J. exp. Biol. 158, 97-116 (1991)
Printed in Great Britain © The Company of Biologists Limited 1991
THE WHOLE-BODY WITHDRAWAL RESPONSE OF
LYMNAEA STAGNALIS
II. ACTIVATION OF CENTRAL MOTONEURONES AND MUSCLES BY
SENSORY INPUT
BY GRAHAM P. FERGUSON* AND PAUL R. BENJAMIN
Sussex Invertebrate Neuroscience Group, School of Biology,
University of Sussex, Brighton BN1 9QG, UK
Accepted 4 February 1991
Summary
The role of centrally located motoneurones in producing the whole-body
withdrawal response of Lymnaea stagnalis (L.) was investigated. The motoneurones innervating the muscles used during whole-body withdrawal, the
columellar muscle (CM) and the dorsal longitudinal muscle (DLM) were cells with
a high resting potential (—60 to — 70 mV) and thus a high threshold for spike
initiation. In both semi-intact and isolated brain preparations these motoneurones
showed very little spontaneous spike activity. When spontaneous firing was seen it
could be correlated with the occurrence of two types of spontaneous excitatory
postsynaptic potential (EPSP). One was a unitary EPSP that occasionally caused
the initiation of single action potentials. The second was a larger-amplitude, longduration (presumably compound) EPSP that caused the motoneurones to fire a
burst of high-frequency action potentials. This second type of EPSP activity was
associated with spontaneous longitudinal contractions of the body in semi-intact
preparations. Tactile stimulation of the skin of Lymnaea evoked EPSPs in the CM
and DLM motoneurones and in some other identified cells. These EPSPs
summated and usually caused the motoneurone to fire action potentials, thus
activating the withdrawal response muscles and causing longitudinal contraction
of the semi-intact animal. Stimulating different areas of the body wall demonstrated that there was considerable sensory convergence on the side of the body
ipsilateral to stimulation, but less on the contralateral side. Photic (light off)
stimulation of the skin of Lymnaea also initiated EPSPs in CM and DLM
motoneurones and in some other identified cells in the central nervous system
(CNS). Cutting central nerves demonstrated that the reception of this sensory
input was mediated by dermal photoreceptors distributed throughout the epidermis. The activation of the CM and DLM motoneurones by sensory input of the
modalities that normally cause the whole-body withdrawal of the intact animal
demonstrates that these motoneurones have the appropriate electrophysiological
properties for the role of mediating whole-body withdrawal.
•Present address: Stazione Zoologica, Villa Comunale, 1-80121 Napoli, Italy.
Key words: mollusc, Lymnaea stagnalis, pond snail, withdrawal behaviour, sensory input.
97
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G. P. FERGUSON AND P. R.
BENJAMIN
Introduction
The effect of sensory input on gastropod behaviour has been investigated in
several species. Most of these studies have examined the role of mechanosensory
and chemosensory input on feeding (Rosen et al. 1979, 1982; Audesirk, 1979;
Audesirk and Audesirk, 1979, 1980a,b; Kemenes et al. 1986) or the effect of
sensory input on the production of withdrawal responses.
In Aplysia californica, sensory neurones involved in local withdrawal responses
of the anterior tentacles (Fredman and Jahan-Parwar, 1977, 1980), gill (Kupfermann and Kandel, 1969; Kupferman et al. 1971, 1974; reviewed by Kandel, 1976,
1979) and tail (Walters et al. 1983a,b) have been identified and their interactions
with interneurones and motoneurones investigated. Similarly, the role of sensory
neurones in inducing escape swimming in Tritonia diomedea has been examined
(reviewed by Getting, 1983).
In Lymnaea, ultrastructural studies have identified six different types of
epidermal sensory cell (Zylstra, 1972; Zaitseva and Bocharova, 1981) and
demonstrated that these are most abundant on the tentacles, lips, front edge of the
foot, pneumostome, mantle edge and dorsal and lateral surfaces of the foot
(Zylstra, 1972). The peripheral nerve pathways involved in transmission of tactile
information to the CNS have been mapped electrophysiologically (De Vlieger,
1968; Janse, 1974, 1976; Janse and Van Swigchem, 1975) and it has been shown
that peripheral nerves have overlapping receptive fields. In Lymnaea, two types of
primary touch-sensitive neurones as well as primary stretch-sensitive neurones and
higher-order sensory neurones sensitive to touch, pressure and stroke or stretch
have been identified (Janse, 1974). Nerve recordings made during tactile stimulation of the skin demonstrated that, with the exception of one type of primary
touch-sensitive neurone (which is only involved in peripheral reflexes), the cell
bodies of these sensory neurones are located within the CNS. Among the higherorder sensory neurones, integration occurred in peripheral ganglia prior to the
sensory information being relayed to the CNS (Janse, 1974). More recently, Janse
and co-workers have examined the control of respiratory behaviour in Lymnaea
and, by intracellular recording, have shown that statocyst cells (Janse et al. 1988)
and other central neurones (Janse et al. 1985; Van Der Wilt et al. 1987) receive
sensory input in response to changes in PQ? and after tactile stimulation of the skin
or pneumostome.
