5
>” \ >
i 2.0-
\ ‘i‘\ ‘.
b t .z 6 .6 3 1.5 5 s
R, 0 .h..
--__
--_ --a_,_
!z --__ --_., I_”
/(.
-m-s, --__
15 3 fj -_
f *z t;
4 ---,
0 . /PI
1.0L 5
2o
f$
0 IO
I 15 Temperature,
I 20
- IO
I 25
“C
FIG. 4. The attenuation factor, membrane resistance and junctional resistance as functions of temperature. The attenuation factors were taken from Fig. 2 and were utilized in calculating l&, and R,. The resistances are plotted as solid lines.
Ret&us cells decreases with cooling and has a &, of 1.55. However, the resistance of the electrotonic junction can be seen to increase only slightly with cooling over the same temperature range. Thus, the Qr,, of 1.05 indicates that Rj is almost temperature independent. When we examine the interactions of action potentials between the coupled Retzius cell pair, we see that an impulse in one cell produces an electrotonically transmitted depolarization in the other cell which usually produces an impulse also. This mutually excitatory coupling through the electrotonic junction usually results in the cell pair having synchronous impulse activity. On some occasions, however, the electrotonic depolarization in the follower neuron is subthreshold and reflects the degree of action potential attenuation by this quotient: action potential (mV)/ electrotonic depolarization (mV). The average attenuation of impulses was 4.9 and ranged between 3.4 and 7.2. Since impulse attenuation data could be collected only
TEMPERATURE
AND
RETZIUS
CELLS
33
occasionally, we did not see any consistent pattern of temperature effects upon impulse attenuation during these experiments. One of the Retzius cells, usually the posterior, has a higher impulse frequency and acts as a pacemaker or driver by its electrotonic depolarization of the follower cell. The delay between the impulse peaks of the driver and follower ranges from 2 to 15 msec and is constant for a particular pair of neurons when their impulse ratio is 1 : 1. This constant latency is suggestive that impulses in the follower are initiated at a uniform level of depolarization from driver impulse electrotonus. However, the impulse ratio is not always 1 : 1 as there are periods of time in which the follower generates fewer impulses than the driver. Two examples of this phenomenon are shown in Fig. 5A. In the left-hand pair of records, the top trace is an intracellular record from the posterior driver cell which is producing impulses at a spontaneous rate of about 5 per sec. It can be seen that the posterior cell generates two to four action potentials and then skips one. This skipping is usually rhythmically patterned even in those rare instances when the follower neuron has four to five times more skips than impulses, an example of which is shown in the right-hand records in Fig. 5A. Skipping is more common in high Mg2+ saline and appears to be a reversible phenomenon since a skipping pair sometimes returns to impulse synchrony. This return to synchrony usually accompanies a reduction in the high Mg2+ concentration. A typical sequence of regular rhythmic skipping is shown in Fig. 5B which consists of a train of four impulses and a skip by the follower. The multiple trace (M) shows these five events in the anterior follower superimposed upon each other. The first impulse in the follower immediately after a skip (1) has the shortest latency to the impulse in the driver and each of the three succeeding follower impulses has an incrementally longer latency. The follower impulse with the longest latency (4), is succeeded by an electrotonic depolarization with no impulse and the entire sequence is then repeated. The follower impulses often appear to have decreasing amplitudes within a sequence of skipping as can be seen clearly in the left-hand records of Fig. 5A. These differences in the recorded amplitudes are produced by the impulses arriving in the follower soma at increasing time intervals along the decaying phase of the electrotonic depolarization (Fig, 5B). Thus, the sums of the action and electrotonic potentials grow smaller during the time course of each skipping sequence. Even though the time between the impulse peaks in the driver and follower cells varies over 25 msec during skipping, the latency between an impulse in either Retzius cell and its electrotonic depolarization of the other neuron is invariant in any particular preparation. Retzius cell action potentials do not overshoot zero membrane potential because their impulses are initiated at some distant locus and enter their neurosomata passively. The resistive and capacitative elements of the axon attenuate the height and increase the time course of each of these impulses. Thus, longer axonal distances will produce greater attenuation and time spread of impulses (Bennett, 1973), and therefore, the constant latency between a Retzius cell impulse and the electrotonic depolarization of its homologue is a measure of the 2
34
CHARLES H. K. WEST AND CHARLFS M. LENT
/JL 4
Lmsec 40
40 mv
C.
FIG. 5A. Two pairs of traces illustrating “skipping” of impulses by the follower cell. In the left pair, the posterior driver (top trace) is firing spontaneously and the follower skips every third to fifth impulse. In the right pair, the follower (top trace) is skipping five impulses for each one generated to the spontaneous firing of the driver. B. A skipping sequence displayed at a higher sweep speed. In the multiple trace (M) the four impulses and the skip in the follower neuron are superimposed. Each of the single frames 1-4 represent a follower impulse in the sequence and 5 shows only the electrotonic depolarization. The photographs were all triggered on the rising phase of the impulses in the posterior driver Retzius cell shown at the bottom of each pair of traces. C. A model of the Retzius cell pair which shows the approximate positions of the electrotonic junction between their neurites and the more distal loci of impulse initiation.
