PROPERTIES OF SYNAPSES MADE BY IVN COMMAND- INTERNEURONES IN THE STOMATOGASTRIC GANGLION OF THE SPINY LOBSTER PANULIRUS INTERRUPTUS

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1 J. exp. Biol. (1982), 97, With 9 figures Printed in Great Britain PROPERTIES OF SYNAPSES MADE BY IVN COMMAND- INTERNEURONES IN THE STOMATOGASTRIC GANGLION OF THE SPINY LOBSTER PANULIRUS INTERRUPTUS BY KAREN A. SIGVARDT* AND BRIAN MULLONEY Department of Zoology, University of California, Davis, California (Received 28 May 1981) SUMMARY 1. The IVN command interneurones synapse directly onto 11 identified neurones in the stomatogastric ganglion: the two pyloric dilators (PDs), the anterior burster (AB), ventricular dilator (VD), the four gastric mill neurones (GMs), the two lateral posterior gastric neurones (LPGNs), and Interneurone 1 (Int 1). 2. The IVN p.s.p.s in PD and AB are biphasic, and consist of a fast depolarizing component followed by a slower hyperpolarizing component. 3. The hyperpolarizing component of this biphasic postsynaptic potential is inhibitory, and appears to be the result of a conductance increase to K + and Cl". 4. The IVN p.s.p. in VD is excitatory and can drive VD one-for-one. 5. The IVN p.s.p.s in GM and LPGN are inhibitory. The amplitude of a single p.s.p. is small but, at high frequency, summation of p.s.p.s holds the postsynaptic membrane potential below threshold. 6. The facilitation characteristics of the p.s.p.s in each neurone are described. 7. The functional significance of these synaptic characteristics is discussed in terms of the modification of motor output caused by a burst of the IVN interneurones. INTRODUCTION Two nerve cells that project from the brain to the stomatogastric ganglion of Panuliris interruptus are now known to be command neurones (Sigvardt & Mulloney, 1981). When these two neurones fire prolonged bursts of impulses, in response either to sensory input or to experimental stimulation, the motor patterns that normally occur in the stomatogastric ganglion change radically (Dando & Selverston, 1972; Sigvardt & Mulloney, 1981). The neurones of the stomatogastric ganglion and their synaptic organization are known in detail (Maynard & Selverston, 1975; Mulloney & Selverston, 1974a, b\ Mulloney, 1977; Selverston & Mulloney, 1974). A description of the synapses in the ganglion made by these command interneurones would make possible an explanation of the changes in the motor pattern that their firing causes. Present address: Dr K. A. Sigvardt, Karolinska Institutet, Department of Physiology III, fcdingovagen i, S Stockholm, Sweden.

2 154 KAREN A. SIGVARDT AND B. MULLONEY This paper describes the p.s.p.s caused by the two IVN interneurones in all th4 stomatogastric neurones with which they synapse. The dynamics of some of these synapses are complex, and contribute significantly to the changes that these interneurones command. METHODS Our methods and the anatomy of the stomatogastric nervous system were described in detail in the companion paper (Sigvardt & Mulloney, 1981). In the experiments reported here, the preparation was isolated completely from the animal and the stomach and consisted of the stomatogastric and oesophageal ganglia connected by the stomatogastric nerve (SGN). The preparation was pinned under saline solution in a petri dish lined with a transparent, inert silicone rubber (Sylgard). Extracellular recordings and stimulations were made with pin electrodes attached to selected peripheral nerves (Mulloney & Selverston, 1974 a). The two command interneurones we studied have axons in the Inferior Ventricular nerve (IVN) (Dando & Selverston, 1972), and only the axons of these two neurones run from the IVN to the stomatogastric ganglion through the SGN (Kushner, 1979; Sigvardt & Mulloney, ibid.). Therefore we could stimulate them at the IVN and record their impulses en passant in the SGN before the impulses caused p.s.p.s in the neurones of the stomatogastric ganglion (Sigvardt & Mulloney, ibid.). Data were recorded directly by photographing an oscilloscope trace, or by using a Gould 220 chart recorder. The results of this paper were obtained from 20 Expts. To record p.s.p.s at different membrane potentials in the postsynaptic neurone, we penetrated the soma of each neurone with two microelectrodes, filled either with 4 M-K acetate or 2-5 M-KC1. We used one electrode to record potential through a Getting M4 or M5 preamplifier and the other to pass the constant currents needed to move the steady-state membrane potential of the neurone to the desired value. These potentials were calibrated with a WP-Instruments Inc. Precision Millivolt Source. The saline solutions used in these experiments contained in mmoles: Ion Normal io-o io-o Control 2K+ High Mg! + Na+ K+ Ca 2+ Mg a + so«- ci- Tris maleate Sucrose Glucose io-o io-o s io-o io-o i IO'O 27 2 The control saline had 5 x normal Ca 2+, and was used to increase the size of the p.s.p. and to reduce spontaneous firing in the postsynaptic neurones. D-tubocurare and picrotoxin were dissolved in control saline. In both control and high Mg 2+, we reduced the NaCl to compensate for the additional Cl~ added as CaCl 2 or MgCl 2 ; the Cl~ concentration was the same within 2 % in the four solutions. To compensate osmotically for the loss of the Na+ in these solutions, we added sucrose to raise

