A SYSTEM OF COMMAND NEURONS IN SNAIL'S

ACTA

NEUROBIOL. EXP.

1979, 39: 97-10'7

A SYSTEM OF COMMAND NEURONS IN SNAIL'S ESCAPE BEHAVIOR P. M. BALABAN Laboratory of conditioned reflexes, Institute of Higher Nervous Activity and Neurophysiology, Moscow, USSR

Abstract. Intracellular stimulation of each of the nine identified neurons released a specific withdrawal reactions. These cells fire in response to strong tactile, light and thermal stimuli releasing escape 'behavior, and are assumed to be command neurons. INTRODUCTION

The term "command neurons" was introduced into the literature from experiments on Crustaceans but was not formally defined (10). Recently Kupfermann and Weiss defined command neurons as both necessary and sufficient for the initiation of a given behavior (7). We found nine cells in the pleuro-parieto-abdominal ganglia complex with the following properties: (i) intracellularly evoked discharge of each cell releases withdrawal reactions; (ii) these cells fire during execution of normal escape behavior evoked by the sensory stimulation, their action potentials preceding withdrawal reactions; (iii) the sensory input of these neurons is polymodal and convergent; (iv) their receptive field extends all over the snail skin; {v) every neuron can act independently from others and in parallel with them. These nerve cells are considered as putative command neurons underlying escape behavior. METHODS

Experiments were done on semi-intact preparation of the snail Helix lucorum L. (Crymea population), species which is close to Helix pomatia in so as far the cellular topography is concerned. The dorsal side of the

suboesophageal complex was exposed. The connections of the central nervous system (CNS) with effectors and receptors were intact. The foot was dissected into two symmetrical parts with CNS and visceral organs placed between. Standard von Frey hairs were used for tactile stimulation. Diameter of the hair tip was 0.1 mm, the pressure applied was 0.15 g (weak stimulus), 0.57 g (moderate stimuli), 0.97 g (strong stimulus). Hairs were attached to the moving part of the coil. Tactile stimuli were applied with the aid of an electromagnetic coil driven by the 100 ms train of pulses. The opening or closure of pneumostome were monitored on an oscilloscope using a photusensitive element. The light was directed through the aperture of the lung cavity representing the pneumostome diameter. CNS was placed in a chamber with constant flow of Ringer solution (in mM: NaCl - 80, KC1 - 4, CaC12 - 7, MgC12 - 5, Tris-HC1 - 5, pH - 7.8). Intracellular stimulation and recording were done with glass microelectrodes filled with 3 M KC1 or K-citrate. Conventional recording and stimulating techniques were used. A depolarizing current was applied through the second intracellular microelectrode, or through the recording electrode via a bridge network. Nine identified neurons in the pleural and parietal ganglia of the snail were studied in 211 recordings. A schematic representation of the dorsal surface of suboesophageal ganglia complex was based on photographs and on histological serial sections. The neurons were numbered according to the map published previously (8). RESULTS

Witltdrawal behavior of the intact snail Withdrawal reactions of the snail can 'be evoked by tactile, thermal, light-off, and gravitational stimuli. Electrical stimulation of the skin, vapour of ethanol also elicit withdrawal behavior. The withdrawal reaction type depends on the intensity of stimulation. (i) A weak tactile stimulus evokes usually a local contraction only in the place of stimulation. CNS extinpation does not alter the latency of this response indicating the participation of peripheral nervous system. (ii) Moderate tactile stimulus of any point on the skin elicits additionally the withdrawal of tentacles and pneumostome closure. Extirpation of CNS eliminates these reactions, unless the stimuli are applied directly at the pneumostome or tentacle. It demonstrates the participation of CNS neurons. (iii) A strong stimulus initiates (besides the types of reactions described above) a contraction of foot muscles, mantle bolster muscles, or even a complete withdrawal of the mollusc into the shell. This type of reactions represents igeneralized reflex,