TSh%PERATURE AND RJXZIUS CELLS
3.5
distance from the locus of impulse initiation in that cell to the neurosoma of its homologue. It can be seen in the traces of Fig. 5B that the latency between the peak of an impulse in the posterior driver neuron and its peak of depolarization in the anterior follower is consistently longer than the latency between the peak of the impulse in the anterior neuron and its depolarization peak in the posterior neuron. Therefore, an impulse initiated in the posterior cell travels further to the recording site in the anterior soma than an anterior impulse travels to the posterior soma. DISCUSSION
Our measurements of the attenuation of d.c. by the electrotonic junction of Retzius cells in Huemopti ranged from 1.55 to 2.27 and these values are similar to those of 1.7-4 (Eckert, 1963) and of 2-3.5 (Hagiwara & Morita, 1962) for Retzius cells in the medicinal leech, iLiirudo. These similar data were all generated by injecting currents with an additional microelectrode. However, when the attenuation of Hamop& Retzius cells is measured by injecting current through the recording microelectrode with a balanced bridge circuit, the d.c. has been shown to be attenuated about three- to fourfold over that seen in this study (A, = 6-7, Lent, 1972; 6-8, Wilson & Lent, 1973). These two techniques also produce consistent differences in the attenuation of impulses. The impulse A, in this study of 34-7.2 is similar to that of 4-7 seen by Hagiwara & Morita (1962) who also impaled one of the cells with an additional microelectrode. Two separate studies on Haemopis Retzius cells showed that impulses were attenuated less when measured without the additional microelectrodes (A, = 2-3, Lent, 1972; 34, Wilson& Lent, 1973). Thus these quantitative differences are not due to species examined, but rather to the techniques. The rising and falling phases of Retzius cell action potentials are most likely produced by two distinct chemical reactions. The similarities of the temperature coefficients of the two processes in leech neurons to those in the squid giant axon (Hodgkin & Katz, 1949) suggest that the rising phase is generated by increased Na+ conductance while the falling phase is generated by increased Kf conductance (Hodgkin & Huxley, 1952). Since the hyperpolarizing after-potential in other leech neurons is produced by K+ conductance (Baylor & Nicholls, 1969) and the Q10 of 2.5 for the after-potential is near that of 2.3 for the falling phase of Retzius cell impulses, we are more confident that the declining phase of action potentials in Retzius cells is produced by K+ conductance. The electrotonic characteristics of Retzius cells have temperature coefficients of 1*05-1.55 which lie within the domain of physical-diffusional processes. The &, of 1.05 for Rj indicates that the electrotonic junction between the Retzius cells is affected minimally by thermal changes, unlike the crayfish septae whose junctional resistance appears to be mediated by a temperature-dependent chemical reaction (Payton et al., 1969). The leech and crayfish junctions also show differing sensitivities to the concentration of Casi- and, therefore, there appear to be at least two fundamentally different types of bidirectional electrotonic junctions in the nervous systems of
36
CHARLES H. K. WENTANDCHARLESM. LENT
invertebrates. The septae of crayfish lateral giant axons represent one type of junction which is easily uncoupled by Ca 2+ depletion and whose resistance has a high temperature dependence. Since these junctions show no selective permeabilities (Bennett et al., 1967) the chemical reaction indicated by the high Q1s probably has no transport function. The reaction more likely establishes and maintains junctional continuity between the component cells. We suspect that the chemical species involved in the maintenance of intercellular continuity is activated by Ca2+ ions. The second type of junction is represented by that between the leech Retzius cells. The resistance of their junctions has a very high threshold to Ca2+ depletion and a low dependence upon temperature changes. These junctions do not appear to be maintained by the activity of a temperature-sensitive chemical reaction. Nicholls & Purves (1972) have recently shown that electrotonic transmission between other neurons in the leech is unaffected by cooling to 4°C. Our observations during periods of skipping impulses suggest that there is a functional asymmetry between the Retzius cells within a single ganglion. The time from the peak of an impulse in the posterior Retzius cell to the peak of its electrotonic depolarization of the anterior cell, is consistently longer than the time between the peaks of an anterior impulse and its depolarization of the posterior cell. This difference in delays implies that the posterior neurosoma is closer to the locus of impulse initiation than the anterior soma. An anatomical study of Retzius cells by dye injection (Lent, 1973) sh owed that the anterior cell has a longer initial segment than the posterior cell. Kristan (1973) used a statistical study of Retzius cell impulse intervals to show that this smaller posterior neuron is less effective in initiating impulses in the anterior cell than is the anterior Retzius cell in initiating posterior impulses. The Mg2+ appears to enhance skipping by abolishing excitatory chemical synaptic inputs to the Retzius cells and effectively lowering their excitability. Thus, the amount of charge transferred electrotonically by each impulse from the smaller posterior cell is increasingly less effective in reaching the threshold of the larger anterior cells until an impulse is skipped and the anterior cell is no longer refractory. The temporal relationships between the impulses in the driver and their electrotonic depolarizations of the follower Retzius neurosoma indicate that impulses are generated on the falling phase of the electrotonus. But since impulses can be initiated only during the rising phase of the electrotonic depolarization, the locus of impulse initiation must be further from the Retzius cell bodies than the electrotonic junction which couples them. If the locus were between the junction and the recording site in the soma, an impulse would never be seen on the falling phase of the electrotonic depolarization. A model of the Retzius cell pair which incorporates these features is illustrated in Fig. SC. Acknowkdgements-We thank C. H. Page, W. B. Kristan and W. Thompson for a critical reading of the manuscript and helpful advice. Part of this work is taken from an M.S. thesis by C. H. K. West submitted to Ohio University.