3 Synapses in the stomatogastric ganglion of the spiny lobstei 155 'osmolarity to within 1 % of that of the normal saline, assuming that all the salts dissociated completely. ABBREVIATIONS PD, pyloric dilator neurone; VD, ventricular dilator neurone; Int 1, interneurone 1; LPGN, lateral posterior gastric neurone; GM, gastric mill neurone; IVN, inferior ventricular nerve; SON, superior oesophageal nerve; SGN, stomatogastric nerve; StG, stomatogastric ganglion. RESULTS The two IVN interneurones synapse with 11 neurones in the stomatogastric ganglion: the three PD-AB neurones and the VD neurone of the pyloric system and Int 1, the two LPGNs and the four GMs of the gastric system. These 11 synapses appear to be direct by two criteria. First, the individual p.s.p.s follow each action potential in the IVN interneurones at a constant latency (Fig. 7 of Sigvardt & Mulloney, 1981) and the latency of the p.s.p.s in all postsynaptic neurones is the same in any given experiment. Second, the p.s.p.s recorded in each neurone follow impulses in the IVN interneurones even at frequencies greater than 50 Hz, and they continue to do so when the extracellular Ca 2+ concentration is raised fivefold. If an interneurone were interposed between the IVN units and these 11 postsynaptic neurones, raising the Ca 2+ concentration would raise this interneurone's threshold and would increase the probability that p.s.p.s would fail to follow IVN impulses (Kehoe, 19726). We observed no failures, and conclude these synapses are direct. The thresholds of these two interneurones to stimulation of the FVN are very similar, and often they can not be stimulated individually. In experiments where the thresholds of the two axons were sufficiently distinct to permit us to study the consequences of stimulating only the lower-threshold unit, we looked for differences in the connexions made by the two IVN units. We did not discover any differences; increasing the stimulus intensity above threshold for the second interneurone simply recruited a second p.s.p. of identical latency and similar other properties in every postsynaptic neurone we examined. In the rest of this paper, we will treat the two as having the same properties. Characteristics of p.s.p.s in different neurones. The IVN interneurones have very different effects on the output of each of the postsynaptic neurones in the stomatogastric ganglion because the properties of each synapse differ. When the IVN is stimulated at low frequencies in normal saline, the p.s.p.s in all neurones except VD are small (Fig. 1). During experiments to characterise the properties of each synapse, we increased the size of the p.s.p.s by perfusing the ganglion with control saline (5 x normal Ca 2+ ; see Methods). Biphasic p.s.p. in PD and AB. Neurons of the PD-AB group have a biphasic p.s.p. following each impulse in the IVN interneurones (Fig. 2). This p.s.p. consists of an initial rapid, excitatory depolarization followed by a slower, long-lasting inhibitory hyperpolarization. The PD-AB neurones are endogenous bursters (Gola & Selverston, 1) whose membrane potentials oscillate periodically through a range as large as