These types of reactions are arranged hierarchically - each one includes all the preceding. Withdrawal reactions can be evoked by different modalities of stimulation in any point of the snail's skin and mantle bolster. Wide receptive fields were characteristic for all types of withdrawal reactions composin.g escape behavior. The closure of the lung cavity in response to noxious stimulation was used for neurophysiological analysis. Usually pneuniostome is continuously opened fur respiration. Its 'closure occurs only when the animal is disturbed (1). Stimulation of any mentioned modality at any point of the body surface evoke pneumostome closure. An increase of stimulus intensity reduces the latency of this reaction, enlarges its amplitude and duration. The stimulus frequency of 6/min and above decreased the amplitude and increased the latency of the reaction (3). Threshold intensities of tactile stimulation for pneumostome closure and tentacle withldrawal coincide. The dynamics of these two reflexes is similar (3). Command neurons in withdrawal ref& The three described types of unconditioned withdrawal reactions remain unchanged in semi-intact preparation. In this preparation the pneumostome was usually closed demonstrating contraction in response to noxious stimuli. Mechanical stimulation of the snails skin evoked responses in most of the studied neurons. It was impossible to find in that way the cells controlling withdrawal response. Intracellular stimulation, resulting in a firing frequency of up to 20 spikes in 40 identified neurons located on the dorsal side of the suboesophageal ganglion complex (Fig. 1) revealed that only nine giant neurons evoke withdrawal reactions demonstrating putative command functions (3, 4, 7). LPa3 and RPa3. These two largest identified neurons are located symmetrically on both sides of the medial part of the parietal ganglia (Fig. 1). Spike discharges evoked by the depolarizing current in cells LPa3 and RPa3 elicit pneumatome closure or its clenching if it is closed, as a component of avoidance reaction (Fig. 2 B, C, D). If the firing frequency is low (3 spike/s), the latency (measuring from the first action potential) of the motor response reaches 1,000 ms (Fig. 2 B). The withdrawal reaction filmed in parallel with oscilloscope recording, was identified as a contraction of pneumostome muscles for a short period of time. An increase of the injected current increases the firing frequency at the command neurons reducing the latency and augmenting the magnitude of reaction (Fig. 2 C). Visual observation in this case revealed that besides pneumostome clenching the high frequency discharge of neurons LPa3 and RPa3 elicited

a contraction of mantle bolster muscles.' Minimum latency of effector reaction was 300 ms for stimulation of RPa3 and 500 ms for stimulation of LPa3.

Fig. 1. Schematic representation of suboesophageal ganglia complex based on' photographs of dissections and histological serial sections; dorsal view. Numbers for neurons are used in accord with the map pub1.ished previously (8). Neurons (4) in parietal ganglia are not seen because of their ventral position. Histological investigations found additionaly two giant cells in right parietal ganglion and one in visceral on the ventral side (dotted line); LP1, left pleural ganglion; LPa, left parietal ganglion; RPa, right parietal ganglion; RP1, right pleural ganglion; V, visceral ganglion (abdominal). Only readily identified cells are numbered.

Fig. 2. Withdrawal reaction of pneumostome closure evoked by intracellular stimulation of cell RPa3 by depolaridng current injected through the second microelectrode. a, current 2 nA; b, 3 nA; c, 6 nA; d, 6 nA after perfusion of CNS with high Mg low Ca Ringer solution. In this and subsequent Figures beginning of motor response is marked by a triangle. Upper curves, photoregistration of movements. Calibration 10 mV, 1 s. Amplification of movement recording was constant.

A semi-intact preparation with CNS placed in a microchamber enables low Ca2+ Ringer solution under to perfuse CNS neurons with high ~ g 2 + osmic pressure balanced by Tris. After 30-40 min of perfusion, the spontaneous excitatory postsynaptic potentials (PSPs) dissappeared. Under such conditions even a strong tactile stimulation could not evoke pneumostome closure. Nevertheless, the intracellular stimulation of neurons LPa3 and/or RPa3 elicited pneumostome closure (Fig. 2 D). No contraction of man$le bolster muscles was evident, however, even if firing frequency reached 20 spikes s-1. Usually a single action potential does not evoke a motor response (Fig. 2 A), In some cases, however, slight contractions of pneumostome muscles correspond to separate spikes observed in the neuron RPa3. This occurred for rare spontaneous spikes (Fig. 3 A) and spikes evoked by depolarizing current (Fig. 3 B). It is noticeable that the latencies of such muscle jerks corresponding to spikes were 300 ms. An increase in spike frequency did not influence this minimum latency, equal for all studied cells. h most cases however, no correlation between individual spikes and muscle contractions was observed. The latency of withdrawal reaction released by intracellular stimulation varied between 300 and 1,000 ms (Figs. 2 and 3). A change of the firing frequency influenced the latency and amplitude of evoked reaction. The total duration of spike discharge, however, does not affect the response (1). LPa2, RPa2 and LPa5. Intracellular stimulation of those three giant cells of parietal ganglia (Fig. 1) also resulted in the withdrawal reactions. Only high firing frequency evoked motor responses with latencies between 1 s and 3 s (Fig. 4). No correspondence of muscle contractions and action potentials was observed. The threshold firing frequency for motor response varied in each experiment. Visual observation showed that a high frequency discharge of neuron LPa5 elicited contractions of muscles located in medial and caudal regions,