TEMPERATURE AND RETZIUSCELLS
37
REFERENCES ASADAY., PAPPASG. D. & BENNETTM. V. L. (1967) Alteration of resistance at an electrotonic junction and morphological correlates. Fedn Proc. Fedn Am. Sots exp. Biol. 26, 330. BAYLORD. A. & NICHOLLSJ. G. (1969) Changes in extracellular potassium concentration produced by neuronal activity in the central nervous system of the leech. J. Physiol., Lond. 203, 555-569. BENNETTM. V. L. (1966) Physiology of electrotonic junctions. Ann. N. Y. Acad. 5%. 137, 509-539. BENNETTM. V. L. (1973) Function of electrotonic junctions in embryonic and adult tissues. Fedn Proc. Fedn Am. Sots exp. Biol. 32, 65-75. BENNETTM. V. L., DUNHAM P. B. & PAPPASG. D. (1967) Ion fluxes through a “tight junction”. J. gen. Physiol. 50, 1094. CHALAZONITIS N., ROMEYG. & ARVANITAKIA. (1967) Resistance de la neuromembrane en fonction de la temperature (neurones d’Aplysia et d’Helix). C.r. Sianc. Sot. Biol. 161, 1625-1628. ECKERTR. (1963) Electrical interaction of paired ganglion cells in the leech. J. gen. Physiol. 46, 573-587. GERASIMOVV. D. & AKOEVG. N. (1967) Effects of various ions on the resting and action potentials of the giant nerve cells of the leech Hirudo medicinalis. Nature, Lond. 214, 1351-1352. HACIWARAS. & MORITA H. (1962) Electrotonic transmission between two nerve cells in leech ganglion. J. Neurophysiol. 25, 721-731. HODGKINA. L. & HUXLEYA. F. (1952) Current carried by sodium and potassium ions through the membrane of the giant axon of Loligo. J. Physiol., Lond. 116, 449-472. HODGKINA. L. & KATZ B. (1949) The effect of temperature on the giant axon of the squid. J. Physiol., Lond. 109, 240-249. KERKUTG. A. & RIDGE R. M. A. P. (1962) Effect of temperature changes on the activity of the neurones of the snail Helix aspersa. Comp. Biochem. Physiol. 5, 283-295. KRISTANW. B. (1973) Characterization of connectivity among invertebrate motor neurons by cross correlation of spike trains. In The Neurosciences Third Study Program. M.I.T. Press, Cambridge, Mass. In press. LARIMERJ. L. (1967) The effects of temperature on the activity of the caudal photoreceptor. Comp. Biochem. Physiol. 22, 683-700. LENT C. M. (1972) Electrophysiology of Retzius cells of segmental ganglia in the horse leech Haemopis marmorata (Say). Comp. Biochem. Physiol. 42A, 857-862. LENT C. M. (1973) Retzius cells from segmental ganglia of four species of leeches : comparative neuronal geometry. Comp. Biochem. Physiol. 44A, 35-40. MARCHIAFAVA P. L. (1970) The effect of temperature change on membrane potential and conductance in Aplysia giant nerve cell. Comp. Biochem. Physiol. 34, 847-852. MURRAY R. W. (1966) The effect of temperature on the membrane properties of neurons in the visceral ganglia of Aplysia. Comp. Biochem. Physiol. 18, 291-303. NICHOLLSJ. G. & BAYLORD. A. (1968) Specific modalities and receptive fields of sensory neurons in CNS of the leech. J. Neurophysiol. 31, 740-756. NICHOLLSJ. G. & PURVESD. (1972) A comparison of chemical and electrical synaptic transmission between single sensory cells and a motoneurone in the central nervous system of the leech. r. Physiol., Lond. 225, 637-656. PAYTON B. W., BENNETTM. L. V. & PAPPASG. D. (1969) Temperature-dependence of resistance at an electrotonic synapse. Science, Wash. 165, 594-597. PAYTONB. W. & LOEWENSTEIN W. R. (1968) Stability of electrical coupling in leech giant nerve cells: divalent cations, propionate ions, tonicity and pH. Biochim. biophys. Acta 150,156-158.
38
CHARLESH. K. WEST AND CHARLESM. LENT
PROSSERC. L. & BROWN F. A. (1961) Comparative Animal Physiology, 2nd Ed., p. 688. Saunders, Philadelphia. STUARTA. E. (1970) Physiological and morphological properties of motor neurones in the central nervous system of the leech. J. Physiol., Lond. 209, 627-646. WILLOWS A. 0. D. (1965) Giant nerve cells in the ganglia of nudibranch molluscs. Comp. Biochem. Physiol. 14, 707-710. WILSON A. H. & LENT C. M. (1973) Electrophysiology and anatomy of the large neuron pairs in the subesophageal ganglion of the leech. Camp. Biochem. Physiol. (In press.) Key Word Index-Retzius cells; temperature; Haemopis marmorata ; giant neurons.