4 i 5 6 KAREN A. SIGVARDT AND B. MULLONEY p_ Normal saline AJL10 mv VD 5 mv GM - / / J 1 s Fig. i. Responses of four stomatogastric neurones to stimulation of the IVN interneurones in normal saline, recorded intracellularly with K-acetate electrodes. Stimulation first at 2-5 Hz, indicated by dots, followed by stimulation at 25 Hz m'arked b> the bar. Response of PD. At 25 Hz, the p.s.p.s are depolarizing but have little effect on the PD burst pattern. Stimulation of the IVN interneurones at 25 Hz disrupts the PD burst pattern and, after the first four stimuli, prevents PD firing. Following this inhibition, the number of PD impulses per burst increases for seveial s. Both PD neurones and the AB neurone respond in the same way. Response of VD. Each IVN e.p.s.p. triggers an impulse in VD, at both 2-5 Hz and 25 Hz. Response of LPGN. At 25 Hz, the IVN i.p.s.p.s in LPGN are small and do not inhibit LPGN effectively. However, at 25 Hz the i.p.s.p.s sum and inhibit LPGN. Characteristically, LPGN shows a strong postinhibitory rebound. Both LPGNs respond in the same way. Response of GM. At 2-5 Hz individual IVN p.s.p.s have little effect on the rate of spontaneous firing of GM. At 25 Hz, however, individual i.p.s.p.s sum rapidly and prevent GM firing. All four GM neurones respond in the same way. 5 mv 20 mv, so the effect of these IVN p.s.p.s depends in part on the potential of the postsynaptic membrane when they arrive and in part on the frequency at which they arrive. At low presynaptic impulse frequencies, the p.s.p.s trigger impulses in the PD-AB neurones during the depolarized parts of the endogenous oscillations, but not during the hyperpolarized parts. These low frequency p.s.p.s have little effect on the endogenous pattern of bursting in PD-AB. When the IVN interneurones fire at higher frequencies, however, the long-lasting inhibitory components of the p.s.p.s sum and firing stops in PD-AB (Figs. 1, 2). After a bout of high-frequency impulses in the presynaptic fibres, PD-AB fire vigorous bursts that return to normal only after several seconds (Fig. i).

5 Synapses in the stomatogastric ganglion of the spiny lobster 157 High Ca 2+ saline PD 10 mv l k_*l t k_ + K_X. 10 mv LPGN Fig. 2. Perfusing the stomatogastric preparation with control saline (5 x normal Ca 8+ ) decreases the excitability of the neurones and increases the amplitude of the postsynaptic potentials. Response of PD. High Ca 2+ does not seem to increase the amplitude of the depolarizing phase of the biphasic synaptic potential. The inhibitory component is now revealed as a slow hyperpolarization following the fast depolarization. Response of VD. IVN p.s.p.s drive VD one-for-one at 2-5 Hz, but at 25 Hz VD fails to fire after the fifth p.s.p. in the train. Response of LPGN. The amplitude of the IVN i.p.s.p. in LPGN increases 20 x in high Ca s+. At 25 Hz stimulation, the LPGN membrane potential is clamped by the synaptic currents at the reversal potential for the p.s.p. Response of GM. The amplitude of the IVN p.s.p. in GM is increased in high Ca 2+. The p.s.p. facilitates even at 2-5 Hz. At 25 Hz, the GM membrane potential is clamped by the synaptic currents at the reversal potential for the p.s.p. Notice the i.p.s.p.s in GM from Int 1 (Selverston & Mulloney, 1974); Int 1 bursts on rebound from inhibition following cessation of IVN stimulation. During a 25 Hz train of impulses in the IVN interneurones, the excitatory and inhibitory components of the p.s.p.s interact. For example, the first three e.p.s.p.s in one 25 Hz train recorded in a PD neurone starting at 56 mv had amplitudes of 5, 3 and 1 mv, even though the membrane potential was moving farther from their reversal potential (Fig. 4). In contrast, the e.p.s.p.s from a 2-5 Hz train recorded at the same initial membrane potential (Fig. 3, control 56 mv) are almost identical. This reduction in the size of the excitatory component is not due to classical synaptic depression because the slower inhibitory component does not decrease proportionately. We think the reduction of the excitatory component occurs because the excitatory current is effectively shunted by the summed inhibitory conductance (Takeuchi, 1977). The net effect of the two components during a high frequency train of p.s.p.s is to excite the PD-AB neurones transiently, and then to inhibit them for the duration fethe train (Fig. 1). EXB 9?