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Fig. 3. Relations between action potentials in cell FbPa3 and contractions of pneumostome muscle (dots). a, spontaneous spikes (lower curves); b, spikes evoked by the depolarizing current injected through the recording electrode using bridge circuit; AC, registration of neuronal activity; DC, registration of

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their movements; total amplitude spikes are is not retouched given. Caliand bration 10 mV, 1 s.

of the left part of the' foot, contractions of mantle ;bolster muscles, and pneumostome closure. Pneumostome closure, .however, resulted from mantle lhlster contraction, and was characterized by a long (up to 2 s) latency. The influence of LPa5 discharge is greater on the foot muscle than on the mantle bolster muscles.

Fig. 4. Low frequency discharge (a) of cell RPa2 (current 3 nA injected through the second electrode) did not evoke any reaction (upper curve). High frequency discharge (b, 6 nA) releases pneumostome closure characterised by great latency. Calibration 10 mV, 1 s.

A high frequency spike discharge of cell LPa2 evoked the same movements as LPa5 but its influence oh mantle 'bolster muscles was greater than on the foot. The high frequency discharge of RPa2 induced by intracellular stimulation elicited a contraction of mantle bolster muscles, a pneumostome closure and, in some experiments the contractions of ipsilateral 'body wall. A low frequency discharge of these neurons did not evoke any reaction (Fig. 4 A). After half an hour perfusion of CNS with high Mgz+ Ringer solution even a high frequency spike discharge of cells Lpa2, RPa2 and LPa5 evoked no reactions. Some unidentified cells seems to mediate the efferent output of these neurons. Pleural ganglia giant cells. Only a high frequency discharge of neurons LPll and RPll (Fig. 1) evoked by intracellular stimulation elicits muscular contractions of ipsilateral body wall. The same contractions can be elicited by strong tactile stimulation of the contialateral part of the foot or of the mantle bolster. Intracellularly induced spike discharge of neurons LP12 and RP12 elicited a tentacle withdrawal respoase and associated contractions of the body near the head region. . Spike activity of nine studied cells seems to be responsible for gll types of withdrawal reactions evoked by natural stimulation in intact animals. We have not stimulated all neurons together, but a simultaneous intracellular stimulation of two neurons resulted in a reduction of latency and an increase of the pneumostome closure amplitude as compared with separate stimulation (Fig. 5). It might be concluded that outputs of both neurons converge on common elements (located most likely in the CNS)

because different pools of interneurons would not decrease the response latency. Endogenous properties. Usually nerve cells LPa3, RPa3, LPa2, LPa5, LP11, RP11, LP12, RP12 are silent despite the subthreshold spontaneous EPSPs (Fig. 6). The firing threshold of these neurons is rather high and

Fig. 5. Pneumostome closure (upper curve) evoked by intracellular activation of neurons LPa2 (middle trace) and RPa3 (lower trace) through independent electrodes. Depolarizing current in all trials 7 nA. a, simultaneous activation; b, c, alternative activation with 10 min interval. Calibration 10 mV, 1 s.

Fig. 6. Synchronous EPSPs in command neurons. Simultaneous recording from cells LPa3 and RPa3 (1, 2 ) ; RPa3 and LPa2 (3); LPa2 and RPa2 (4, 5). The start of a long depolarization through the recording electrode is marked with an arrow; elecyrical stimulation of right pallial nerve is also marked with an arrow (5). Spikes retouched. Calibration 10 mV, 1 s.