leech C.N.S.;
electrotonic
coupling;
EFFECTS OF TEMPERATURE UPON THE ELECTROPHYSIOLOGY OF RETZIUS CELLS IN THE LEECH CHARLES Department
H. K. WEST*
and CHARLES
M. LENT?
of Zoology and Microbiology, Ohio University, Athens, Ohio 45701, U.S.A. (Received 6 March 1973)
Abstract-l. The Qia temperature coefficients of several electrophysiological parameters of Retzius cells were examined by microelectrode recording in segmental ganglia from the horse leech, Huemopis marmorata (Say), at temperatures between 5 and 25°C. 2. The action potential characteristics of Retzius cells have temperature coefficients which are suggestive of processes mediated by chemical reactions: duration, 3-l ; rate of rise, 3%; rate of decline, 2-3 ; and amplitude of the afterpotential, 2-5. 3. The membrane and electrotonic characteristics have coefficients which are suggestive of physical or diffusional processes: membrane resistance varies with temperature (Qia, 1.55) while the attenuation factor (QrO, 1.25) and resistance of the electrotonic junction (Qi,,, l-05) vary inversely with temperature. 4. An analysis of the impulses in either Retzius cell and of the electrotonic depolarization of the homologous neuron indicates that the cell pair is functionally asymmetric and their loci of impulse initiation are more distant from their neurosomata than their electrotonic junction.
INTRODUCTION ALTERATIONS
in temperature have significant effects upon neurobiological processes at levels ranging from membrane biophysics to behavioral thermoregulation. A widely used measure of effects induced by temperature changes is the QI,, or temperature coefficient of van’t Hoff which is the ratio of the rate of a process at a given temperature to its rate at a temperature 10°C lower. This coefficient indicates whether the rate-limiting step in a biological function is mediated by physical means or by a chemical reaction. This is because processes such as diffusion which proceed by the physical movements of their molecules have QIO values of l-2 as a result of thermal agitation. However, processes involving chemical reactions usually have co-efficients of 2-35 because as the temperature increases, the relative number of * Present address : Department of Physiology, Michigan State University, East Lansing, Michigan 48823. t Present address: Department of Zoology, University of Oklahoma, Norman, Oklahoma 73069. 27
28
CHARLJZSH. K. WEST ANDCHARLESM. LENT
molecules possessing the critical energy of activation increases exponentially (Prosser & Brown, 1961). Various electrical properties of neurons fall into two different classes of QIO. The membrane resistance and resting potential of many neurons are altered by temperature changes and have coefficients between 1 and 2 (Murray, 1966 ; Chalazonitis et al., 1967; Marchiafava, 1970), while the duration of action potentials usually increases at lower temperatures with coefficients greater than 2 (Kerkut & Ridge, 1961; Willows, 1965 ; Larimer, 1967). Hodgkin& Katz (1949) showed that the rising phase of action potentials in the giant axon of the squid has a QIO of 3.2 while its fall has a value of 2.0, and they concluded that two distinct chemical species with different energies of activation mediate these two phases of the impulse. Payton et al. (1969) studied the electrotonic junctions between the segments composing the lateral giant axons in crayfish and they showed that the resistance of these junctions increases at lower temperatures with a QrO of 3.1 even though the resistance of the non-junctional membrane has a coefficient of only 1.5. They hypothesized that the membranes of these septal junctions might be altered by lower temperatures. The pair of large Retzius cells located within each segmental ganglion of leeches is also coupled by a bidirectional electrotonic synapse (Hagiwara & Morita, 1962; Eckert, 1963). The junction between the Retzius cell pair is unaffected by low Ca2+ concentrations (Gerasimov & Akoev, 1967; Payton & Lowenstein, 1968) and may therefore be qualitatively different from crayfish junctions which are readily uncoupled by low Ca2+ concentrations (Asada et al., 1967). This paper reports on an investigation of the temperature coefficients of many electrical properties of Retzius cells including impulse parameters, membrane resistance and electrotonic coupling. The resistance of Retzius cell electrotonic junctions is essentially independent of temperature, and we conclude, therefore, that these junctions are maintained by a process different from that in crayfish septal junctions. MATERIALS
AND METHODS
Horse leeches, Haemopis marmorata (Say), which had been maintained at 5 & 2°C in a S-10% solution of leech physiological saline (Nicholls & Baylor, 1968) were immobilized for dissection by chilling to about 1°C. The techniques for dissection and the general morphology of the segmental ganglia have been described (Nicholls & Baylor, 1968; Lent, 1972). The isolated ganglion was secured in the recording chamber by its connectives and/or roots in order to minimize movements from spontaneous contractions of the muscles located in the neural sheath. The Retzius cells were viewed with lateral illumination for penetration with microelectrodes. The S-ml recording chamber was situated upon a thermoelectric cold stage by which the bath temperature was controlled. The temperature was monitored by means of a thermistor fully immersed in the saline near the preparation. An inlet to the chamber was connected through branching plastic tubes to Mariotte bottles containing aerated salines. In some experiments designed to block chemical synaptic activity, the saline contained 20 mM Mga+ which presumably interferes with the release of transmitters from the nerve terminals (Stuart, 1970).
