Neurophysiological correlates of tactile stimulus-induced whole-body eversion, a novel type of behavior in the snailHelix pomatia L

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Brain 16'ac,~r~h. ~ 12 i. 1~)t~3) 16 2 c 1993 Elsevier Science Publishers t3.V. All rights reser~'cd 0006 ,~9t~3/03 $06.()!~

BRES 18821

Neurophysiological correlates of tactile stimulus-induced whole-body eversion, a novel type of behavior in the snail Helix pomatia L. Gy6rgy Kemenes a, Igor S. Zakharov

b, Agnes Vehovszky

a and Katalin S.-R6zsa "

" Balaton Limnological Research Institute of the Hungarian Academy of Sciences, Tihany (Hungary) and h Institute of Higher Nervous Actiuity and Neurophysiology of the Academy Of Sciences of Russia, Moscow (Russian Federation) (Accepted 9 December 1992)

Key words: Tactile stimuli; Eversion; Pneumostome opening; Efferent neuron; Snail

Repeated weak tactile stimuli to the exposed skin of the foot of withdrawn snails (Helix pomatia L.) result in a rapid whole-body eversion response not previously described. We studied the neurophysiological correlates of this novel type of behavior in semi-intact preparations consisting of the foot, pneumostome and mantle collar attached to the CNS. The pneumostome opening component of the eversion response is retained in semi-intact preparations and can be triggered by a series of weak tactile stimuli to the foot. The same stimuli also strongly excite a giant neuron (LPd7) in the pedal ganglion. This cell has axon branches in the anal and left pallial nerve which innervate the pneumostome and mantle collar. Intracellular stimulation of LPd7 causes the pneumostome to open. Bursts of spikes in LPd7 also lead to contractions of a specific group of muscles in the mantle collar innervated by the left pallial nerve. Both responses are retained when the CNS is bathed in high Mg2+/0 Ca 2+ saline but the mantle response has a much shorter latency than the opening of the pneumostome. We conclude that LPd7 is an efferent neuron which causes opening of the pneumostome via a peripheral pathway in the mantle collar and may also be a motoneuron of muscles in the mantle collar. Besides its pneumostome-opener and putative mantle motoneuron function, the LPd7 cell also triggers movements of the foot which are also part of the eversion sequence in intact snails. The LPd7 cell receives excitatory input from neurons which trigger withdrawal reactions in active snails. The excitation is due to peripheral interactions between the two cell types and leads to opening of the closed pneumostome following brief aversive stimuli. We suggest that LPd7 is part of a novel putative neuronal network underlying the described eversive reaction to tactile stimuli.

INTRODUCTION

the general physiological mechanisms

underlying the

e m e r g e n c e has b e e n g i v e n by S o m m e r v i l l e 19. The

neuroethological

analysis

of various

aversive

It is a c o m m o n o b s e r v a t i o n t h a t i n c r e a s e d h u m i d i t y

b e h a v i o r a l r e a c t i o n s in m o l l u s c s has c o n t r i b u t e d g r e a t l y

or direct contact with water

to t h e u n d e r s t a n d i n g

s p o n s e in inactive, w i t h d r a w n t e r r e s t r i a l snails, b u t n o

associative

learning

of the neural processes

basis o f s i m p l e

(reviews

by

results

in e v e r s i o n re-

B y r n e 4,

i n f o r m a t i o n is a v a i l a b l e o n m o r e s p e c i f i c stimuli t h a t

C a r e w a n d SahleyS). B e h a v i o r a l c o m p o n e n t s a n d n e u -

might bring about such an eversion response. On the

rophysiological mechanisms of unconditioned and con-

o t h e r h a n d , s e n s i t i z a t i o n m e c h a n i s m s l e a d i n g to b e h a v -

d i t i o n e d w i t h d r a w a l r e a c t i o n s h a v e also b e e n d e s c r i b e d in t h e snail Helix in s t u d i e s b a s e d o n o b s e r v a t i o n s o f

ioral a r o u s a l in active a n i m a l s h a v e a l r e a d y b e e n studi e d in s e v e r a l m o l l u s c a n species ( A p l y s i a 13'24

intact active animals and on electrophysiological exper-

L y m n a e a ~2,20, Helix ~L). In t h e s e e x p e r i m e n t s f o o d stim-

i m e n t s w i t h s e m i - i n t a c t p r e p a r a t i o n s ~'3.

uli w e r e u s e d to b r i n g t h e a n i m a l s f r o m a ' n o r m a l ' level o f r e s p o n s i v e n e s s to an ' a r o u s e d ' level iT. In Aplysia

M u c h less a t t e n t i o n has b e e n p a i d to t h e c o u n t e r part of the aversive reaction, namely the eversion process by w h i c h t h e i n a c t i v e snail e m e r g e s f r o m t h e shell a n d b e c o m e s a c t i v e again. So far o n l y a d e s c r i p t i o n o f

californica c o m p l e x t a c t i l e stimuli, like tail p i n c h a n d h a n d l i n g , also h a d a n a r o u s i n g e f f e c t ~3, a n d s i m i l a r l y to f o o d - i n d u c e d a r o u s a l , t h e u n d e r l y i n g m e c h a n i s m was

Correspondence: G. Kemenes. Present address: Sussex Centre for Neuroscience, School of Biological Sciences, University of Sussex, Falmer. Brighton, BN1 9QG, UK. Fax: (44) (273) 678 433.