Studies of the processing of photic information have demonstrated that
behavioural responses to shadows are mediated by dermal photoreceptors, rather
than the eyes (Stoll, 1972), that only responses to light being switched on are
recorded from the optic nerves (Stoll, 1973) and that a dermal light-sensitive
system is responsible for non-ocular orientation behaviour (Van Duivenboden,
1982).
In the previous paper (Ferguson and Benjamin, 1991) the muscles mediating
whole-body withdrawal of Lymnaea and their innervation by a network of
motoneurones were described. This paper examines the effects of the sensory
modalities that induce withdrawal behaviour (photic light off and tactile sensory
Sensory activation of snail withdrawal
99
stimuli) on the activity of these motoneurones. In semi-intact preparations, it is
shown that these sensory inputs cause activation of the CM and DLM motoneurones and a contraction of the muscles used during withdrawal (the CM and
the DLM). This demonstrates that the CM and DLM motoneurones have the
appropriate electrophysiology to mediate whole-body withdrawal. Preliminary
reports of some of these results have appeared elsewhere (Benjamin et al. 1985;
Ferguson and Benjamin, 1985).
Materials and methods
Animals were obtained as described in the previous paper (Ferguson and
Benjamin, 1991). They were maintained under a 12h:12h light:dark cycle for a
minimum of 1 week prior to experiments and given the minimum disturbance
compatible with being fed regularly.
Light off and tactile stimuli were presented to the semi-intact animal by
switching off the light source illuminating the preparation or prodding the
epidermis with a rounded glass probe or a cat's whisker. The whisker had a
diameter of 0.3 mm and delivered a pressure of approximately 2.5 gmm" 2 to the
area of skin stimulated (calibrated using a pan balance). In some experiments this
hair was mounted on a piezoelectric crystal. The voltage change caused by
deformation of the crystal when the animal was stimulated was fed directly into an
oscilloscope, allowing the latency of the response to be measured. Electrophysiological experiments were conducted in Hepes (Sigma) buffered saline, using
conventional recording techniques, isolated brains and a semi-intact preparation
similar to that described in the previous paper (see Fig. 1, Ferguson and
Benjamin, 1991). The semi-intact preparation consisted of a deshelled snail
bisected by longitudinal cuts along the dorsal and ventral midlines of the headfoot. The CNS was pinned out between the two halves of the body wall. All nerves
were left intact.
Results
Spontaneous inputs received by CM and DLM motoneurones
The electrophysiology of all the electrotonically coupled CM and DLM
motoneurones (and of the visceral and right parietal ganglion cells described in the
previous paper, but whose motoneuronal role is unknown) was very similar. They
showed no obvious spontaneous activity, had resting potentials between —60 and
—70 mV and were normally silent. Two types of excitatory postsynaptic potential
(EPSP) were recorded both in isolated brains (Fig. 1A) and in semi-intact
preparations (Fig. 1B,C). The most frequently recorded input (100% of preparations) was a unitary EPSP which varied in amplitude from 2 to 15 mV, and in
which individual potentials were often summated. When this input occurred it was
usually recorded synchronously in motoneurones in different ganglia of the brain.
An example of this is given in Fig. 1A (arrows), where a right cerebral A cluster
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G. P. FERGUSON AND P. R. BENJAMIN
Right cerebral A cluster
DLM motoneurone
40 mV
Left parietal
DLM motoneurone
0.8 s
Left DLM
Left cerebral A cluster
DLM motoneurone
40 mV
Left CM
Left pedal G cluster
CM motoneurone
Left CM
40mV
4s
Left cerebral A cluster
DLM motoneurone
Fig. 1. Spontaneous inputs received by the CM and DLM motoneurones were of two
types. A unitary excitatory postsynaptic potential (EPSP) (arrows, A) which rarely
caused spike initiation and a large-amplitude EPSP which always caused a burst of
action potentials. The large-amplitude EPSP was received synchronously by all CM
and DLM motoneurones and was present in isolated (A) and semi-intact (B and C)
preparations. In semi-intact preparations the bursts of spikes in the CM and DLM
motoneurones was accompanied by high-frequency excitatory junctional potentials
(EJPs) in the CM and DLM and a longitudinal contraction of the head-foot (visual
observation).