TEMPERATURE
AND RETZIUS
CELLS
29
Glass microelectrodes with tip resistances of 15-40 MS2 were pulled from stock tubing containing three to four glass fibers that serve as a wick to draw the 3 M KC1 conducting solution into the pipette tip. The Retzius cells in a ganglion were each impaled with a pipette and their intracellular potentials were displayed upon either a dual beam or a storage oscilloscope. Hyperpolarizing currents were passed into one of the neurons by a third microelectrode. A single microelectrode for simultaneous recording and stimulation by passing current through a balanced bridge circuit is an unsatisfactory procedure for measuring d.c. attenuation when the temperature is changed. The resistance of a 25 MR electrode in 20°C saline increases by about 1 Mn/‘C of cooling. The electrode resistance constitutes one of the four legs in the bridge circuit and temperature alterations produced imbalances for which we found it difficult to compensate with consistency. Hyperpolarizing currents were used to measure d.c. attenuation and the resistances of the neurons, as depolarizations generate impulses which confound the measurement of membrane potential. The Qr,, coefficients were calculated by the van’t Hoff equation which is valid over all temperature ranges :
Q,, = (Kl/K&lo’t,-tr, where K, is the measure of the process at the higher temperature, tI (in “C) and K,, the measure at the lower temperature, tr (Prosser & Brown, 1961). For those processes which varied inversely with temperature, their inverse values were used in order to have all temperature coefficients with values greater than one.
RESULTS
The effects of temperature upon the characteristics of the non-overshooting action potentials recorded in the neurosomata of Retzius cells are summarized in Fig. 1. It can be seen in the top panel that the duration of these impulses is Panel B shows that impulse duration varies increased at lower temperatures. The Qr,, for the impulse duration is 3.1 (7-S25°C) inversely with temperature. and this coefficient is the same regardless of whether the temperature is changed by heating or cooling. In other words, the impulse duration is related to the temperature per se and not of the direction of temperature changes; therefore, impulse duration shows no hysteresis, as is true for all the electrophysiological characteristics measured in this study. The increase in the impulse duration at lower temperatures can be accounted for by the decreases in the rates of the rising and falling phases of the impulse, dV/dt. The effects of temperature upon these two phases are illustrated in Fig. 1C and D which show that the rising slope (Qr,,, 3.8) is more affected by temperature than is the falling slope (QiO, 2.3). The hyperpolarizing after-potential is an additional impulse characteristic which is affected by temperature (Fig. 1D). As the figure indicates, magnitude of the hyperpolarization varies inversely with temperature and it has a QIO of 2.5. When we examine the electrotonic and membrane characteristics of the Retzius cell pair, we see that injecting a current into either of the cells produces a potential, Y1, and a proportionally smaller potential, V,, in its bilaterally homologous cell. The ratio of these potentials, VJV,, is the attenuation factor, A,. It can be seen in Fig. 2 that Vz is a linear function of V, for hyperpolarizing currents of up to
CHARLES H. K, WEST AND CHARLESM. LENT
30
FIG, 1. The characteristics of action potentials recorded in Retzius cells from as functions of temperature. A. Traces of individual action potenB. Impulse duration as a function of tials at six experimental temperatures. temperature showing an inverse relationship (data from five experiments). C. The rising phase of Retzius cell impulses as a function of temperature. D. The falling phase of Retzius cell impulses as a function of temperature (data in C and D were obtained from the same two cells in a single ganglion). E. Magnitude of the hyperpolarizing after-potential of Retzius cell impulses as a function of temperature (for C, D and E: -V-, cooling; - - -A- - -, heating).
H. mamorata
The A, is l-55 at WC, increases of 2-27 at 55 mV at each of five temperatures. 7GY’C, has a QIO of 1.25 and is shown as a function of temperature in Fig. 4. The high Mga+ had no apparent effects upon the d.c. attenuation. However, the A, is not an adequate measure of the resistance of the electrotonic junction, I?,, since it is also affected by the membrane resistance, R,, of the Retzius cell pair. A simple relationship between I$ and the experimentally measured parameters can be derived from an equivalent circuit for coupled cells
TEMPERATURE
0
0
15
AND
RRTZIUS
30 VI,
31
CELLS
45
60
mV
FIG. 2. V, as a linear function of V, at five different temperatures. The attenuation of these hyperpolarizations is represented by the slope of the line at each temperature (data from a single experiment, similar results were obtained in four other experiments).
R9 R9
1 ti FIG. 3. A schematic diagram of the equivalent circuit for electronically coupled cells is shown. I is the current source to cell 1 (modified after Bennett, 1966). in Fig. 3. The injected current, I, is divided between the two arms of the parallel circuit and we assume that all elements are linear and that R,. = Rm,. Therefore, which is illustrated
I=
VI/%
+ WR,
+ %I
(1)
CHARLES H. K. WEST AND CHARLIB M. LENT
32
and since &II = (VI f ~~)~(~~+ &z),
(2)
we can substitute (2) into (1) to eliminate Rm, solve for R, and simplify to this equation for calculating junctional resistance : R, = (VT - &?)/(I
* V,).
(3)
Since Y, = VI- vs, Ij = I, and Ij = VJR,, we can also calculate Rm by setting it equal to Ys/Ij. The calculated values for Rj and R, are shown as functions of temperature in Fig. 4 where it can be seen that I?, rises from 8.7 MQ at 75°C to 18*6yvZsZat 25°C in a roughly linear fashion. Thus, the membrane resistance of 2.5
>” \ >
i 2.0-
\ ‘i‘\ ‘.
b t .z 6 .6 3 1.5 5 s
R, 0 .h..