17 behavioral sensitization 24. Complex tactile stimuli also play an important arousing role in the initial phase of the mating behavior of helicid snails 8. We hypothesized that tactile stimuli that proved sensitizing in active animals might also bring the general level of activity from a suppressed (withdrawn snail) to a normal or even aroused level (everted, actively crawling snail). We examined the effect of tactile stimulation on withdrawn snails, and we found that repeated weak tactile stimuli to the small exposed area of the skin of the foot still visible in withdrawn snails resulted in opening response of the pneumostome followed by eversion behavior. This behavioral response has never been described previously. Using the same stimuli in semi-intact preparations, we then tried to relate the recorded muscle movements and neuronal activity to various elements of this novel type of behavior. As a first step we carried out an extensive search in the pedal ganglia for putative efferent eversion neurons that fulfil the following criteria. 1. They are excited by weak tactile stimuli applied to the skin of the foot and by electrical stimulation of the appropriate sensory nerve connecting the foot to the CNS in semi-intact preparations. 2. They send axons through nerves innervating the pneumostome a n d / o r the mantle collar and their action potentials can be recorded one for one from these nerves. 3. Their activation triggers opening movement of the pneumostome a n d / o r movements of the foot. Putative motoneurons for the opening of the pneumostome during respiration have previously been found in the visceral ganglion of the CNS of Helix 2. These opener motoneurons receive direct inhibitory inputs after strong tactile and other noxious stimuli to the skin but they are not excited by weak tactile stimuli 2 so they do not fulfil the above criteria. So far we have only found one cell that fulfils all the above criteria and can therefore be regarded as member of a putative network of eversion neurons. This neuron is a previously identified giant cell in the left pedal ganglion (LPd7 9,21). In the present paper first we give a behavioral description of the eversion response evoked by tactile stimuli in intact, inactive Helix. Then we examine how an important component of this behavior, the opening movement of the pneumostome is retained in semi-intact preparations and can be triggered by the same serial weak tactile stimuli that were used in intact snails. We report that these stimuli excite the LPd7 neuron and intracellular stimulation of LPd7 leads to opening movement of the pneumostome, comparable

to that evoked by tactile stimuli in intact snails or semi-intact preparations. We provide data indicating that LPd7 may be a motoneuron which sends efferent signals to muscles of a particular area of the mantle collar. We have previously reported 2~ that the LPd7 neuron was excited upon intracellular stimulation of members of a system of so called withdrawal command neurons t. Since withdrawal and eversion are mutually exclusive behaviors we wished to elucidate the mechanism and possible role of excitation of the LPd7 'eversion' neuron by the 'withdrawal' cells. We found that the activity of the LPd7 neuron is influenced by the LPa and RPa neurons via peripheral interactions rather than central connections and this may contribute to the opening movement of the pneumostome which often follows the closure evoked by brief aversive stimuli in active snails. Elucidation of neuronal pathways taking part in the activation of a withdrawn snail will also facilitate better understanding of more general sensitization processes. MATERIALS AND METHODS Behavioral experiments with intact withdrawn snails For the behavioral analysis, we used snails from a batch of 200 adult Helix pomatia specimens. T h e snails were collected locally on the Tihany peninsula in Hungary and maintained in the laboratory for several weeks prior to testing. T h e size of the batch was chosen to be about twice as large as the designed sample size so as to allow for loss of animals over the pre-experimental period and yet to be able to test at least a h u n d r e d snails of similar, controlled pre-testing history. Initially, the snails were fed lettuce ad libitum. The lettuce leaves were washed with plenty of water, so the snails were actually living on a 'bed' of moist leaves which kept the environment in their tanks humid. As a result, the snails were constantly active during this longer pre-experimental period. However, several days prior to the experiments, all lettuce was removed from the tanks and as a result, the snails were being kept unfed and in a dry environment. During this immediate pre-experimental period all the snails had withdrawn into their shells, and in the withdrawn state the p n e u m o s t o m e was constantly closed. For testing, we fixed individual snails by the shell to a horizontal holder so that the aperture of the shell was in a vertical position facing the experimenter. A solenoid-operated calibrated tactile stimulator unit was fixed in a manipulator and positioned so that by each pressing of a switch a single tactile stimulus of 4 g / m m 2 pressure could be applied. Alternatively, a hand-held light brush could be used for the same purpose. We applied the tactile stimulus to the skin of the foot not covered by the lobes of the mantle collar. Altogether we tested 112 snails of the original batch of 200 in groups from 20 to 60 either with single tactile stimuli or with series of 30 or 60 tactile stimuli given with 3 Hz frequency. We measured the latency of the following two behavioral responses: (1) opening of the pneumostome, which is a key event in the behavioral sequence leading to eversion 19, and (2) full eversion of the snail. We defined the latency of the p n e u m o s t o m e opening reaction as the time in seconds between the beginning of the tactile stimulation and the occurrence of the response. The latency of the full eversion was defined as the time between the last stimulus in the series and the full extension of the large tentacles at the end of the eversion sequence.

18 F'-..

Fig. 1. The semi-intact Helix preparation used in present study. The preparation allows application of the tactile stimulus to various parts of the body, movement recording from the foot (IMP. CONV., impedance converter) and the pneumostome and other mantle collar areas (DIFF. AMP., differential amplifier, right), intracellular recording from up to 4 neurons of the CNS (AMP.) and extracellular recording from nerves (DIFF. AMP., left). The central compartment containing the CNS is isolated from the rest of the preparation and can be perfused separately with high MgZ+/Ca2+-free saline. Using this preparation we investigated the neurophysiological mechanisms involved in the opening reaction of the pneumostome to tactile stimulation of the foot and to intracellular stimulation of the LPD7 neuron. If the snails did not emerge from the shell within 5 min after the last stimulus we regarded them as non-responding. The number of these snails was also recorded in the tests. When the snails became fully everted, we removed them from the holder and placed them on the dry bottom of an empty tray where they could crawl around freely. The duration of the post-testing active period was also measured and compared after 30 and 60 tactile stimuli applied in the original test. We used this as a measure of the level of general arousal. We used unpaired, one-tailed t-tests for the statistical analysis of the latency and post-test active period data. The percentage of responding snails was statistically compared between the groups by a test of the significance of the difference between percentages.

