Download Identification of Mechanoafferent Neurons in Terrestrial Snail

J Neurophysiol
87: 2364 –2371, 2002; 10.1152/jn.00185.2001.
Identification of Mechanoafferent Neurons in Terrestrial Snail:
Response Properties and Synaptic Connections
ALEKSEY Y. MALYSHEV AND PAVEL M. BALABAN
Institute of Higher Nervous Activity and Neurophysiology, Moscow 117485, Russia
Received 6 March 2001; accepted in final form 8 January 2002
INTRODUCTION
Well-coordinated interactions between sensory and motor
neuronal elements are essential for the orderly production of
behavioral outputs in all animal species. The identification of
both motor and sensory components and characterization of
their synaptic and functional interactions is critical for understanding the cellular basis of animal behavior. Individual sensory neurons mediating various behaviors and their synaptic
connections have been identified and described in a variety of
invertebrate species, such as crustaceans (Calabrese 1976a,b;
Fricke et al. 1982; Wiese 1976; Wine 1977), insects (Basarsky
and French 1991; Callec et al. 1971; Davis and Murphey 1993;
French et al. 1993; Wolf and Burrows 1995), annelids (Kristan
1982; Nicholls and Baylor 1986), and mollusks (Audesirk
1979; Byrne et al. 1974; Clatworthy et al. 1994; Inoue et al.
Address for reprint requests: P. M. Balaban, Institute of Higher Nervous
Activity and Neurophysiology, Butlerova 5A, Moscow 117485, Russia
(E-mail: [email protected]).
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1996; Janse 1974; Mellon 1972; Olivo 1970; Rosen et al. 1982;
Spray et al. 1980a,b; Walters et al. 1983). Different behavioral
and neural aspects of the withdrawal reactions have been
extensively studied in pulmonate snails (Arshavsky et al.
1994a; Sakharov and Rozsa 1989). Whole-body withdrawal
motoneurons activated by multimodal sensory inputs have
been identified in freshwater snail Lymnaea (Ferguson and
Benjamin 1991a,b) and Planorbis (Arshavsky et al. 1994b,c).
Terrestrial snail Helix lucorum is a useful model for
studying the cellular basis of different behaviors. Neural
mechanisms of avoidance behavior had been intensively
studied during last two decades, and the efferent part of the
withdrawal neural circuit in Helix has been described in
details (Balaban 1979, 1983; Balaban et al. 1987; Zakharov
and Balaban 1987). However, almost nothing is known
about the sensory neuronal elements recruited in avoidance
reactions and sensory inputs to the withdrawal interneurons
involved in triggering the withdrawal responses (Balaban
1979). Several putative mechanosensory cells presynaptic to
withdrawal interneurons were described earlier; however,
their receptive fields were not well defined and assigned to
liver and other internal organs located under the shell (Arakelov et al. 1989). In this study, we have identified for the
first time a group of mechanosensory neurons with receptive
fields on the foot of the snail H. lucorum that monosynaptically activate interneurons for the withdrawal behavior.
These neurons, termed pleural ventrolateral neurons (PlVL),
presumably mediated part of the withdrawal response of the
snail via activation of the withdrawal interneurons.
METHODS
Animals
The experiments were performed on the mature specimens of H.
lucorum L. weighting 30 –35 g. Snails were originally collected in
Crimea and maintained in an active state in the laboratory.
Behavioral experiments
Experiments on freely behaving snail were performed as described
earlier (Balaban and Bravarenko 1993). Video records of experiments
from a standard video tape recorder were digitized on the IBM-PCcompatible computer and then analyzed using video analysis software
PhysVis (Kenyon College). The anterior tip of upper tentacles (or the
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0022-3077/02 $5.00 Copyright © 2002 The American Physiological Society
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Malyshev, Aleksey Y. and Pavel M. Balaban. Identification of
mechanoafferent neurons in terrestrial snail: response properties
and synaptic connections. J Neurophysiol 87: 2364 –2371, 2002;
10.1152/jn.00185.2001. In this study, we describe the putative mechanosensory neurons, which are involved in the control of avoidance
behavior of the terrestrial snail Helix lucorum. These neurons, which
were termed pleural ventrolateral (PlVL) neurons, mediated part of
the withdrawal response of the animal via activation of the withdrawal
interneurons. Between 15 and 30 pleural mechanosensory neurons
were located on the ventrolateral side of each pleural ganglion. Intracellular injection of neurobiotin revealed that all PlVL neurons sent
their axons into the skin nerves. The PlVL neurons had no spontaneous spike activity or fast synaptic potentials. In the reduced “CNSfoot” preparations, mechanical stimulation of the skin covering the
dorsal surface of the foot elicited spikes in the PlVL neurons without
any noticeable prepotential activity. Mechanical stimulus-induced action potentials in these cells persisted in the presence of high-Mg2⫹/
zero-Ca2⫹ saline. Each neuron had oval-shaped receptive field 5–20
mm in length located on the dorsal surface of the foot. Partial
overlapping of the receptive fields of different neurons was observed.