6 158 KAREN A. SIGVARDT AND B. MULLONEY Control 2X (K + ) o Is 10 mv -88 J -98. V.W Fig. 3. Response of PD to 2-5 Hz stimulation of the IVN interneurones, at different initial membrane potentials in control saline, in contiol saline with elevated internal [Cl"], and in high potassium (2 x [K + ] o ) saline. Steady state membrane potentials are given in mv to the left of each record. Left. The depolarizing phase of the response causes PD to fire when PD is depolarized above rest ( 46 mv). The slow hyperpolarizing phase increases in amplitude when PD is depolarized and decreases as the membrane is hyperpolarized. The reversal potential for this synaptic potential is near 76 mv. Middle. Increasing the external potassium ion concentration shifts the reversal potential for the inhibitory component to near 56 mv. Measurements were made 15 min after changing to high K + solution. Right. Increasing the internal chloride ion concentration also shifts the reversal potential for the inhibitory component in the depolarizing direction. Cl~ was injected inco the neurone with a KCl-filled microelectrode, with which the neurone was hyperpolarized to about nomv for 15 min. The p.s.p.s were recorded immediately after this period of hyperpolarization. This prolonged hyperpolarization changed the resting potential from 46 to 58 mv, but one-half hour later resting potential had returned to 46 mv. Excitatory p.s.p. in VD. VD normally fires bursts of impulses that alternate with the bursts in the PD-AB neurones because PD-AB inhibit VD effectively (Maynard & Selverston, 1975). Impulses in the IVN interneurones cause large e.p.s.p.s in VD (Figs. 1, 2); each of these p.s.p.s usually causes an impulse in VD throughout the normal range of IVN impulse frequencies (Sigvardt & Mulloney, 1981). When the IVN interneurones fire at low frequencies, additional impulses are simply intercalated in VD's characteristic train of bursts (Fig. 1). When the IVN interneurones fire at high frequencies, bursting is suppressed in the PD-AB neurones and VD then fires a train of impulses each of which is triggered by a p.s.p. from the IVN interneurones (Fig. 1). In high Ca 2+ saline, VD may fail to follow the IVN interneurones impulsefor impulse (Fig. 2), but in normal saline VD continues to fire during a long train of IVN p.s.p.s.

7 Synapses in the stomatogastric ganglion of the spiny lobster 159 Control 2X (K + ) out High (CD P J llomv 1 s Fig. 4. Response of PD to 25 Hz stimulation of the IVN interneurones at different initial membrane potentials under three experimental conditions. Steady-state membrane potentials are given in mv to the left of each recoid. Left. In control saline, reversal potential for the summed p.s.p. after 2 s of 25 Hz stimulation (arrows) lies between 66 and 76 mv. Notice that the excitatory components of the p.s.p.s get smaller as the stimulus train progresses. Middle. A twofold increase in the external concentration of K + changes the reversal potential of these p.s.p.s to 56 mv. Measurements were made after 15 min of perfusion with high K+ saline. Right. Increasing the internal concentration of Cl" also changes the reversal potential of these p.s.p.s to less than 58 mv. These measurements were made immediately after a 15 min hyperpolarization of the neurone using a KCl-filled microelectrode. Inhibitory postsynaptic potential in LPGN. The postsynaptic response of LPGN appears to be inhibitory when the amplitude of the p.s.p. is increased by increasing extracellular calcium (Fig. 2). The i.p.s.p. is long-lasting, and stimulation of IVN at 25 Hz produces a rapid summation to a potential that is probably the reversal potential for the p.s.p. There may be an initial depolarizing component in normal saline (Fig. 1), but this component is overwhelmed by the inhibitory component in high-calcium saline and during high-frequency stimulation in normal saline. Since the excitatory component is so small, the biphasic p.s.p. can be described functionally as an i.p.s.p. The IVN to LPGN synapse facilitates; at 2-5 Hz the i.p.s.p. is about 0-5 mv (Fig. 1) but at 25 Hz the third stimulus produces a 2-0 mv i.p.s.p. and the response increases to a maximum of about 4 mv. Its facilitation characteristics are such that it usually takes several stimuli to prevent firing of LPGN when LPGN is firing tonically. Inhibitory postsynaptic potential in GM. The GM neurone receives inhibitory input from the IVN interneurone (Figs. 1, 2). Individual i.p.s.p.s are small and therefore have no effect on the cell'sfiring pattern when they occur at low frequencies. The timecourse of the i.p.s.p. is slow, and summation to a subthreshold level is rapid at high frequency (Figs. 1, 2). This cell shows little or no postinhibitory rebound following IVN input. Jphasic p.s.p. in Interneurone 1. Interneurone 1 (Int 1) is a critical component in letwork that generates the gastric rhythm (Mulloney & Selverston, 19746). The