Sensory activation of snail withdrawal
101
DLM motoneurone and the left parietal DLM motoneurone were recorded
simultaneously. This input was usually not strong enough to depolarize the cell to
the threshold for action potential initiation.
The second type of EPSP was always received synchronously by all electrotonically coupled cells. This compound EPSP input caused a rapid depolarization of up
to 40 mV in amplitude and a high-frequency burst of 20-40 action potentials,
lasting 1-2s. Within the burst, there was no absolute synchrony of action
potentials in particular pairs of cells (Fig. 1A) and different cells were often
excited for slightly different periods (Fig. IB). In semi-intact preparations,
activation of CM and DLM motoneurones by this synaptic input was correlated
with a rapid longitudinal contraction of the body, and a burst of large-amplitude,
high-frequency excitatory junctional potentials (EJPs) was recorded from the CM
and DLM simultaneously with the activity in the motoneurones (Fig. 1B,C). This
input was observed in less than 5 % of isolated brains and reduced preparations
and, when present, had no fixed periodicity, occurring between once every 10 s
and once or twice an hour. The reason for the variability in the frequency of
occurrence of this strong EPSP input and its behavioural significance are unclear.
However, the fact that this input occurred in the isolated nervous system indicates
that there are centrally located interneurones capable of producing these strong
bursts of spikes in the CM and DLM motoneurones independently of sensory
input to the snail. Whether this second type of EPSP input comes from the same
interneurone(s) as the first is not clear because in neither case have the neurones
responsible been identified.
Sensory inputs received by CM and DLM motoneurones
Effects of tactile stimuli
The most effective tactile stimuli for activating CM and DLM motoneurones
were repeated gentle proddings of the skin of the animal. These stimuli were
normally presented by touching the skin with a flexible cat's whisker as this
provided a standard stimulus to the animal (although it should be noted that the
area of skin being stimulated could show slightly different levels of contraction
during repeated stimulation). Each prod evoked a burst of summating EPSPs
within the motoneurones, which usually depolarized them sufficiently to produce
action potentials (Fig. 2). The maximum amplitude of the compound EPSP varied
in different cells, but was usually between 5 and 35 mV. The response to each prod
was phasic and usually only lasted for 1 or 2 s. Successive stimuli caused
reactivation of the motoneurones (e.g. Fig. 2A).
All motoneurones showed similar excitatory responses to tactile stimulation of
the skin. Fig. 2 shows the general features of the effect of tactile stimulation of the
ipsilateral lip on CM and DLM motoneurones and on the activity of the CM and
DLM. These three recordings (from different preparations) show that after tactile
stimulation synchronous synaptic inputs were evoked in two left cerebral A cluster
K)LM motoneurones (Fig. 2A), in left cerebral A cluster DLM and CM moto-
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G. P. FERGUSON AND P. R. BENJAMIN
Left DLM
Left CM
Left cerebral A cluster
DLM motoneurone
60 mV
Left cerebral A cluster
CM motoneurone
Left cerebral A cluster
DLM motoneurone
r
Left CM •
Left cerebral A cluster
DLM motoneurone
LI II
t
tt ! t ft -77
fit
tt
Touch left lip
Touch left lip
60 mV
Sc
rfl
Left DLM
Left pedal G cluster
CM motoneurone
|60mV
Left cerebral A cluster
DLM motoneurone
Left CM1.2s
t
t
Touch left lip
t
Fig. 2
t
tttt
Sensory activation of snail withdrawal
103
Fig. 2. Effects of tactile stimulation on CM and DLM motoneurones. Touching the
left lip caused simultaneous activation of two cerebral A cluster DLM motoneurones
(A), left cerebral A cluster DLM and CM motoneurones (B) and a left cerebral A
cluster DLM and a left pedal G cluster CM motoneurone (C). Repeated stimulation of
the lip caused synchronous short-duration bursts of spikes or EPSPs in the motoneurones, EJPs in the CM and DLM (clipped in top trace of C) and a longitudinal
contraction of the CM and DLM. (A, B and C are from different preparations.)
neurones (Fig. 2B) and in a left pedal G cluster CM motoneurone and a left
cerebral A cluster DLM motoneurone (Fig. 2C). (For locations of motoneurones
see Fig. 4, Ferguson and Benjamin, 1991.) In all three examples, tactile stimulation of the lip caused a depolarization of the motoneurones, usually resulting in
action potential initiation. High-frequency compound EJPs recorded extracellularly from the DLM and CM coincided with spike activity in the motoneurones and
the duration of this activity was very similar in muscles and motoneurones. Even
when a prod failed to produce an action potential in a particular motoneurone EJP
activity was still recorded in the muscle, suggesting that other motoneurones were
active at the same time. An example of this is most clearly given in Fig. 3A. Here,
the largest cerebral A cluster DLM motoneurone was recorded with a smaller
cerebral A cluster DLM motoneurone. Both cells received synchronous EPSP
input, but only the smaller cell was depolarized sufficiently to produce action
potentials. This suggests that the threshold for activation may be higher for the
larger cerebral A cluster cells than for the smaller ones. Some of the EPSPs
received by the larger cell had a large amplitude and were probably electrotonic
EPSPs produced by action potentials in other cells (arrowed, Fig. 3A).