Intracellular stimulation of the PlVL neurons produced excitatory
inputs to the parietal and pleural withdrawal interneurons, which are
known to control avoidance behavior. The excitatory postsynaptic
potentials (EPSPs) in the withdrawal interneurons were induced in 1:1
ratio to the PlVL neuron spikes, and spike-EPSP latency was short and
highly stable. These EPSPs also persisted in the high-Mg2⫹/highCa2⫹ saline, suggesting monosynaptic connections. All these data
suggest that PlVL cells were the primary mechanosensory neurons.
MECHANOAFFERENT NEURONS IN TERRESTRIAL SNAIL
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anterior tip of the head, in cases when tentacles were completely
retracted) was monitored, and its movements were plotted as a function of time.
Isolated CNS preparation
Reduced “CNS-foot” preparation
The reduced “CNS-foot” preparation consisted of the CNS connected with a foot dissected sagittaly in two halves positioned with the
skin of the dorsolateral surface upwards. Half of the foot was placed
in a separate compartment of the experimental chamber to prevent
skin mucus to affect neuronal activity. All pedal cutaneous nerves
remained intact. Ganglia were desheathed as it is described for an
isolated CNS preparation. Tactile stimuli were delivered by means of
electrical-mechanical tapper with fixed pressure intensity 1 g (tip
diameter 0.05 mm). Central and peripheral synaptic connections were
blocked by perfusing preparation with zero-Ca2⫹/high-Mg2⫹ saline (0
mM Ca2⫹/25 mM Mg2⫹).
Electrophysiology and dye injection
Intracellular recordings from either an isolated brain or reduced
CNS-foot preparation were made using standard electrophysiological
techniques. Identified withdrawal premotor interneurons of the pari-
FIG. 2. Morphology of PlVL neurons. A: all stained PlVL cells projected
toward periphery via pleuropedal connective and then into the nervus cutaneus
pedalis secundus. B: morphology of the Pl4 neuron. The Pl4 cell in addition
had central projection into the pleurocerebral commissure.
etal ganglia (Pa2 and Pa3) and pleural ganglia (Pl1) were penetrated
with glass microelectrodes filled with 2 M potassium acetate (tip
resistance, 10 –15 M⍀). For recording from PlVL neurons, electrodes
with a fine tip (25–30 M⍀) were used. Intracellular signals were
recorded with preamplifiers (Neuroprobe 1600, A-M Systems), digitized, and stored on computer (Digidata 1200A A/D converter and
Axoscope 8.0 software, both from Axon Instruments). All EPSP
recordings were filtered with a 10-Hz low-pass filter built into the
Axoscope program.
For morphological investigation of recorded neurons, a 5% neurobiotin solution in 2 M potassium acetate was iontophoretically injected via the recording electrodes with 1-nA positive current pulses
for 30 –50 min and after fixation of tissue was visualized with TexasRed-labeled avidin (Vector Laboratories, Burlingame, CA). The preparations were then examined with an Axioscope fluorescence microscope and Bio-Rad MRC 600 laser scanning confocal microscope.
Double-labeling experiments
FIG. 1. Schematic diagram of the ventral surface of the left pleural ganglion. Pleural ventrolateral (PlVL) cells (●) are situated on the ventrolateral
surface of each pleural ganglion. Morphologically identifiable pleural withdrawal interneuron (Pl1) is shown.
J Neurophysiol • VOL
For double-labeling experiments, pleural ventrolateral neurons
were injected with neurobiotin as described in the preceding text. Pa3
and Pl1 neurons in the same preparation were injected by using the air
pressure system (PV830 Pneumatic PicoPump, WPI) with 3% Lucifer
yellow in 0.1 M KCl in an approximate volume 10 pl. The preparations were then immunocytochemically processed with the rabbit
anti-Lucifer yellow polyclonal antiserum (Chemicon International).