only 10-30 mV depolarization induced their spike discharges. A rapid habituation of spike response and a consequent hsbituation of motor responses to successive intracellular stimulation is characteristic for these cells. The lack of spontaneous activity emphasized the importance of each action potential evoked in the cells by applied stimulation (9). Input characteristics. The receptive fields of nine studied neurons are extremely wide. Tactile stimulation of any part of the snail skin evoked in all nine cells compound EPSPs with 90-100 ms latency. Spike generation occurred only when the intensity of stimulus exceeded threshold of mediated centrally withdrawal reactions. A tactile stimulus of moderate intensity evoked phasic firing usually in ipsilateral neurons only. A strong stimulus evoked spike responses in all nine cells, accompanied by generalized withdrawal reaction. Every neuron has in its wide receptive field a specific area the stimulation of which results in a maximum discharge. Ipsilateral stimulation evoke greater response in cells than contralateral stimulation. The habituation of spike response to ipsilateral in respett of neuronal location stimulation was slower than to contralateral stimulation. Thus the first tactile stimulation of the left part of the foot evoked two spikes in neurons LPa3 and RPa3 (Fig. 7 A). The forth stimulus applied with 10 interval evoked only two action p~tentialsin cell LPa3 but no spikes in contralateral cell RPa3 (Fig. 7B). The pneumostome area was an exceptive: a moderate tactile stimulation of the pneumostome evoked in cells LPa3 and RPa3 a maximum spike discharge (up to 6 synaptically induced action potentials), followed by activation of pacemaker spikes. Specific area for neurons LPa5, RPa2, LPa2, LPll and RPll was the ipsilateral part of the foot. Cells LP12 and RP12 responded with a maximum discharge if ipsilateral rostra1 part of the foot was stimulated.

Fig. 7. Pneumostome closure (upper curve) and reactions of cells LPa3 (middle trace)- and RPa3 (bottom trace) evoked by moderate intensity tactile stimulation (arrow) of the left side of the foot. a, first stimulation; b, fourth in a sequence of stimuli (frequency 0, 1 Hz). Spikes are retouched, total amplitude not shown. Calibration 10 mV, 1 s.

Nine command neurons respond also with spike discharge to switching off the light, local skin heating and electrical stimulation of the skin. Action potentials in these neurons always precede withdrawal responses. Wide receptive fields and convergence of noxious stimulation of different modalities are characteristic features of th6se neurons. Spontaneous subthreshold EPSPs are highly synchronous in all studied cells. Ninety percent of EPSPs are synchronous in neurons from parietal and nearly 50010 in cells from pleural ganglia. Common interneurons are presumably responsible for this (Fig. 6). Neither chemical nor electric connections exist between any pair within the nine studied neurons. Even a high frequency discharge of one cell, evoking a motor response, brings no change in activity of either neuron. The absence of connections and highly synchronous inputs of these cells suggest their parallel functioning. Close correspondence between spike discharges and behavioral responses during the habituation procedure indicates that cells LPa2, LPa3, LPa5, RPa2, RPa3, LP11, LP12, RP11, RP12 are responsible for neuronal mechanisms of the withdrawal responses (2). DISCUSSION

Spike discharges evoked in each of the nine investigated neurons result in motor reactions, representing the components of escape behavior. These neurons, however, are not motoneurons. The minimum latency of muscle contraction evoked by their discharge is 300 ms. Taking into account a maximum time propagation 120-140 ms, it may be concluded that central command is mediated by peripheral neurons (6). This conclusion is reinforced by the fact that peripheral neurons located in the Helix pneumostome muscles are found histologically. There is no direct correlation of spontaneous pneurnostome movements and command neurons activity. Intracellular discharge of the command neurons elicits a coordinated contraction sequence of different muscles. The action potentials of the command neurons preceding withdrawal reaction are not required during performance of the motor response. Extremely wide receptive fields of command neurons coincide with receptive field of behavioral withdrawal reactions. The command neurons are characterized by a convergence of tactile, thermal and light stimulation demonstrating a multimodal mode of integration. A high firing threshold prevents their spiking to many sensory stimuli which reach them. Each neuron act as a device for detectiig dangerous stimuli and triggering withdrawal reactions (5). The cells LPa3 nad RPa3 are command neurons for unconditioned reflex of pneumostome closure. Cells LP12 and RP12 are command units 4