Electrophysiological experiments with semi-intact foot-CNS-mantle collar preparations made from withdrawn snails" To study the possible neurophysiological correlates of the behavioral phenomena observed, we used over 50 'whole-body' Helix preparations (Fig. 1). These were similar to those described by Maximova and Balaban is. Snails from the behaviorally tested groups were kept in a dry environment for several days prior to dissection. Before the experiments we kept the snails for 30 min in a refrigerator at 4°C. We dissected the animals on ice in order to anaesthetize them as well as to try to preserve the internal state of the withdrawn snails as much as possible for the electrophysiological experiments. We placed the CNS into a central circular compartment within a Sylgard-lined dish. The nerves were gently guided into 5 slots in the wall of the plexiglas circle and held in place with a soft rubber band

(Fig. l). The left and right side of the foot and the visceral hLimI) with the mantle collar and pncumostome facing upward were a l ranged around the central compartment. The pneumostome or dif ferent areas of the mantle were positioned on lhe highly polished flail tip of a vertical plexiglas rod inserted into the bottom of the' experimental dish and they could be illuminated from below. The arrangement of the preparation allowed tactile stimulation of lhc foot as well as simultaneous recording of movements of the pncu mostome, mantle collar and fl)ot. The skin on the dorsal surface ol the left foot was chosen as tile stimulus site. This allowed stimulation and movement recording to be performed at relatively distant parts of the body and thereby avoid interference from passive movements of the preparation caused by the tactile stimulus. For stimulation of the foot, we used the solenoid-operated tactile stimulator unit of the behavioral tests. For movement recording, at bipolar electrode connected to a Biocom. Inc. impedance converter (MOD-2991) and an electro-optical movement indicator device e~ were used (Fig. I). The bipolar electrode was measuring the impedance changes caused by muscle contractions in the saline in the immediate vicinity of the skin of the fllot or mantle. We tlsed tile electro-optical movement indicator to measure changes in the intensity of transmitted light clue to opening or closure of the pneu mostome or muscle contractions in the mantle. In one type of experiment the sensor of the electro-optical device was above the centre of the pneumostome. In a second type of experiment we positioned the sensor above the boundary of pneumostome and it could detect large active opening movements of the pneumostome as well as smaller passive movements caused by contractions in more distant mantle collar areas. In a third type of experiment the sensor of the electro-optical device or the bipolar electrode was positioned above different parts of the mantle collar to record muscle movements in response to intracellular stimulation of the LPd7 neuron (see below). Prior to experiments with a particular preparation we made visual observations on the pneumostome with parallel movement recording. This was to correlate upward and downward deflections of the oscilloscope and chart-recorder traces with opening or closure of the pneumostome. We used standard electrophysiological methods to record intracellularly from up to 3 neurons of the CNS. In the experiments with the semi-intact preparations two types of neurons were examined systematically. These were the giant LPd7 cell in the left pedal ganglion '~'2~ and the LPa2, LPa3, RPa2 and RPa3 giant neurons of the left and right parietal ganglia, respectively. In preliminary experiments we found that LPd7 is capable of triggering opening of the pneumostome in semi-intact preparations and responds to tactile stimulation of the foot. The parietal ganglion cells have been described as command neurons for withdrawal reactions, including closure of the pneumostome t. The CNS was constantly perfused with fresh Itelix saline. The foot and the visceral hump were only half immersed in saline and the exposed surfaces were kept moist by saline-soaked tissue paper pieces. The composition of the saline was as follows (mM): NaCI, 80: KCI, 4; CaCI 2, 10; MgCI 2, 5" Tris-HCl buffer, 4, to give a final pH of 7.4. The perfusion system could be switched to perfuse high Mg 2~ (50 raM), Ca2+-free saline over the CNS alone. We used the high M g 2 + / 0 Ca 2+ saline to study whether the output of the LPd7 neuron was influenced by uncoupling synaptic pathways within the CNS. Previously we had succesfully used this saline in the isolated Helix CNS to block identified central synapses, such as the ones between an identified pedal cell and members of a cluster of neurons in the right parietal ganglion 22. In semi-intact preparations, after perfusing the CNS for more than 30 min with high Mg 2 ~/0 Ca 2 saline, any inputs to LPd7 could only be attributed to peripheral synapses or direct sensory function of the cell. We monitored the activity of the left and right pallial and the intestinal nerves by extracellular bipolar platinum electrodes connected to a differential amplifier (Fig. 1). tn some experiments we cut the left pedal skin nerve distally to the CNS and we stimulated it by suction electrodes. In ttelix nerves the physiological range for extracellular potentials is between I and 15 ,ttV (see Fig. 5b). In present experiments the amplitude of the electric stimuli applied to the left pedal skin nerve was just above threshold and produced 5 to

19 After dehydration in graded alcohols and clearing in methyl salicylate, the wholemount preparations were mounted on slides in Canada balsam and photographed. This staining aided the unambiguous identification of the LPd7 neuron and the parietal cells from preparation to preparation.

10 /~V extracellular signals recorded from the same nerve more proximally to the CNS. This enabled us to study afferent inputs of various cell types and their resemblance to the inputs caused by tactile stimulation of the foot. The left pedal skin nerve was chosen because it innervates the foot surface which was stimulated in the behavioral and physiological experiments as well Is. To further analyze the putative motoneuronal function of the LPd7 neuron, we measured muscle contraction latency and intensity at different lobes of the mantle collar (Fig. 1), while systematically increasing the LPd7 spike frequency from 1 to up to 21 spikes per second. The inputs received by the LPd7 neuron from the LPa2 withdrawal command cell (one of the neurons that trigger closure of the pneumostome 1) were studied in preparations with intact nerves as well as in preparations where the left and right pallial, intestinal and anal nerves were all severed. The aim of these experiments was to find out whether the withdrawal command cell excited LPd7 through central or peripheral connections. For the statistical analysis of the effect of LPa2 activation on LPd7 activity, we measured instantaneous LPd7 spike frequencies before, during and immediately after intracellular stimulation of the LPa2 neuron in 3 different experiments (5, 7 and 13 replicates, respectively). This analysis was performed on preparations with intact nerves as well as 10 min and 2 h after severing the nerves in the same preparations. We considered any experimentally caused changes in cellular or muscle activity significant only if they were found in at least 75% of the total number of replicates within a particular type of experiment and in a minimum of three independent experiments with different semi-intact preparations. After electrophysiological experiments the identified neurons were filled intracellularly with Ni2+-lysine solution 7. To develop the chemical reaction for staining, rubeanic acid solution was used according to the method employed by Quicke and Brace I6.