Preparations were scanned with Bio-Rad MRC 600 laser confocal
microscope with two different filters for Texas Red and FITC.
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Animals were anesthetized by injection of isotonic MgCl2 (⬃15%
of the animal weight). The central ganglionic ring was removed from
the animal and pinned to a silicone-elastomer (Sylgard)-coated dish.
Connective tissue sheath was partially removed using fine forceps and
scissors. To facilitate further desheathing, ganglia were treated with
Protease (0.25 mg/ml; Type XIV, Sigma) for 30 min at room temperature and washed out, and then the fine sheath was completely
removed. To expose ventrolateral surface of pleural ganglion, we
usually transected the pedal commissure and rotated each pedal ganglion 180° outward. The CNS was bathed in a saline solution that
contained (in mM) 100 NaCl, 4 KCl, 7 CaCl2, 5 MgCl2, and 10
Tris-HCl buffer (pH 7.8). Electrophysiological recordings were performed 60 min after the dissection.
To examine the monosynaptic connections, we used high-Mg2⫹/
high-Ca2⫹ solution containing 28 mM Ca2⫹ and 20 mM Mg2⫹ to
raise firing threshold and reduce the chances of activating interneurons (Berry and Pentreath 1976).
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RESULTS
Electrophysiological characteristics of PlVL neurons
During the pilot search of neurons monosynaptically connected with premotor withdrawal interneurons, several presynaptic cells were discovered in the pleural ganglia on the ventrolateral surface. In each experiment, after identifying the cell
as presynaptic, we investigated its morphology, electrophysiological characteristics, and properties of the connections with
several withdrawal interneurons.
Morphological characteristics of the pleural ventrolateral
neurons
FIG. 3. Responses of PlVL cells to mechanical stimulation of the skin. A: mechanical stimulus applied to the skin covering
lateral surface of snail body elicited in the Pl4 cell an action potential. Note that action potentials appeared only at the onset and
the end of mechanical stimulus (ON and OFF responses). Increase of duration did not change the ON-OFF character of the PlVL
neurons response. Mechanical stimulation also produced depolarizing synaptic responses in pleural withdrawal interneuron (Pl1)
coincident in time with spikes in Pl4 cell. B: mechanical stimulation with brief (20 ms) mechanical stimuli (1) applied with
frequency of 10 Hz produced spikes in Pl4 cell approximately with the stimulation rate. Pleural interneuron showed fast adapting
response in such conditions.
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Between 15 and 30 PlVL monosynaptically connected to
withdrawal interneurons were located on the ventrolateral surface of each pleural ganglion between pleuropedal and pleurocerebral connectives (Fig. 1). Most of PlVL cells had a soma
diameter of ⬃20 –30 ␮m. One cell from this group was bigger
then others (soma diameter, ⬃40 ␮m) and could be visually
identified; this cell has been designated as Pl4 neuron. Intracellular injection of neurobiotin revealed that all stained PlVL
neurons (n ⫽ 12) sent their axons through the pleuropedal
connective and pedal ganglion into the skin nerve (nervus
cutaneus secundus; Fig. 2A). In addition, the Pl4 neuron had a
second axon branch that projected into the cerebral ganglion
through the pleurocerebral connective (n ⫽ 5, Fig. 2B). In two
cases, axons from the pleural sensory neurons were traced to
the skin.
The results described in the following text were based on
examination of 263 PlVL cells in 52 preparations. The PlVL
neurons were electrically silent, showing no spontaneous activity or fast synaptic potentials both in isolated brain and
reduced CNS-foot preparations. The average membrane potential of the PlVL cells was 57.5 ⫾ 3.1 mV. A gentle mechanical
touch to the skin covering dorsal surface of the foot elicited
spikes in the PlVL neurons without any noticeable prepotential
activity (Fig. 3A). It is important to stress that action potentials
appeared only at the beginning and the end of the mechanical
stimulation. Mechanical stimulus was applied in wide range of
duration (0.02–10 s). Such stimulation also produced a depolarizing response in ipsilateral pleural and all parietal interneurons for withdrawal (Pa2 and Pa3). Application of brief mechanical stimuli (20 ms) with high frequency (ⱕ10 Hz)
produced spike responses in the PlVL neurons to each stimuli
showing no habituation or depression (Fig. 3B). Mechanical
stimulus-induced action potentials in these cells persisted in
high-Mg2⫹/zero-Ca2⫹ saline (Fig. 4). At the same time, depolarizing responses in the pleural withdrawal interneurons were
completely abolished. These data suggest that PlVL neurons
were primary mechanoreceptors. However, the possibility still
exists that these cells receive excitatory inputs via electrical
synapses or highly encapsulated chemical synapses in the
periphery.