8 i6o KAREN A. SIGVARDT AND B. MULLONEY o Control, with 50 mm-ca 2+ 2X (K + ) out A High (CDm -25" Fig. 5. Plot of amplitude of postsynaptic response in PD (ordinate) against membrane potential (abscissa) in high Ca 8+ saline ( ), 2 x [K + ] o saline ( ), and following intracellular injection of Cl~ (A). The reversal potential (the point at which the line crosses the abscissa) shifts in the depolarizing direction in both 2 x [K + J o and increased ([Cl~]i, which indicates that the inhibitory component of the biphasic p.s.p. in PD is due to a conductance increase to both K + and Cl~. Each regression line was drawn as a least-squares fit to the data of Fig. 4. IVN interneurones synapse directly with Int 1 (Fig. 7 of Sigvardt & Mulloney, 1981). When the IVN interneurones fire at low frequencies, the postsynaptic response is excitatory; additional impulses are intercalated in the ongoing train of Int 1 impulses whenever the IVN interneurones trigger a p.s.p. At higher frequencies, a long-lasting inhibitory component becomes apparent, probably because of temporal summation during the high-frequency train. This inhibitory component effectively prevents firing in Int 1 until the FVN interneurones stop and Int 1 recovers. This inhibition of Int 1 can also be inferred from the changing frequency and amplitude of the i.p.s.p.s caused by Int 1 in GM (Fig. 2). Ionic basis of the biphasic potential in PD Biphasic potentials like these in PD must arise from the integration of more than one synaptic current. In the PD-AB neurones, the excitatory and inhibitory currents overlap in time, and we do not have pharmacological methods to isolate them. Therefore, to study the contributions of different ions to the inhibitory current, we measured the size of the p.s.p. under conditions that held the excitatory current constant. Then, although each p.s.p. had a contribution from the excitatory current, differences between experimental and control p.s.p.s recorded at the same holding p ^ ^

9 Synapses in the stomatogastric ganglion of the spiny lobster 161 J measure changes in the contributions of the inhibitory currents themselves. The time-to-peak of the hyperpolarizing part of the p.s.p. was not constant under different experimental conditions when the IVN interneurones were stimulated at 2-5 Hz (Fig. 3) so we measured the size of the summed p.s.p. immediately following a 2 s, 25 Hz train of stimuli to IVN (Fig. 4). This summed compound p.s.p. had an excitatory component, but this component was comparatively constant, and so could be accounted for in the analysis of i.p.s.p.s. If K+ is the charge carrier for the inhibitory current, a two-fold change in the external concentration of K + should change the size of the i.p.s.p.s measured at a controlled starting voltage by 17 mv. We measured the size of the summed p.s.p. following a 25 Hz train of IVN stimuli (arrows in Fig. 4), and plotted these measurements as a function of the holding potential at which each p.s.p. was measured (Fig. 5). The reversal potential for the control p.s.p.s was 68-3 mv, and the reversal potential changed to 52-6 mv when external K + increased twofold. The difference between the predicted 17 mv and measured 15 mv is within the limits of accuracy of our recording apparatus. In these neurones, a pure K+ current in normal saline should have a reversal potential of 80 mv (Marder & Paupardin-Tritsch, 1978 a), so the difference between this and the measured reversal potential reflects the contribution of other currents. If Cl~ also acts as a charge-carrier for the inhibitory current, then altering the external or internal Cl~ concentration should also change the size of the i.p.s.p. These experiments are complicated by the presence in these neurones of a significant nonsynaptic resting Cl~ conductance (Marder & Paupardin-Tritsch, 1978a) that will allow Cl~ to redistribute across the membrane rapidly. Therefore we injected CL~ through a microelectrode into an individual neurone by prolonged hyperpolarization (Kehoe, 1972a), and measured the size of the summed p.s.p. at the end of a 2 s, 25 Hz, train of IVN stimuli (Fig. 4), immediately after the injection was complete. It was not possible to measure the amount of Cl~ injected into the neurone, and so we can not predict the corresponding change in the reversal potential of a purely Cl~ carried i.p.s.p., but Figs. 4 and 5 show that increasing the internal Cl~ concentration changed the size and reversal potential of this summed, compound p.s.p. We conclude that Cl~ also contributes to the inhibitory current at this synapse. The agreement between the measured and predicted change in reversal potential when K+ concentration changed two-fold does not mean that only K+ acts as a charge carrier because Cl~ could redistribute across the membrane during the 15 min intervals between the change in K+ concentration and stimulation of the IVN interneurones. The excitatory component of this p.s.p. increased in size as the neurone was hyperpolarized. It did not reverse in the range of potentials below threshold, and it did not appear to be the product of an electrical synapse because it was blocked reversibly by a low Ca 2+, high Mg 2+ external medium (Fig. 6). We suggest that this component is an inward Na + current, but we have not explored this question further. Ionic basis of e.p.s.p. in VD. The synapse of the IVN interneurones with VD appears to be purely excitatory. If VD is hyperpolarized, the IVN p.s.p. fails to evoke an impulse in VD, and the underlying e.p.s.p. is revealed (Fig. 7). The p.s.p. showed decrement with repeated stimulation but reached a steady-state amplitude after stimuli. This e.p.s.p. is probably due to an increase in Na + conductance, because