RESULTS

Opening of the pneumostome and whole-body eversion response evoked by tactile stimuli in intact, withdrawn snails A single tactile stimulus to the exposed skin of the foot resulted in a weak local contraction of the mantle collar. This was never accompanied by any reaction of either the closed pneumostome or the whole body of the withdrawn snail. Serial tactile stimulation, on the other hand, caused strong complex movements of the mantle collar followed immediately by opening movement of the pneumostome in 100% of the snails tested (n = 112). The minimum number of tactile stimuli to achieve this varied between 6 and 12, and the mean latency of the opening response of the pneumostome was 3.4 s (_+ 1.2 S.D.) (Fig. 2). After 30 tactile stimuli only 26% of the snails became fully everted, and even these animals reacted

weak, frequent tactlle stimulus (4 gram'2; 3Hz) I I

I

I

I

I

I

I

I

1

0

10

20

30

40

50

60

70

I latency of pneumostome

ol~rm0

1 80 sec I

latency of full eversion (59.4+22.2 SD sec)

(3.4+1.2 SD see) Fig. 2. The sequence of behavioral events leading to full eversion of the snail Helix pomatia L. following serial tactile stimulation of the foot skin of the withdrawn animal. The sequence begins with the short-latency opening of the pneumostome, continues with the emergence of the tail and the anterior part of the foot and is completed by the full extension of the tentacles. The latency of the full eversion was measured from the offset of the stimulus series so that the effects of series consisting of different numbers of single stimuli could be compared. This figure shows the means (_+ S.D.) of latency data measured when the series consisted of 60 stimuli and when nearly 100% of the snails responded with emergence (see Fig. 3).

20 with a long m e a n latency (107 s (_+36 S.D.), Fig. 3). In the snails that did e m e r g e the o p e n i n g r e a c t i o n of the p n e u m o s t o m e always p r e c e d e d the e m e r g e n c e from the shell. T h e s e q u e n c e of b e h a v i o r a l events from the first o p e n i n g m o v e m e n t of the p n e u m o s t o m e was as follows: the strong initial o p e n i n g m o v e m e n t of the p n e u m o s t o m e was first followed by i r r e g u l a r closingo p e n i n g cycles. D u r i n g these, the tail of the animals, followed by the m o r e a n t e r i o r p a r t s of the foot, s t a r t e d e m e r g i n g from b e t w e e n the lobes o f the m a n t l e collar. T h e last to e m e r g e was the head, a n d finally, the two pairs of t e n t a c l e s w e r e fully e x t e n d e d . W h e n after 30 stimuli the e v e r t e d snails were p l a c e d on a tray, they w i t h d r e w into the shell within 4 - 5 min (Fig. 3). A f t e r increasing the n u m b e r of stimuli in the series to 60, the p r o p o r t i o n of everting snails i n c r e a s e d to 96% a n d the latency of the r e s p o n s e (59.4 s (+_ 22.2 S.D.)) was significantly s h o r t e r than after 30 stimuli ( P < 0.001, Fig. 3). T h e e v e r t e d snails r e m a i n e d active for a significantly l o n g e r p e r i o d , up to 2 5 - 4 5 m i n u t e s after full eversion, t h a n in the tests with 30 tactile stimuli ( P < 0.001, Fig. 3). This active state was c h a r a c t e r i z e d by i n t e n s e l o c o m o t i o n , f o o d - s e a r c h i n g p a t t e r n a n d s p o n t a n e o u s l y o c c u r r i n g f e e d i n g rasps.

Muscle and neuronal responses in semi-intact preparations correlating with elements of the euersion sequence 1. Actiuation of the pneumostome and LPd7 by sensory inputs from the foot. A single w e a k tactile stimulus a p p l i e d to the s a m e spot on the dorsal surface of the skin of the left half of the foot that was also s t i m u l a t e d in the intact snails did not cause o p e n i n g m o v e m e n t of the p n e u m o s t o m e in s e m i - i n t a c t p r e p a r a t i o n s (Fig. 4a). Serial tactile s t i m u l a t i o n a p p l i e d at 3 H z f r e q u e n c y for a m i n i m u m of 3 s, however, led to o p e n i n g of the p n e u m o s t o m e (Fig. 4b) in 76% of the e x p e r i m e n t s . T h e latency of the r e s p o n s e to s t i m u l a t i o n of the left foot v a r i e d b e t w e e n 4 a n d 8 s. This m e a n s that the minim u m n u m b e r of tactile stimuli to evoke p n e u m o s t o m e r e a c t i o n was a b o u t 10, well within the r a n g e f o u n d in intact snails (6 to 12). In the c o u r s e of a p r e l i m i n a r y e l e c t r o p h y s i o l o g i c a l m a p p i n g study we f o u n d that LPd7, a previously identified n e u r o n in the right p e d a l ganglion 9'21 r e c e i v e d tactile inputs from the foot. In p r e s e n t e x p e r i m e n t s we f o u n d that serial tactile s t i m u l a t i o n c a u s e d s t r o n g e r activation in LPd7 t h a n a single stimulus d i d (Fig. 4a,b). T h e previously i d e n t i f i e d p n e u m o s t o m e closure triggering n e u r o n s ~, on the o t h e r h a n d , r e s p o n d e d with s u b t h r e s h o l d E P S P s a n d d e p o l a r i z a t i o n to b o t h single and serial tactile stimuli (Fig. 4a,b). Electrical s t i m u l a t i o n of the left p e d a l skin nerve r e s u l t e d in excitatory r e s p o n s e s on both L P d 7 a n d the

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Fig. 3. The build-up of the eversion response following increasing numbers of tactile stimuli. A single tactile stimulus (1) has no effect on the withdrawn snails. After increasing the number of stimuli to 30 and 60, respectively, the number of responding snails (empty bars, % of total) increases, the latency of the full eversion (shaded bars. means+_S.D.) decreases and the active period following the emergence (solid bars, means_+S.D.) becomes more prolonged. Asterisks indicate values that are significantly different after 60 tactile stimuli from those after only 30 tactile stimuli (one-tailed t-tests to compare means of latency and active period values between the groups: P < 0.001; test for the significance of the difference between two percents to compare the percentage of everting snails between the groups: P < 0.001).

w i t h d r a w a l triggering n e u r o n s (Fig. 5a). This excitation ( s u b t h r e s h o l d for the p a r i e t a l ceils, s u p r a t h r e s h o l d for LPd7) was similar to that achieved by tactile stimulation of the foot a r e a i n n e r v a t e d by this nerve l~, and this was the s a m e a r e a that was s t i m u l a t e d in the e x p e r i m e n t s with intact snails. a pneumostome

pneumostome ', ; 4 \

lose topen

LPd7 RPa3

RPa3 .