MECHANOAFFERENT NEURONS IN TERRESTRIAL SNAIL
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FIG. 4. Effect of blockade of synaptic transmission in preparation. A:
responses of PlVL and Pl1 neurons to the mechanical stimulus while preparation was bathed in normal Ringer solution. B: superfusion and maintenance
of this preparation (60 min) in high-Mg2⫹/0-Ca2⫹ saline blocked response in
withdrawal interneuron but failed to block spike response in PlVL neuron to
the same mechanical stimulus.
FIG. 6. Comparison of responses to mechanosensory and chemical stimulation of the skin. A: sensory selectivity of PlVL cells. Tactile stimulation of
skin induced subthreshold depolarization in pleural withdrawal interneuron
(Pl1) and spike response in PlVL cell in the CNS-foot preparation. Chemical
stimulation of the same area did not produce action potentials in PlVL neuron
but elicited a strong spike response in Pl1 cell. B: chemical stimulation
produced stronger withdrawal response in freely behaving snails than mechanosensory stimuli. Mechanograms of withdrawal responces induced by mechanical stimulus (black arrows, dashed line) and by chemical stimulus (white
arrow, solid line) are plotted together. Presented snail contour is illustrating the
withdrawal response ratio.
body into the shell lasting tens of minutes (Fig. 6B). Thus
chemical stimulation of skin produced much more stronger
withdrawal response in the freely behaving snail than mechanosensory stimuli. This observation is in full accordance with
electrophysiological data described in the preceding text.
PlVL neurons monosynaptically activate interneurons of
avoidance behavior
FIG.
5. Receptive fields of PlVL neurons. Results are from a single animal.
䡠 䡠 䡠 , receptive field borders. Skin stripes pattern is shown for orientation.
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To test the hypothesis that the PlVL neurons can activate the
withdrawal interneurons, we performed a series of experiments
with simultaneous intracellular recordings from PlVL and
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To identify the individual receptive fields, PlVL neurons
were impaled one after another in the same CNS-foot preparation, and receptive fields of each neuron were delineated by
applying mechanical stimuli to sites over entire body surface.
We found that each neuron had an oval-like shaped receptive
field located on the body surface (Fig. 5). The receptive field of
an identifiable Pl4 neuron was situated behind the rear tentacles
and was ⬃20 mm in length, whereas receptive fields of other
PlVL neurons were smaller (⬃5–12 mm). Partial overlapping
of receptive fields of different neurons was often observed. We
did not find any electrical or chemical interactions between
different PlVL cell. Mechanical stimulation of the skin on the
outer edge of a receptive field did not produce a hyperpolarization of neurons (data are not shown), as it was described in
some other mollusks (Getting 1976; Mellon 1972; Spray et al.
1980; Walters et al. 1980).
Stimulation of the skin inside the receptive fields of PlVL
neurons with various chemical agents, such as concentrated
NaCl saline, 0.5 N solution of HCl and NaOH, failed to elicit
spikes in these cells (n ⫽ 5 in 5 preparations) even though such
a stimulation produced vigorous response in interneurons of
avoidance behavior (Fig. 6A). This observation suggested that
PlVL cells were specific mechanoafferent neurons, while other
sensory neurons transferred inputs of chemical modality.