10 162 KAREN A. SIGVARDT AND B. MULLONEY Control High Mg 2+, low Ca 2+ VD, PD I 5mV 20 ms Fig. 6. High Mg s+, low Ca i+ saline blocks the IVN postsynaptic potentials in PD and VD. Top trace: SON; second trace: SGN; third trace: VD; bottom trace: PD. Top. Control saline shows normal response in VD and PD with 17 msec delay from IVN axonal impulse in SGN (arrow). Bottom. High Mg J+, low Ca 8+ saline blocks the response. The impulse in VD was spontaneous, not synaptically driven. the e.p.s.p. increased in amplitude as the membrane was hyperpolarized, never reversed and was unaffected by increases in external potassium ion concentration (Fig. 7) or internal Cl~ injection. However, we did not test the Na + hypothesis directly. Blocking agents and the IVN p.s.p.s in PD and VD The identity of the transmitter released by the IVN command interneurones is not known, but the pharmacology of receptors for various possible transmitters has been described for different neurones of the StG (Marder & Paupardin-Tritsch, 1978a, b). Therefore, we used two different pharmacological agents in an attempt to separate the different ionic currents on the p.s.p.s in PD and VD. Picrotoxin, io~ 4 M, in control saline had no effect on transmission at these synapses (Fig. 8). This concentration is known to block transmission at certain other synapses in the ganglion (Bidaut, 1980). 5 x io~ 5 M D-Tubocurare (D-TC) did not affect transmission, but 5 x io~ 4 M blocked the inhibitory component completely and ^

11 Synapses in the stomatogastric ganglion of the spiny lobster 163 2x do, Control -50, VD <r~. J-- J^^-J^ _~Jv ^ J.J..XXX , VJ J 1 s 10 mv 100^ Fig. 7. The IVN e.p.s.p. in VD at different levels of membrane potential in control saline and high potassium saline (2 x [K + ] o ). Left. As the membrane of VD is hyperpolarized, the neurone fails to fire and the underlying IVN e.p.s.p. is revealed. The e.p.s.p. decrements somewhat but at rest potential the e.p.s.p. still remains large enough to fire the cell. Right. Changing the external potassium concentration has no effect on the p.s.p. (compare the p.s.p.s at 90 mv). the excitatory component (Figs. 8, 9). The reduction of the excitatory component accurred in both PD and VD (Fig. 9). Biphasic synoptic potentials DISCUSSION The effect of the biphasic p.s.p. in PD depends on the burst structure of the IVN j^rneurones. When the IVN units fire at a low-frequency, the p.s.p. is excitatory

12 164 KAREN A. SIGVARDT AND B. MULLONEY PD Wash' ^WttUlWlMltt)lUUMii)»ii'^ -** l0mv 1 s Fig. 8. The effect of picrotoxin and curare on the biphasic IVN synaptic potential in PD. Stimulation frequency 2-5 Hz (dots) and 25 Hz (bar). The biphasic p.s.p. in control saline is unaffected by io~* M picrotoxin. 5 x io~ 4 D-tubocurare blocks the inhibitory component but the depolarizing component remains. The effect of D-TC is reversible. and can cause the cell to fire if its membrane potential is near threshold. At the beginning of a high-frequency IVN burst, the excitatory components of the IVN p.s.p.s can cause the cell to fire. After the first few p.s.p.s the inhibitory components sum to short-circuit the excitatory components and prevent the cell from firing (i.e. the postsynaptic response effectively changes sign). Another synapse with a frequency-dependence similar to that of the IVN-PD synapse is the synapse from Lio to L7 in Aplysia (Wachtel & Kandel, 1967, 1971). Lio produces an e.p.s.p. in L7 at low frequency that converts to an i.p.s.p. at higher frequencies. However their results suggest a different mechanism for the conversion than the one we propose for the IVN-PD synapse; at the L10-L7 synapse the excitatory receptor desensitizes while the inhibitory receptor becomes sensitized to the transmitter. The biphasic p.s.p. described by Berry & Cottrell (1975) is always functionally inhibitory because the excitatory component is initially small compared to the inhibitory component and the inhibitory component facilitates. Several biphasic or dual-action synapses have been described in molluscan nervous systems (Berry & Cottrell, 1975; Gardner & Kandel, 1972; Kehoe, 1969, 1972a, b; Levitan & Tauc, 1975; Wachtel & Kandel, 1967, 1971). They are interesting because, following the release of a single transmitter, the postsynaptic cell has a multicomponent response that results from the activation of more than one type of receptor in the postsynaptic membrane. In most of these cases, the biphasic potential consists of a depolarizing phase and a hyperpolarizing phase, although the synaptic potential described by Kehoe (1969, 1972a) has two inhibitory components - a rapid i.p.s.p. superimposed on a slow, long-lasting hyperpolarizing wave. The postsynaptic receptors that underlie these dual-action synapses vary in ways that include the ^J