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for the tentacles withdrawal reaction. Neurons LPa2, RPa2, LPa5, LPll and RPll are less specific command neurons. Acting in parallel, all nine neurons induce an activation responsible for escape behavior. Schematic representation of the unconditioned reflex of the pneumostome closure circuit is shown in Fig. 8. Sensory information from all receptive surfaces passes to sensory block 1. Every command neuron receives information from sensory elements. The synaptic inputs coming to a particular command neuron from various receptive field regions differ from one another. Thus a stimulus of constant intensity evokes complex EPSPs of different amplitude when applied to different parts of the receptive surface. Such input characteristics of the command neurons are indicated by different size of the triangles. The neurons LPa3 and RPa3 are connected with the peripheral motor neurons directly and through the central motor elements (block 2). The cells LPa2, RPa2 and LPa5 regulate the motor response via the central motor elements only. High frequency firing of RPa2 released contraction of mantle bolster muscles together with pneumostome closure. A moderate tactile stimulus did not evoke high frequency discharge in RPa2 and consequently no generalized behavioral reaction. Few action potentials in cell RPa3 were sufficient, however, for pneumostome closure. A moderate tactile stimulation evoking several spikes in cell RPa3 or LPa3 results in reactions of some effectors only. It shows that under such conditions only specific command neurons for these effectors are acting. Strong stimulus elicits a sfnchronous discharge in all command neurons and a generalized escape reaction.

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Fig. 8. Schematic representation of unconditioned withdrawal reflex (pneumostome closure) neural structure. Receptive field (left and right parts of the foot with mantle bolster between them) is shown on the top. 1, pool of sensory neurons (presumably primary) receiving information from the whole receptive field and sending it to the command neurons. Effectiveness of input is shown by different size of triangles. 2, hypothetical pool of central motor cells connected with the peripheral net or muscles 3. Pn, pneumostome. Command neurons LPa3 and RPa3 are connected directly to the peripheral net and can simultaneously influence central motor cells. Neurons RPa2, LPa2 and LPa5 can evoke pneumostome closure only via central motor cells. Cells from pool (2) can participate in other avoidance reactions as well.

The neurons LP12 and RP12 are mainly responsible for the tentacles withdrawal. For representation of total sequence of escape behavior all command neurons should lbe taken into consideration. Described cells satisfy the "sufficiency" criterion of Kupfermann and Weiss, act during execution of withdrawal reactions evoked by an adequate stimulation, but the necessity criterion is fullfilled in only a limited number of cases (7). We consider a really necessary condition for command function is the convergence of sensory information necessary and sufficient for a cer.tain behavioral act. I am grateful to Professor E. N. Sokolov for careful guiding this work. REFERENCES 1. BALABAN, P. M. 1976. Functional organisation and neural structure of unconditioned reflex of the snail. In J. Salanki (ed.), Neurobiology of invertebrates. Gastropoda brain. Akademiai Kiado, Budapest, p. 561-566. 2. BALABAN, P. M. 1978. Habftuation and sensitisation in command neurons of the avoidance reflex of the snail (in Russian). Zh. Vyssh. Nervn. Deyat. Im. I. P. Pavlova 28: 356-363. 3. BALABAN, P. M., and LITVINOV, E. G. 1977. Command neurons of unconditioned reflex of the snail (in Russian). Zh. Vyssh. Nervn. Deyat. Im. I. P. Pavlova 27 : 538-544. 4. BOWERMAN, R. F. and LARIMER, J. L. 1976. Command neurons in crustaceans. Comp. Biochem. Physiol. 54A: 1-5. 5. BULLOCK T. H. 1961. The problem of recognition in an analiser made of neurons. In W. A. Rosenblith (ed.), Sensory communications. MIT Press, Boston, p. 717-724. 6. HOYLE G., and WILLOWS, A. 0. D. 1973. Neuronal basis of behavior in Tritonia. 11. Relationship of muscular contractions to nerve impulse pattern. J. Neurobiol. 4: 239-254. 7. KUPFERMANN, I. and Weiss, K. R. 1978. The command neuron concept. Behav. Brain Sci. 1 : 3-39. 8. LITVINOV, E. G. and BALABAN, P. M. 1973. Endogenous potentials in the organisation of the neuronal spike response to sensory stimulus. (in Russian). Zh. Vyssh. Nervn. Deyat. Im. I. P. Pavlova 23: 1313-1315. 9. SOKOLOV, E. N. 1973. The role of neuronal pacemaker potential in the mechanisms of behavior (in Russian). Zh. Vyssh. Nervn. Deyat. Im. I. P. Pavlova 23 : 1241-1243. 10. WIERSMA, C. A. G. and IKEDA, K. 1964. Interneurons commanding swimmeret movements in the crayfish Procambarus clarkii (Girard), Comp. Biochem. Physiol. 12: 509-525.

Accepted 15 December 1978 p. M. BALABAN, Institute of Higher N ~ v O U S Activity and Neurophysiology, Academy of Sciences of the USSR, Butlerova la, Moscow, USSR.