To compare behavioral effects of mechanical and chemical
stimulation, we performed a series of experiments in intact
freely moving snails (n ⫽ 6). We found that short (300 ms)
mechanical stimuli applied to the receptive field of Pl4 neuron
induced transient, partial retraction of the upper tentacles
(ⱕ70% of their initial length), whereas chemical stimulation of
the same region with a drop of 0.5 N NaOH induced complete
withdrawal of tentacles immediately followed by retraction of
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A. Y. MALYSHEV AND P. M. BALABAN
withdrawal interneurons. Each induced spike in any PlVL
neuron was found to produce EPSPs in relation 1:1 in the
ipsilateral Pl1, Pa2, and Pa3 and contralateral Pa2 and Pa3
neurons (Fig. 7, A and B). The average amplitude of EPSPs in
pleural interneurons elicited by first spike in PlVL cells was
5.8 ⫾ 2.1 mV (n ⫽ 12), whereas amplitudes of EPSPs in
parietal interneurons were in general smaller (3.9 ⫾ 1.7 mV,
n ⫽ 10). A short burst of spikes induced in PlVL was found to
elicit spike response in Pl1 neuron but only subthreshold
EPSPs in all parietal withdrawal interneurons (Fig. 7C).
PlVL neuron-induced EPSPs in the withdrawal interneurons
persisted in high-Mg2⫹/high-Ca2⫹ saline (Fig. 7D). A high
stability of spike-EPSP latency represented an additional con-
firmation of monosynaptic nature of the recorded connections.
Figure 7E shows six superimposed traces of spikes in PlVL and
responses in Pl1 neurons with constant latency 8 ms (Fig. 7E).
These data suggest that the connections between PLVl cells
and withdrawal interneurons appear to be monosynaptic.
Synaptic connections between the PlVL neurons and withdrawal interneurons showed a progressive depression with a
repeated stimulation. Such depression has been observed reliably over a wide range of interstimulus intervals: from 1 to
300 s. Figure 8 summarizes the results of experiments in which
individual PlVL neurons (7 cells in 7 preparations) were repeatedly activated with brief intracellular pulses at 60-s intervals. Averaged results of behavioral experiments in intact
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FIG. 7. Synaptic connections between PlVL cells and parietal and pleural interneurons for withdrawal behavior. A: intracellulary
induced spikes in LPlVL cell produced EPSPs in ipsilateral pleural and parietal withdrawal interneurons. Note that EPSP amplitude
in Pl1 neuron is almost 2 times bigger than in Pa3 cell. B: Pl4 neuron excited both ipsi- and contrlateral Pa3 neurons. C: short burst
of spikes induced in PlVL cell was able to produce spike response in pleural withdrawal interneuron but only subthreshold response
in parietal interneuron. D: synaptic connection between PlVL cell and pleural interneuron persisted in high-Mg2⫹/high-Ca2⫹
solution. E: superposition of 6 traces of spikes in PlVL and responses in Pl1 neurons demonstrated high stability of latency of this
synaptic connection.
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MECHANOAFFERENT NEURONS IN TERRESTRIAL SNAIL
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snails are also presented in Fig. 8 (n ⫽ 11). Tactile stimulus
was delivered to the head region, 4 –5 mm behind the ommatophores (approximately to the receptive field of Pl4 cell; Fig. 5).
The initial increase in the behavioral response amplitude
was apparently related to the sensitization process (Balaban
1983; Zakharov and Balaban 1987). The following dynamics of habituation of the behavioral responses and depression of EPSPs amplitude in the withdrawal interneurons
were very similar.
Location of synapses between mechanoafferent neurons and
withdrawal interneurons
DISCUSSION
Comparison of PlVL cells to other mechanoafferent neurons
In the present study, we identified mechanosensory neurons
that produced monosynaptic excitatory inputs to the interneurons for the whole-body withdrawal behavior. Based on their
partial morphological, electrophysiological, and functional
similarities with other mechanosensory neurons described in
various molluscan species, we believe that PlVL neurons are
primary mechanosensory cells. In particular, PlVL neurons
exhibited morphological and electrophysiological features that
were somewhat similar to the mechanosensory neurons previ-
FIG. 8. Dynamics of behavioral responses to tactile stimulation in intact
snails (n ⫽ 11; E) and depression of monosynaptic connections between PlVL
and withdrawal interneurons (●) with repeated 1/min stimulation. In behavioral
experiments, mean amplitude of ommatophore withdrawal in percents of initial
are shown. In neurophysiological experiments, mean responses ⫾ SE to
intracellular stimuli (each activating a single spike) applied at regular 60-s
intervals are plotted (n ⫽ 7 cells in 7 preparations). EPSPs are normalized to
the 1st EPSP elicited.