--__
--_ --a_,_
!z --__ --_., I_”
/(.
-m-s, --__
15 3 fj -_
f *z t;
4 ---,
0 . /PI
1.0L 5
2o
f$
0 IO
I 15 Temperature,
I 20
- IO
I 25
“C
FIG. 4. The attenuation factor, membrane resistance and junctional resistance as functions of temperature. The attenuation factors were taken from Fig. 2 and were utilized in calculating l&, and R,. The resistances are plotted as solid lines.
Ret&us cells decreases with cooling and has a &, of 1.55. However, the resistance of the electrotonic junction can be seen to increase only slightly with cooling over the same temperature range. Thus, the Qr,, of 1.05 indicates that Rj is almost temperature independent. When we examine the interactions of action potentials between the coupled Retzius cell pair, we see that an impulse in one cell produces an electrotonically transmitted depolarization in the other cell which usually produces an impulse also. This mutually excitatory coupling through the electrotonic junction usually results in the cell pair having synchronous impulse activity. On some occasions, however, the electrotonic depolarization in the follower neuron is subthreshold and reflects the degree of action potential attenuation by this quotient: action potential (mV)/ electrotonic depolarization (mV). The average attenuation of impulses was 4.9 and ranged between 3.4 and 7.2. Since impulse attenuation data could be collected only
TEMPERATURE
AND
RETZIUS
CELLS
33
occasionally, we did not see any consistent pattern of temperature effects upon impulse attenuation during these experiments. One of the Retzius cells, usually the posterior, has a higher impulse frequency and acts as a pacemaker or driver by its electrotonic depolarization of the follower cell. The delay between the impulse peaks of the driver and follower ranges from 2 to 15 msec and is constant for a particular pair of neurons when their impulse ratio is 1 : 1. This constant latency is suggestive that impulses in the follower are initiated at a uniform level of depolarization from driver impulse electrotonus. However, the impulse ratio is not always 1 : 1 as there are periods of time in which the follower generates fewer impulses than the driver. Two examples of this phenomenon are shown in Fig. 5A. In the left-hand pair of records, the top trace is an intracellular record from the posterior driver cell which is producing impulses at a spontaneous rate of about 5 per sec. It can be seen that the posterior cell generates two to four action potentials and then skips one. This skipping is usually rhythmically patterned even in those rare instances when the follower neuron has four to five times more skips than impulses, an example of which is shown in the right-hand records in Fig. 5A. Skipping is more common in high Mg2+ saline and appears to be a reversible phenomenon since a skipping pair sometimes returns to impulse synchrony. This return to synchrony usually accompanies a reduction in the high Mg2+ concentration. A typical sequence of regular rhythmic skipping is shown in Fig. 5B which consists of a train of four impulses and a skip by the follower. The multiple trace (M) shows these five events in the anterior follower superimposed upon each other. The first impulse in the follower immediately after a skip (1) has the shortest latency to the impulse in the driver and each of the three succeeding follower impulses has an incrementally longer latency. The follower impulse with the longest latency (4), is succeeded by an electrotonic depolarization with no impulse and the entire sequence is then repeated. The follower impulses often appear to have decreasing amplitudes within a sequence of skipping as can be seen clearly in the left-hand records of Fig. 5A. These differences in the recorded amplitudes are produced by the impulses arriving in the follower soma at increasing time intervals along the decaying phase of the electrotonic depolarization (Fig, 5B). Thus, the sums of the action and electrotonic potentials grow smaller during the time course of each skipping sequence. Even though the time between the impulse peaks in the driver and follower cells varies over 25 msec during skipping, the latency between an impulse in either Retzius cell and its electrotonic depolarization of the other neuron is invariant in any particular preparation. Retzius cell action potentials do not overshoot zero membrane potential because their impulses are initiated at some distant locus and enter their neurosomata passively. The resistive and capacitative elements of the axon attenuate the height and increase the time course of each of these impulses. Thus, longer axonal distances will produce greater attenuation and time spread of impulses (Bennett, 1973), and therefore, the constant latency between a Retzius cell impulse and the electrotonic depolarization of its homologue is a measure of the 2
34
CHARLES H. K. WEST AND CHARLFS M. LENT
/JL 4
Lmsec 40
40 mv
C.
FIG. 5A. Two pairs of traces illustrating “skipping” of impulses by the follower cell. In the left pair, the posterior driver (top trace) is firing spontaneously and the follower skips every third to fifth impulse. In the right pair, the follower (top trace) is skipping five impulses for each one generated to the spontaneous firing of the driver. B. A skipping sequence displayed at a higher sweep speed. In the multiple trace (M) the four impulses and the skip in the follower neuron are superimposed. Each of the single frames 1-4 represent a follower impulse in the sequence and 5 shows only the electrotonic depolarization. The photographs were all triggered on the rising phase of the impulses in the posterior driver Retzius cell shown at the bottom of each pair of traces. C. A model of the Retzius cell pair which shows the approximate positions of the electrotonic junction between their neurites and the more distal loci of impulse initiation.