,

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LPa2

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LPa2 I single

tactile

I tactile serial ~

10 mV

(RPa3,LPa2)

20rnV ( L P d 7 ) 6sec

Fig. 4. Comparison of the effects of single and serial tactile stimuli in semi-intact preparations, a: a single tactile stimulus (arrow) applied to the dorsal surface of the skin of the left foot does not cause movements of the pneumostome (top trace), weakly excites the LPd7 neuron (second trace from the top) and results in subthreshold depolarization of the RPa3 and LPa2 neurons (two bottom traces), b: serial tactile stimulation (between arrows) of the foot causes the pneumostome to open, strongly excites the LPd7 cell, but the depolarizing effect on the withdrawal cells is still subthreshold.

21

2. The efferent role of LPd7 in the opening response of the pneumostome and in movements of the foot. Action potentials of the LPd7 neuron were found 1 for 1 among potentials recorded extracellularly from the left pallial nerve (Fig. 5b). This nerve innervates an area lying anterior to the pneumostome ~s in the mantle collar (Fig. 8). In addition, LPd7 has an axon branch in the anal nerve 9 which also innervates the vicinity of the pneumostome ]s. We also found that activation by intracellular current injection of LPd7 (Fig. 6a) consistently triggered opening movement of the pneumostome in semi-intact preparations when all the nerves connecting the visceral hump to the CNS were intact (Fig. 6b). This result is exactly the opposite of what we found in a previous study 2] when the left pallial nerve was cut and only the anal and right pallial nerves were intact and LPd7 was stimulated. In this case the response was always closure of the pneumostome. Since the preparation we used in present study is more intact than the one used previously and contains all the nerves with axon branches from LPd7, we should no longer think of LPd7 as a pneumostome-closer cell, as our previous paper showed 2x, but should now think of it as a pneumostome-opener cell. In similar preparations, with or without the left pallial nerve, current injection into the withdrawal triggering neurones always resulted in closure movement of the pneumostome (Fig. 6c,d and Vehovszky et aI.21). Upon intracellular stimulation of the LPd7 neuron, movements of the mantle collar always appeared before the opening movement of the pneumostome. This

was particularly apparent in semi-intact preparations in which the CNS was perfused with high Mg2+/0 Ca 2+ saline (Fig. 7a). These movements of the mantle collar appeared with short latency after the LPd7 stimulation (Fig. 7b) and persisted after bathing the CNS for over 60 min in high Mg2+/0 Ca 2+ saline (Fig. 7c). Systematical alteration of the position of the bipolar movement recording electrode or the sensor of the electro-optical device revealed that with the same stimulus parameters contractions appeared with the shortest latency and largest amplitude in a particular area labelled 1 on Fig. 8. In this area already as little as 4 spikes evoked by intracellular stimulation in the LPd7 neuron were enough to cause measurable movements (Fig. 8d). The mean latency of the muscle response in Area 1 to LPd7 spikes occurring with 1 Hz frequency was 1109 ms (_+98 S.D., n = 5). With increasing LPd7 spike frequency, the latency of the muscle response decreased (Fig. 8a,b) while the amplitude of the movement (measured in arbitrary units) simultaneously increased (Fig. 8a,b). For comparison, in the mantle collar area labelled 2 in Fig. 8, high-frequency intracellular stimulation of the LPd7 cell resulted in a weaker response of much longer latency than in Area 1. The equation that describes the dependence of the latency of movement (1) in Area 2 on the frequency of spikes (u) in LPd7 is F(u) = 1202 - 5.1~, (r = 0.2) as opposed to F ( u ) = 1 1 0 9 - 7 5 . 5 u ( r = 0 . 8 ) in Area 1 ( r = regression coefficient). This shows that the latency of the response decreases much more sharply in Area 1 than in Area 2 with increasingly more frequent spikes

b

!!jlij!!j|fr!l!altJll!l!!g!!!!

!int.n.

i

nl

i

i

i

ii

i

i=

I

~

_

LPd7

LPd7

r.pa .n. o i

RPa3

LPd7 ,I.naH.n. ::::::::::

LPa3 LI~I7

J __.i lOmV 6=mc

current

L

s.~ SpV(nerves) 20 mV (cell)

Fig. 5. Input and output pathways of the LPd7 neuron, a: electrical stimulation (asterisks) of the left pedal skin nerve by suction electrodes produces activation of LPd7 and weak subthreshold inputs on RPa3 and LPa3. Note the close resemblance of these inputs to the sensory inputs the same cells receive after tactile stimuli (compare with Fig. 4a). b: spikes from the LPd7 cell can be recorded 1 for 1 on the left pallial nerve (l.pall.n.) hut are not present among the potentials of the intestinal or right pallial nerve (int.n. and r.pall.n.).

22 in the LPd7 bursts. O t h e r areas of the m a n t l e collar w e r e all similar to A r e a 2 in that the latency of their responses much

to stimulation of LPd7 w e r e

less d e p e n d e n t

upon

longer and

the f r e q u e n c y of LPd7

spikes than that o f the r e s p o n s e in A r e a 1. In e x p e r i m e n t s w h e r e s p o n t a n e o u s foot c o n t r a c t i o n s did not occur (n = 6), the i n t r a c e l l u l a r stimulation of the LPd7 r e s u l t e d not only in m a n t l e collar contrac-

tions and subsequent opening of the pneumostome but also in one or a few contractions of fl)ot muscles (Fig. 9a). In the semi-intact preparations spontaneous bursts of the LPd7 cell were also often followed by opening movements of the pneumostome (Fig.9b) or by rhythmic contractions of the foot muscles (Fig. 9c).

3. Inputs receiced by LPd7 J?om the withdrawal triggering neurons. When all the nerves connecting the CNS

b

/z~

pneumostome

v ¥open

12omv

LPd7 _r---L current

d

_

5sec pneumostome

Topen

LPa2

120mY _r--L current

5sec

Fig. 6. The position of the LPd7 and LPa2 neurones and the different effect of the stimulation of these cells on the pneumostome, a,c: the soma and main axon branches of the LPd7 and LPa2 neurons following intracellular labelling with nickel-lysine. LPd, left pedal; LPI, left pleural; LPa, left parietal; V, visceral ganglion, b: depolarization of LPd7 leads to opening of even the spontaneously closing pneumostome, d: depolarization of LPa2 results in closure of the pneumostome.