J Neurophysiol • VOL
FIG. 9. Possible site of a synaptic contact between PlVL and the withdrawal interneuron. The PlVL neuron was stained with neurobiotin and visualized with Texas Red (appears white). Ipsilateral withdrawal interneuron
(LPa3) was injected with Lucifer yellow, then processed with anti-Lucifer
antibodies, and then developed with FITC-conjugated secondary antibodies
(appears black). Confocal microscopy study revealed that neuropile of the
pleural ganglion is the only place in the CNS where processes of both cells are
located together. Processes of the LPa3 neuron are marked by an arrow. Only
few optical slices are presented (total thickness is ⬃40 ␮m).
ously characterized in Aplysia (Byrne 1980a,b; Byrne et al.
1978; Walters et al. 1983), Tritonia (Getting 1976), and Lymnaea (Inoue et al. 1996). Like Aplysia VC mechanosensory
neurons, Tritonia S cells, and Lymnaea RPD3, the PlVL neurons have its somata located in the central ring ganglia and
have both peripheral and central (in case of Pl4 cell) projections. When mechanical stimulation was applied to the skin
inside the receptive field of each PlVL neuron, neurons fired
action potentials in the absence of observable prepotentials
(Fig. 4). These responses persisted in the presence of various
solutions known to block indirect polysynaptic transmission.
Similar to other mechanoafferent and sensory interneurons in
invertebrates (Byrne 1982; Byrne et al. 1974; Callec et al.
1971; Walters et al. 1983; Zucker 1972), synaptic connections
between the PlVL neurons and withdrawal interneurons
showed a progressive depression with repeated stimulation.
The PlVL neurons and sensory neurons in other mollusks also
have such common characteristics as the lack of impulse activity without mechanical stimulation and relatively low
thresholds for their activation. However, in contrast to the
mechanoafferent cells found in Aplysia (Walters et al. 1983)
and Lymnaea (Inoue et al. 1996), which demonstrated slowly
adapting responses to the maintained mechanical stimulation,
the PlVL neurons showed single spike ON-OFF response in such
conditions. It makes them more similar to the T cells of the
leech (Nicholls and Baylor 1968), Tritonia S cells (Getting
1976), and mechanoafferent cells in razor clam (Olivo 1970).
But in contrast to Tritonia S cells, the PlVL neurons were not
activated by chemical stimuli. Unlike other mechanosensory
cells (Getting 1976; Spray et al. 1980), the PlVL neurons lack
a hyperpolarizing responses to stimuli applied outside of the
excitatory receptive field. It is interesting to note that other
putative sensory neurons (Pa7, 9, 11) described in Helix (Logunov and Balaban 1978) that innervate the liver and organs
located inside the shell exhibited hyperpolarizing responses to
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To determine the location of synapses between PlVL and
withdrawal interneurons, we performed double-labeling experiments. PlVL and withdrawal interneurons were stained with
different fluorescent dyes in the same preparation (see METHODS). We found that processes of PlVL cells and both Pa3 and
Pl1 neurons were present in the same area only in the neuropile
of the pleural ganglion (Fig. 9). Such synapse location can
partially explain the difference in EPSP amplitude in parietal
and pleural withdrawal interneurons: distance from possible
synapse zone to soma is minimal for the Pl1 cell in which we
recorded largest EPSPs.
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A. Y. MALYSHEV AND P. M. BALABAN
mechanical stimulation of the skin and the mantle, whereas
short mechanical stimulation of visceral organs elicited bursts
of spikes lacking prepotentials in those cells (Arakelov et al.
1989). Unfortunately, authors did not apply maintained mechanical stimulation, so we cannot compare the adaptation
response properties of PlVL and Pa7,9,11 neurons.
Functional role of PlVL neurons
This work was supported by grants from the Russian Foundation for Basic
Research and International Association for the Promotion of Cooperation with
Scientists from the New Independent States of the Former Soviet Union
(INTAS) Grant 99-1481.
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Mechanosensory neurons in mollusks in some cases make
direct excitatory chemical connections to the motor neurons,
contributing to withdrawal (Walters et al. 1983), or may activate interneurons mediating whole-body withdrawal behavior
(Arshavsky et al. 1994b; Inoue et al. 1996). Known structure of
the network underlying withdrawal behavior in Helix includes
nine giant premotor withdrawal interneurons receiving convergent multimodal sensory input and recruiting the appropriate
motor neurons (Balaban 1983; Balaban and Zakharov 1992). It
has been previously shown that intracellular stimulation of
parietal interneurons (Pa3) elicited pneumostome closure as a
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