13 Synapses in the stomatogastric ganglion of the spiny lobster 165 Control PD 5 X 10-5 MdTC 10 mv Fig. 9. Effect of D-tubocurare on the response of PD and VD to IVN stimulation. Top trace: PD. Bottom trace: VD. IVN stimulation at 2-5 Hz and 25 Hz. Control saline shows normal response at 2-5 Hz (dots) and 25 Hz (bar) - a biphasic p.s.p. in PD and e.p.s.p. in VD. The response in PD and VD is unaffected by 5 x io~ 6 M D-TC. 5 x io~ 4 M D-TC blocks the inhibitory component of the PD biphasic response and the e.p.s.p. onto VD. conductance change, threshold for activation, kinetics, and facilitation or desensitization properties. As a result, the biphasic potential may be very sensitive to the pattern of firing of the presynaptic interneurone; its amplitude and effect depend on the temporal characteristics of the burst. These biphasic potentials thus permit more integrative complexity than conventional synaptic potentials that may vary in amplitude but not in sign. Functional consequences of synaptic properties The two IVN interneurones are command neurones that originate centrally and provide a pathway for specific alteration of the motor output of the ganglion. A burst in the IVN interneurones is initiated by various types of sensory input including input from mechanoreceptors in the pyloric region of the stomach (Sigvardt & Mulloney, 1981). In isolated preparations, where no sensory fields remain attached, these fibres will often fire prolonged bursts spontaneously (Selverston-e* al. 1976). In both situations, the bursts have similar structure; they start with impulses occurring at about KIz, rapidly accelerate to over 50 Hz and then slow to a stable plateau of about 10 Hz

14 166 KAREN A. SIGVARDT AND B. MULLONEY (Sigvardt & Mulloney, ibid., Fig. 5). Bursts may last as long as 20 s. Because of this( variation in frequency during a burst, the dynamic properties of the different synapses are significant determinants of the responses of the postsynaptic neurones during and after normal IVN bursts. For example, low-frequency impulses in the IVN interneurones will each trigger impulses in VD but allow PD to burst without interruption, and will not affect the ongoing activity of the neurones of the gastric system. At the other end of the scale, a sudden high-frequency burst in the IVN interneurones will drive a high-frequency burst in VD but inhibit completely PD, GM, LPGN and Int 1. The IVN interneurones are a convenient experimental pathway that can be used to perturb the ganglion's spontaneous activity in a controlled way. Ayers & Selverston (1977, 1979) stimulated the IVNs at frequencies ranging from 0-59 to 1-52 Hz to produce e.p.s.p.s in PD at periods just shorter than the inherent period of PD bursts, in an attempt to demonstrate the cellular mechanism for the entrainment of coupled oscillators described by von Hoist (1939). In the stimulus regime used by Ayers & Selverston, the excitatory component of the biphasic IVN p.s.p. in PD predominates (cf Fig. 1) and PD oscillations can be entrained by these cyclic excitatory inputs. Strong excitatory input to any oscillatory system will entrain that system in a manner that is largely a function of the difference between the period of the input and the period of the free-running oscillator (e.g. Stein, 1976). However, the spontaneous and sensory-initiated bursts have structures very different from the experimental trains used in these entrainment experiments. Ionic mechanism of i.p.s.p. in PD It is probable that the inhibitory component of the biphasic p.s.p. in PD is the result of an increase in conductance to both K+ and Cl~ because manipulations of the concentrations of each had a large effect on the reversal potential of this component. Both increasing extracellular K+ and increasing intracellular Cl~ shifted the reversal potential in the depolarizing direction, as is predicted from the Nernst relation if each ion can act as a charge-carrier here. Although the results support qualitatively the hypothesis of an increased conductance to both ions, it is not possible to consider the results quantitatively because of the high resting Cl~ conductance of stomatogastric neurones. Any manipulation of the intracellular or extracellular concentration of one of the ions simultaneously affects the resting membrane potential and the equilibrium potentials of K + and Cl~. The dual-component synapse described by Kehoe (1969, 1972 a) is a result of an increase in both Cl~ and K+ conductance, but the Cl~ mediated and K+ mediated phases have different latencies and different durations, and so cause a p.s.p. that has two inhibitory phases rather than a single inhibitory phase. The other dual excitatory-inhibitory p.s.p.s whose ionic mechanisms have been described (Wachtel & Kandel, 1971; Gardner & Kandel, 1972) have an excitatory component that is Na + dependent and an inhibitory component that is Cl~ dependent. Pharmacological properties of the biphasic p.s.p.s Marder & Paupardin-Tritsch (1978 a) have described the pharmacological properties of the responses of some of the stomatogastric neurones to various transmitt^