23

a

a

(CNS in high Mg2+/0Ca2* saline)

&close pneumostome

mantle collar

pneumostome opening mantle ~ l l a r / ~ movement /

(CNS in normal saline)

i

i

i

:

\

foot LPd7

"=-

-"-

._~ LPo7

5 sec

Ull

~

"~-

Itl IK II IIIIIIIIL

LPd7 spontaneous bursts

__120mY 12sec Fig. 7. The effect of activation of LPd7 on the mantle collar and the pneumostome, a: depolarization of the LPd7 soma results in shortlatency movements of the mantle collar, followed by opening of the pneumostome. The CNS, but not the mantle collar, was bathed in high M g 2 + / 0 Ca 2+ saline. The secondary bursts on LPd7 that follow the muscle movements are evoked by inputs propagating back from the periphery along the axon branches of LPd7. b: in preparations with the CNS in normal saline, intracellular stimulation of LPd7 leads to contractions of mantle collar muscles, c: the effect of stimulation of LPd7 on the mantle collar muscles persists after bathing the CNS for over 60 min in high M g 2 + / 0 Ca 2+ saline. In the experiments shown in b and c only movements of the mantle collar were recorded.

foot LPd7 j----q. current

_..J20mV 5sec

/~_-~ _ _ ~ .

~ LPd7 spontaneous burst

140mV 20sec

Fig. 9. The temporal relationship of evoked and spontaneous activity of the LPd7 neuron with pneumostome and foot movements, a: in quiescent preparations increased spike activity in LPd7 triggers foot as well as pneumostome movements, b,c: spontaneous bursts of the LPd7 cell are followed by opening movement of the pneumostome and rhythmic contractions of the foot muscles, respectively.

"~"

to the mantle collar and pneumostome were intact, a burst of spikes evoked by intracellular current injection in the soma of the LPa2 led to an activity increase in

~lOmV 6sec

C

c (CNS in high Mg2+/0Ca2* saline) mantle collar ~-~, ~ -

current

pneumostome / ~ ~open / . ~

the LPd7 neuron (Fig. 10a). In this case, the pneumostome first started to close, and this was rapidly followed by an opening movement (Fig. 10a, top trace). When these nerves were severed, however, intracellularly evoked bursts of spikes in LPa2 did not excite the LPd7 neuron (Fig. 10b). After analyzing instantaneous LPd7 spike frequency values, we found that cutting the nerves had a shortduration frequency increasing effect on the spontaneous APs of LPd7 (Fig. 11, middle graph). Two hours

b

a mantle collar 1

mantle

collar 1

O~3a i i

,

'i V - 7 H z

LPd7

_]'---L current

_Jloomv

LPd7

I sec

J L

_j loomv

current

lsec

d

C mantle

9.5Hz

mantle collar 2

collar .

.

.

.

mantle

collar 1

#_.LL o,7a IV-eHz !

i i i i

(7 3 spikes

IL =

pne~mostome

LPd7_J---- L LPd7 current ~lOOmV 1$ec

.J]_

current J

i£

4 spikes

~1200mV I sec

Fig. 8. Dependence of the latency and intensity of muscle contractions on the frequency of spikes in LPd7. a,b: after increasing LPd7 spike frequency (u) by injecting more current into the soma, the latency (1, 600 ms at u = 7 Hz) of the movement recorded at Area 1 of the mantle collar decreases to 0.751 (450 ms at u = 9.5 Hz) and the amplitude (a, arbitrary units) of the movement increases to 1.3 a. c: in comparison with Area 1, Area 2 of the mantle collar responds after a longer latency (2.51, 1,500 ms at u = 8 Hz) and with a weaker movement (0.7 a) to activation of LPd7. d: at Area 1 as little as four LPd7 spikes produce a visible contraction.

24 after the nerves had b e e n s e v e r e d g r a p h ) the s p o n t a n e o u s activity of cantly ( P < 0 . 0 0 1 ) w e a k e r t h a n in intact nerves (Fig. 11, t o p graph). In severed nerves the L P d 7 cell did not

(Fig. 11, b o t t o m LPd7 was signifip r e p a r a t i o n s with p r e p a r a t i o n s with r e s p o n d to stimu-

3 ] L P d 7 instant . . . . . . .

pike freq . . . . y ( H Z ) ~ beforej . . . . . . . .

,¢ ,, y

' j

lation of L P a 2 (Fig. 11, m i d d l e and b o t t o m graphs). T h e LPa2 n e u r o n was n e v e r excited u p o n i n t r a c e l l u l a r stimulation of LPd7. DISCUSSION

-~-- + lOmin

O u r b e h a v i o r a l e x p e r i m e n t s s h o w e d that a sufficient n u m b e r of r a p i d l y p r e s e n t e d w e a k tactile stimuli could bring inactive, w i t h d r a w n snails into a b e h a v i o r a l l y active state that r e s e m b l e d a r o u s a l states previously d e s c r i b e d in o t h e r molluscs a n d Helix as well tt'2°'25. Sixty stimuli reliably e v o k e d this r e s p o n s e in practically all the snails t e s t e d (96%). T h e most a p p a r e n t c h a n g e in the b e h a v i o r that was directly t r i g g e r e d by t h e serial tactile s t i m u l a t i o n was e m e r g e n c e from t h e shell in exactly t h e s a m e s e q u e n c e of events that was d e s c r i b e d e a r l i e r in s p o n t a n e o u s l y e m e r g i n g snails ~9. T h e key event of the spontaneous e m e r g e n c e (as d e s c r i b e d by S o m m e r v i l l e 19) is the initial o p e n i n g of t h e p n e u m o s t o m e . This is o f t e n followed by strong b r e a t h i n g m o v e m e n t s which a r e t h o u g h t to g e n e r a t e an i n c r e a s e d p r e s s u r e in the c e p h a l o p e d a l h a e m o c o e l which p r o p e l l e s the a n t e r i o r p a r t of the b o d y out o f the shell ~'~. In the e m e r g e n c e p r o c e s s t r i g g e r e d by tactile stimuli in o u r e x p e r i m e n t s also the o p e n i n g of the p n e u m o s t o m e was the first r e a c t i o n o b s e r v e d after the o n s e t of the stimulus series (Fig. 2). W e can not exclude that

b (nerves are intact)

~

(nerves severed +lOmin)

close pneumostome voPoPen

pneumostome

LPd7

LPd7

1

.........

kPa2

_J-L current

;

_120mV lsec

.... /

LPa2

_r-L / 2 0 m y current lsec

Fig. 10. Peripheral excitatory connection between LPa2 and LPd7. a: in semi-intact preparations with intact anal, intestinal and pallial nerves stimulation of LPa2 results in excitation of LPd7 and in a closing-opening cycle of the pneumostome, b: after severing these nerves stimulation of LPa2 fails to excite LPd7.