TSh%PERATURE AND RJXZIUS CELLS
3.5
distance from the locus of impulse initiation in that cell to the neurosoma of its homologue. It can be seen in the traces of Fig. 5B that the latency between the peak of an impulse in the posterior driver neuron and its peak of depolarization in the anterior follower is consistently longer than the latency between the peak of the impulse in the anterior neuron and its depolarization peak in the posterior neuron. Therefore, an impulse initiated in the posterior cell travels further to the recording site in the anterior soma than an anterior impulse travels to the posterior soma. DISCUSSION
Our measurements of the attenuation of d.c. by the electrotonic junction of Retzius cells in Huemopti ranged from 1.55 to 2.27 and these values are similar to those of 1.7-4 (Eckert, 1963) and of 2-3.5 (Hagiwara & Morita, 1962) for Retzius cells in the medicinal leech, iLiirudo. These similar data were all generated by injecting currents with an additional microelectrode. However, when the attenuation of Hamop& Retzius cells is measured by injecting current through the recording microelectrode with a balanced bridge circuit, the d.c. has been shown to be attenuated about three- to fourfold over that seen in this study (A, = 6-7, Lent, 1972; 6-8, Wilson & Lent, 1973). These two techniques also produce consistent differences in the attenuation of impulses. The impulse A, in this study of 34-7.2 is similar to that of 4-7 seen by Hagiwara & Morita (1962) who also impaled one of the cells with an additional microelectrode. Two separate studies on Haemopis Retzius cells showed that impulses were attenuated less when measured without the additional microelectrodes (A, = 2-3, Lent, 1972; 34, Wilson& Lent, 1973). Thus these quantitative differences are not due to species examined, but rather to the techniques. The rising and falling phases of Retzius cell action potentials are most likely produced by two distinct chemical reactions. The similarities of the temperature coefficients of the two processes in leech neurons to those in the squid giant axon (Hodgkin & Katz, 1949) suggest that the rising phase is generated by increased Na+ conductance while the falling phase is generated by increased Kf conductance (Hodgkin & Huxley, 1952). Since the hyperpolarizing after-potential in other leech neurons is produced by K+ conductance (Baylor & Nicholls, 1969) and the Q10 of 2.5 for the after-potential is near that of 2.3 for the falling phase of Retzius cell impulses, we are more confident that the declining phase of action potentials in Retzius cells is produced by K+ conductance. The electrotonic characteristics of Retzius cells have temperature coefficients of 1*05-1.55 which lie within the domain of physical-diffusional processes. The &, of 1.05 for Rj indicates that the electrotonic junction between the Retzius cells is affected minimally by thermal changes, unlike the crayfish septae whose junctional resistance appears to be mediated by a temperature-dependent chemical reaction (Payton et al., 1969). The leech and crayfish junctions also show differing sensitivities to the concentration of Casi- and, therefore, there appear to be at least two fundamentally different types of bidirectional electrotonic junctions in the nervous systems of
36
CHARLES H. K. WENTANDCHARLESM. LENT
invertebrates. The septae of crayfish lateral giant axons represent one type of junction which is easily uncoupled by Ca 2+ depletion and whose resistance has a high temperature dependence. Since these junctions show no selective permeabilities (Bennett et al., 1967) the chemical reaction indicated by the high Q1s probably has no transport function. The reaction more likely establishes and maintains junctional continuity between the component cells. We suspect that the chemical species involved in the maintenance of intercellular continuity is activated by Ca2+ ions. The second type of junction is represented by that between the leech Retzius cells. The resistance of their junctions has a very high threshold to Ca2+ depletion and a low dependence upon temperature changes. These junctions do not appear to be maintained by the activity of a temperature-sensitive chemical reaction. Nicholls & Purves (1972) have recently shown that electrotonic transmission between other neurons in the leech is unaffected by cooling to 4°C. Our observations during periods of skipping impulses suggest that there is a functional asymmetry between the Retzius cells within a single ganglion. The time from the peak of an impulse in the posterior Retzius cell to the peak of its electrotonic depolarization of the anterior cell, is consistently longer than the time between the peaks of an anterior impulse and its depolarization of the posterior cell. This difference in delays implies that the posterior neurosoma is closer to the locus of impulse initiation than the anterior soma. An anatomical study of Retzius cells by dye injection (Lent, 1973) sh owed that the anterior cell has a longer initial segment than the posterior cell. Kristan (1973) used a statistical study of Retzius cell impulse intervals to show that this smaller posterior neuron is less effective in initiating impulses in the anterior cell than is the anterior Retzius cell in initiating posterior impulses. The Mg2+ appears to enhance skipping by abolishing excitatory chemical synaptic inputs to the Retzius cells and effectively lowering their excitability. Thus, the amount of charge transferred electrotonically by each impulse from the smaller posterior cell is increasingly less effective in reaching the threshold of the larger anterior cells until an impulse is skipped and the anterior cell is no longer refractory. The temporal relationships between the impulses in the driver and their electrotonic depolarizations of the follower Retzius neurosoma indicate that impulses are generated on the falling phase of the electrotonus. But since impulses can be initiated only during the rising phase of the electrotonic depolarization, the locus of impulse initiation must be further from the Retzius cell bodies than the electrotonic junction which couples them. If the locus were between the junction and the recording site in the soma, an impulse would never be seen on the falling phase of the electrotonic depolarization. A model of the Retzius cell pair which incorporates these features is illustrated in Fig. SC. Acknowkdgements-We thank C. H. Page, W. B. Kristan and W. Thompson for a critical reading of the manuscript and helpful advice. Part of this work is taken from an M.S. thesis by C. H. K. West submitted to Ohio University.