15 Synapses in the stomatogastric ganglion of the spiny lobster 167 (substances. PD shows K+ dependent and Cl~ dependent inhibitory responses to both glutamate and GABA. However, all of these responses except the GABA Cl~ response are blocked by picrotoxin, which did not block the response of any StG neurone to IVN interneurones. Therefore, it is unlikely that the IVN interneurones release GABA or glutamate. Acetylcholine causes a depolarizing response in PD and VD that is blocked by D-tubocurare. Although we were unable to block completely the excitatory component of the biphasic p.s.p. in PD, Marder reports that the IVN e.p.s.p. in PD is blocked by curare and in our study the IVN e.p.s.p. in VD was partially blocked by curare. Marder & Paupardin-Tritsch (19786) also discovered a biphasic response to acetyl-/?-methylcholine that has an early excitatory component and a longer-lasting, K+ dependent inhibitory component. Acetylcholine is the putative transmitter for several of the stomatogastric neurones including PD and VD, and all of these neurones cause i.p.s.p.s in other stomatogastric neurones (Marder, 1974, 1976). Thus, although acetylcholine remains a candidate for the transmitter released by the IVN interneurone, the identity of the transmitter is still unknown. We thank Hilary Anderson, Duncan Byers, Donald H. Edwards, Jr, Gad Geiger, Richard Nassel, Dorothy H. Paul and Kate Skinner for criticizing drafts of this paper. This research was supported by USPHS Grant NS 12295, by a USPHS Postdoctoral Fellowship to K.A.S. and by the Alfred P. Sloan Foundation. REFERENCES AYERS, J. L. & SELVERSTON, A. I. (1977). Synaptic control of an endogenous pacemaker network. J. Physiol., Paris 73, AYERS, J. L. & SELVERSTON, A. I. (1979). Monosynaptic entrainment of an endogenous pacemaker network: a cellular mechanism for von Hoist's Magnet Effect. J. comp. Physiol. 129, BERRY, M. S. & COTTRELL, G. A. (1975). Excitatory, inhibitory and biphasic synaptic potentials mediated by an identified dopamine-containing neurone. J. Physiol, Lond. 244, BIDAUT, M. (1980). Pharmacological dissection of pyloric network of the lobster stomatogastric ganglion using picrotoxin. J'. Neurophysiol. 44, DANDO, M. R. & SELVERSTON, A. I. (1972). Command fibres from the supraoesophageal to the stomatogastric ganglion in Panulirus argus. J. comp. Physiol. 78, GARDNER, D. & KANDEL, E. R. (1972). Diphasic postsynaptic potential: a chemical synapse capable of mediating conjoint excitation and inhibition. Science, N. Y. 176, GOLA, M. & SELVERSTON, A. I. (1981). Ionic requirements for bursting activity in lobster stomatogastric neurones. J. comp. Physiol. (in the Press). KEHOE, J. S. (1969). Single presynaptic neurone mediates a two-component postsynaptic inhibition. Nature, Lond. 221, KEHOE, J. S. (1972a). Ionic mechanism of a two-component cholinergic inhibition in Aplysia neurones. J. Physiol., Lond. 225, KEHOE, J. S. (1972b). The physiological role of three acetylcholine leceptors in synaptic transmission in Aplysia. J. Physiol, Lond. 225, KUSHNER, P. D. (1979). Location of interganglionic neurones in the stomatogastric system of the spiny lobster. J. Neurocytol. 8, LEVITAN, H. & TAUC, L. (1975). Polyphasic synaptic potentials in the ganglion of the mollusc, Navanax. J. Physiol., Lond. 248, 35~44- MARDER, E. (1974). Acetylcholine as an excitatory neuromuscular transmitter in the stomatogastric system of the lobster. Nature, Lond. 251, MARDER, E. (1976). Cholinergic motor neurones in the stomatogastric system of the lobster. J. Physiol., Lond. 257, H R, E. & PAUPARDIN-TRITSCH, D. (1978a). The pharmacological properties of some crustacean

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