2-

rl

y~:~ +2hrs

1-

1st

5th

lOth

15th

20th

25th

sec

Fig. 11. C o m p a r i s o n of s p o n t a n e o u s and LPa2-evoked activity in

LPd7 in CNS preparations with and without peripheral connections. For the statistical analysis, the post-stimulus data obtained during and after stimulation of LPa2 were pooled separately and compared with pooled data from the pre-stimulus period. The asterisks indicate significant differences between the post- and prestimulus activity of the LPd7 cell (P < 0.05, two-tailed t-tests). Top graph: in semi-intact preparations with intact nerves the instantaneous spike frequency values (means_+S.D.) of the LPd7 cell are significantly higher during (solid bars) and immediately after (shaded bars) current injection into the LPa2 soma than before activation of LPa2 (open bars). Middle graph: severing the anal, intestinal and pallial nerves has a short-term excitatory effect on the spontaneous activity of LPd7. During this period the high-level spontaneous activity cannot bc further increased by injecting current into the LPa2 cell. Bottom graph: two hours after the nerves have been scvcred the spontaneous activity (open bars) of LPd7 is low but still can not be affected by stimulation of the LPa2 cell (solid and shaded bars).

the actual eversion b e h a v i o r that followed this initial p n e u m o s t o m e o p e n i n g was b r o u g h t a b o u t by the direct s u m m a t i n g effect of successive tactile stimuli. However, the increasingly longer arousal state following e m e r g e n c e after 30 and 60 stimuli, respectively (Fig. 3) indicates that a b e h a v i o r a l sensitization process may have also b e e n involved. W e suggest that h e r e the first tactile stimuli m a y have h a d the initial sensitizing effect n e c e s s a r y to p r o d u c e a g r a d e d eversion r e s p o n s e to s u b s e q u e n t tactile stimulation. W h e t h e r or not the actual eversion r e s p o n s e was just due to the s u m m a t e d effect of the r e p e a t e d tactile stimuli or the s u g g e s t e d sensitization m e c h a n i s m was also playing a role, rem a i n s to b e e l u c i d a t e d . T h e b e h a v i o r a l function of the cversion r e s p o n s e e v o k e d by tactile stimuli in the snail is not yet clear. W e suggest t h a t in the b e g i n n i n g of the m a t i n g p e r i o d it might play a role in the activation of still inactive, w i t h d r a w n snails by a l r e a d y active ones.

25 In the semi-intact preparations (Fig. 1) the key event of the behavioral sequence of eversion, opening of the pneumostome, could be reliably evoked by the same stimuli that were used in the behavioral tests. In these preparations serial tactile stimulation produced opening reaction of the pneumostome comparable in latency, duration and intensity to that observed in intact snails (Fig. 4). The same serial stimuli also excited the LPd7 neuron but were subthreshold for the command neurons that trigger withdrawal reactions ~ (Fig. 4). This confirms that (1) LPd7 receives inputs through the pathways that carry this type of mechanosensory information from the periphery to the CNS and (2) LPd7 is not a member of the withdrawal network described by Balaban I and therefore the behavioural response to this type of stimulus is not an aversive reaction. The inputs received by the two different cell types after electrical stimulation of the appropriate sensory nerve were also excitatory for LPd7 and subthreshold for the withdrawal cells (Fig. 5a). The two types of neuron appear to be 'tuned' differentially to inputs resulting from the same tactile stimuli and received via the same sensory pathway from the foot to the CNS. Bursts of spikes evoked intracellularly in the LPd7 soma led to opening response of the pneumostome (Figs. 6b, 7a and 9a). This was similar to that observed in intact snails and semi-intact preparations after serial tactile stimulation of the foot. Since LPd7 fulfils all the criteria for being an efferent 'eversion' neuron (see Introduction), we suggest that in withdrawn snails LPd7 takes part in the mediation of the eversion response to serial tactile stimuli. Preliminary evidence has shown that the pneumostome-opening response persists when LPd7 is hyperpolarized during tactile stimulation which indicates that LPd7 is not the only central neuron that mediates this type of response and excludes the possibility that LPd7 is a command-type neuron. The tactile stimulus-evoked behavioral response is probably the output of a system of putative 'eversion' neurons of which LPd7 is only one element. We also observed that bursts of LPd7 preceded spontaneous movements of the pneumostome and foot in semi-intact preparations (Fig. 9b,c). Activation by current injection of the LPd7 cell also led to movements of the foot (Fig. 9a). These observations give further support to the hypothesis that the LPd7 neuron is a member of a centrally located network of putative 'eversion' neurons whose integrated output is responsible for the complex pattern of emergence involving movements of the pneumostome and foot. Our previous experiments showed that when the left pallial nerve was cut, but the anal nerve that also carries a branch of the LPd7 axon 9 remained intact,