TEMPERATURE AND RETZIUSCELLS
37
REFERENCES ASADAY., PAPPASG. D. & BENNETTM. V. L. (1967) Alteration of resistance at an electrotonic junction and morphological correlates. Fedn Proc. Fedn Am. Sots exp. Biol. 26, 330. BAYLORD. A. & NICHOLLSJ. G. (1969) Changes in extracellular potassium concentration produced by neuronal activity in the central nervous system of the leech. J. Physiol., Lond. 203, 555-569. BENNETTM. V. L. (1966) Physiology of electrotonic junctions. Ann. N. Y. Acad. 5%. 137, 509-539. BENNETTM. V. L. (1973) Function of electrotonic junctions in embryonic and adult tissues. Fedn Proc. Fedn Am. Sots exp. Biol. 32, 65-75. BENNETTM. V. L., DUNHAM P. B. & PAPPASG. D. (1967) Ion fluxes through a “tight junction”. J. gen. Physiol. 50, 1094. CHALAZONITIS N., ROMEYG. & ARVANITAKIA. (1967) Resistance de la neuromembrane en fonction de la temperature (neurones d’Aplysia et d’Helix). C.r. Sianc. Sot. Biol. 161, 1625-1628. ECKERTR. (1963) Electrical interaction of paired ganglion cells in the leech. J. gen. Physiol. 46, 573-587. GERASIMOVV. D. & AKOEVG. N. (1967) Effects of various ions on the resting and action potentials of the giant nerve cells of the leech Hirudo medicinalis. Nature, Lond. 214, 1351-1352. HACIWARAS. & MORITA H. (1962) Electrotonic transmission between two nerve cells in leech ganglion. J. Neurophysiol. 25, 721-731. HODGKINA. L. & HUXLEYA. F. (1952) Current carried by sodium and potassium ions through the membrane of the giant axon of Loligo. J. Physiol., Lond. 116, 449-472. HODGKINA. L. & KATZ B. (1949) The effect of temperature on the giant axon of the squid. J. Physiol., Lond. 109, 240-249. KERKUTG. A. & RIDGE R. M. A. P. (1962) Effect of temperature changes on the activity of the neurones of the snail Helix aspersa. Comp. Biochem. Physiol. 5, 283-295. KRISTANW. B. (1973) Characterization of connectivity among invertebrate motor neurons by cross correlation of spike trains. In The Neurosciences Third Study Program. M.I.T. Press, Cambridge, Mass. In press. LARIMERJ. L. (1967) The effects of temperature on the activity of the caudal photoreceptor. Comp. Biochem. Physiol. 22, 683-700. LENT C. M. (1972) Electrophysiology of Retzius cells of segmental ganglia in the horse leech Haemopis marmorata (Say). Comp. Biochem. Physiol. 42A, 857-862. LENT C. M. (1973) Retzius cells from segmental ganglia of four species of leeches : comparative neuronal geometry. Comp. Biochem. Physiol. 44A, 35-40. MARCHIAFAVA P. L. (1970) The effect of temperature change on membrane potential and conductance in Aplysia giant nerve cell. Comp. Biochem. Physiol. 34, 847-852. MURRAY R. W. (1966) The effect of temperature on the membrane properties of neurons in the visceral ganglia of Aplysia. Comp. Biochem. Physiol. 18, 291-303. NICHOLLSJ. G. & BAYLORD. A. (1968) Specific modalities and receptive fields of sensory neurons in CNS of the leech. J. Neurophysiol. 31, 740-756. NICHOLLSJ. G. & PURVESD. (1972) A comparison of chemical and electrical synaptic transmission between single sensory cells and a motoneurone in the central nervous system of the leech. r. Physiol., Lond. 225, 637-656. PAYTON B. W., BENNETTM. L. V. & PAPPASG. D. (1969) Temperature-dependence of resistance at an electrotonic synapse. Science, Wash. 165, 594-597. PAYTONB. W. & LOEWENSTEIN W. R. (1968) Stability of electrical coupling in leech giant nerve cells: divalent cations, propionate ions, tonicity and pH. Biochim. biophys. Acta 150,156-158.
38
CHARLESH. K. WEST AND CHARLESM. LENT
PROSSERC. L. & BROWN F. A. (1961) Comparative Animal Physiology, 2nd Ed., p. 688. Saunders, Philadelphia. STUARTA. E. (1970) Physiological and morphological properties of motor neurones in the central nervous system of the leech. J. Physiol., Lond. 209, 627-646. WILLOWS A. 0. D. (1965) Giant nerve cells in the ganglia of nudibranch molluscs. Comp. Biochem. Physiol. 14, 707-710. WILSON A. H. & LENT C. M. (1973) Electrophysiology and anatomy of the large neuron pairs in the subesophageal ganglion of the leech. Camp. Biochem. Physiol. (In press.) Key Word Index-Retzius cells; temperature; Haemopis marmorata ; giant neurons.
leech C.N.S.;
electrotonic
coupling;