the pneumostome response to LPd7 stimulation was closure rather than opening 2~. Dual action efferent neurons producing both suppressive and facilitative effects in the periphery were described in Aplysia by Lukowiak and Peretz ~4. They found that the CNS modulates the peripheral nervous system by means of facilitative control in one of the nerves carrying a motoneuron branch and suppressive control in another nerve carrying a different axon branch of the same motoneuron. We suggest that in Helix the LPd7 cell sends information through its branch in the anal nerve which causes the pneumostome to close, whereas the signals sent through its branch in the left pallial nerve may lead to opening. When both nerves are intact, the facilitative control apparently overrides the suppressive one. Therefore, in intact snails we should think of LPd7 as a pneumostome-opener cell. The opening reaction of the pneumostome to intracellular stimulation of LPd7 persisted when the CNS was bathed in high Mg2+/0 Ca 2+ saline (Fig. 7a). This showed that central synaptic connections were not involved in the mediation of the response. Although axon branches of LPd7 run in the left pallial nerve and anal nerve which innervate the vicinity of the pneumostome, the relatively long latency of the pneumostome response indicates that connections of LPd7 with so far unidentified local neurons in the mantle collar might play the most important role in the LPd7-triggered pneumostome response. We therefore suggest that LPd7 is not a motoneuron of the pneumostome itself but exerts its consistent pneumostome opening effect via a peripheral pathway localized in the mantle collar. This putative peripheral network might also play an important role in the pneumostome response to serial tactile stimuli mediated by LPd7 and possibly by other LPd7-type neurons which have not been identified yet. Interactions between the central and peripheral nervous systems of Aplysia have been shown to play an important role in the mediation of the gill withdrawal reflex evoked by tactile stimulus 14. These interactions are also thought to be important in the mediation of facilitated behavioral responses TM comparable to the facilitated pneumostome response in Helix. The fact that serial tactile stimuli lead to emergence followed by a behaviorally aroused state shows that the putative system of 'eversion' neurons might be part of a multi-cell executive arousal system similar to that proposed to exist in Aplysia 25. This network in Helix appears to operate independently from the parietal neurons which trigger pneumostome closure in active snails. This is probably due to differences in spontaneous activity levels and spike thresholds 2~ as well as to

2~ differential sensitivity of the two cell types to sensory inputs. Motor n e u r o n s for the m a n t l e have been described in the

pedal

ganglia of the

opistobranch

mollusc

tions influence the activity of the LPd7 cell by peripheral interactions rather than central c o n n e c t i o n s (Figs. l(I and 11). It is not yet clear w h e t h e r this interaction is due to proprioceptive input from the p n e u m o s t o m c to

A r m i n a californica ~'. T h e s e cells had very similar axonal

the LPd7 cell or to synaptic c o n n e c t i o n s b e t w e e n the

b r a n c h i n g p a t t e r n to that of LPd7 in Helix, also received sensory inputs u p o n tactile stimulation of the

peripheral b r a n c h e s of the axons of kPd7 and the c o m m a n d n e u r o n s . We have evidence that the LPd7

m a n t l e and foot, a n d they were capable of p r o d u c i n g

cell receives excitatory inputs upon tactile stimulation

both m a n t l e a n d foot m o v e m e n t s . A l t h o u g h we did not

of the p n e u m o s t o m e itself 2~ but it is also truc thai

record m o n o s y n a p t i c ejp-s from m a n t l e collar muscle fibers, the c o n n e c t i o n b e t w e e n LPd7 a n d m a n t l e collar

both the LPd7 and the withdrawal c o m m a n d cells send efferents to the m a n t l e collar. We suggest that the

A r e a 1 seems to fulfil most of the criteria established

excitatory effect of the withdrawal c o m m a n d cells on

for proving the m o t o n e u r o n function of a nerve cell~°.

LPd7 might c o n t r i b u t e to the o p e n i n g m o v e m e n t of the p n e u m o s t o m e which often follows the closure evoked

We suggest that beside being a m e m b e r of an efferent network taking part in the o p e n i n g of the p n e u mostome, LPd7 may be a m o t o n e u r o n of one or more

by brief aversive stimuli in active snails (our u n p u b lished observations).

muscles in A r e a 1 of the m a n t l e collar. It can not be exluded, however, that LPd7 is presynaptic to a peripheral m o t o n e u r o n in the mantle. T h e m a n t l e m o t o n e u ron function of LPd7 can only be e l u c i d a t e d by directly recording muscle cell ejp-s or record muscle responses when the periphery is b a t h e d in solutions (e.g. H1DI) which reduce p e r i p h e r a l n e u r o n a l r e c r u i t m e n t . How-

Acknowledgements. We thank Drs. A. Getperin and T. Carew for discussions of the material of this paper presented at the 2nd International Congress of Neuroethology, Berlin, September, 1989 and Dr. C. Ellion for reading the manuscript. We appreciale the technical assistance of Ms. K. Komfiromi and Mr. B. Bal~zs. This work was supported by an OTKA grant to K.S.-R.. Gy.K. and ,~.V. and a HAS/USSRAS travel fellowship to I.S.Z.

ever, these tests arc not easy to perform on the CNSvisceral h u m p system, given the complexity a n d the

REFERENCES

often strong s p o n t a n e o u s m o v e m e n t s of the musculature of the m a n t l e a n d the fact that the putative peripheral n e u r o n e s or axon terminals are deeply emb e d d e d in m u c u s - c o a t e d tissue, r e n d e r i n g the H I D I solution less effective in raising the firing threshold of p e r i p h e r a l cells. T h e role of the LPd7 n e u r o n in i n f l u e n c i n g movem e n t s of the p n e u m o s t o m e might be more plastic t h a n its putative m a n t l e m o t o n e u r o n function, a n d the actual o u t p u t of the cell to the p n e u m o s t o m e might d e p e n d on a variety of i n t e r n a l factors. T h e typical reaction to tactile stimuli in acth'e snails is closure of the p n e u m o s t o m e . T h e i n t e r n a l states of withdrawn and actively crawling snails can be very different, and the snails may give opposite responses to the same stimulus d e p e n d i n g on this state. Earlier observations on Aplysia also indicated that, d e p e n d i n g on behavioral states or physiological conditions, a mildly aversive stimulus can either facilitate or inhibit a particular response ~-~. In active snails LPd7 may be acting in parallel with the withdrawal c o m m a n d cells, while in withdrawn snails it appears to c o n t r i b u t e to the completion of the behavioral s e q u e n c e of whole-body eversion triggered by weak serial tactile stimuli. T h e inform a t i o n carried by different axon b r a n c h e s of LPd7 may play an i m p o r t a n t role in switching b e t w e e n these different functions. T h e c o m m a n d n e u r o n s that trigger withdrawal reac-

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18 19

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