A different type of experiment is shown in Fig. 3B. Here the latency of the
mechanoreceptor response was measured. The tactile stimulus was applied to the
left lip of the snail using a cat's whisker attached to a piezoelectric crystal. This
allowed the latency of the response in the cerebral A cluster neurone to be
measured more accurately than using the hand-held probe. The top trace of
Fig. 3B shows the voltage change recorded from the piezoelectric crystal upon
tactile stimulation of the animal, and the bottom trace shows an action potential
evoked in a left cerebral A cluster DLM motoneurone. The record indicates a
delay of about 140 ms from the start of the mechanical stimulus to the start of the
action potential in the motoneurone. This value was typical of all motoneurones
tested.
The convergence of sensory input from different areas of the body surface onto
particular motoneurones is demonstrated in Fig. 4. Different parts of the body
were stimulated whilst recording DLM motoneurone activity. The effects of
stimulating the left lip or the left mantle edge whilst recording from a left cerebral
A cluster DLM motoneurone and the left parietal DLM motoneurone are shown.
Stimulating both body areas produced synchronized excitation of both motoneurones. When the lip was stimulated (Fig. 4Ai) the cerebral cell fired action
|Otentials before the parietal cell became active. Stimulation of the mantle edge
104
G. P. FERGUSON AND P. R. BENJAMIN
Piezoelectric
stylus
Smaller left
cerebral A cluster
DLM motoneurone
60mV
DLM motoneurone
30 mV
Left cerebral A cluster
DLM motoneurone •
Left DLM
0.4s
t !t
1
t t
6s
Touch left lip
Fig. 3. Details of the tactile response. The largest cerebral A cluster DLM motoneurones were less excitable than other cells in the cluster (A). Tactile stimulation of
the left lips rarely caused them to fire action potentials. However, they received EPSPs
synchronously with other motoneurones in the cluster and EJPs in the DLM. Some of
these EPSPs were probably electrotonically conducted spikes from other active
motoneurones (arrows). When the cat's whisker used for tactile stimulation was
mounted on a piezoelectric stylus (B), touching the skin produced a voltage change
(top trace) from which the latency to action potential initiation could be measured. The
latency in B was 140 ms.
(Fig. 4Aii) caused synchronous activation of both cells. These two different body
areas are innervated by different nerves, the lips by the lip and superior cervical
nerves and the mantle edge by the parietal nerves (Janse, 1974). Thus, there must
be considerable sensory convergence onto the DLM motoneurones from different
parts of the body, as the two cells were excited after stimulation of both the lip and
the mantle edge.
There was widespread reception of tactile input by cells ipsilateral to the site of
stimulation and, within these cells, the input evoked similar EPSP and spike
activity. However, responses on the contralateral side were much weaker. This is
shown in Fig. 4B where simultaneous recordings were made from a left and right
cerebral A cluster DLM motoneurone and the left CM. Tactile stimuli were
delivered in turn to the left lip and then the right lip. More spike activity and a
larger underlying EPSP were evoked within the cerebral A cluster motoneurone
when the stimulus was delivered to the ipsilateral side of the body compared with
that applied to the contralateral side, although stimulation of the contralateral sidM
Sensory activation of snail withdrawal
105
An
Ai
Left cerebral A cluster
DLM motoneurone
60mV
Left parietal
DLM motoneurone
!
t t
Touch left mantle
Touch left lip
t
6s
Left CM
Right cerebral A cluster
DLM motoneurone
60mV
Left cerebral A cluster
DLM motoneurone
3s
Fig. 4. Effect of the site of tactile stimulation on CM and DLM motoneurones.
Touching lip and mantle skin areas known to be innervated by several different nerves
(Janse, 1974) caused simultaneous excitation of ipsilateral motoneurones in parietal
and cerebral ganglia (Ai and Aii, same cells), suggesting that tactile inputs are widely
distributed throughout the CNS. When both sides of the body were stimulated
separately (B), the response was stronger on the ipsilateral side than on the
contralateral side.
106
G. P . FERGUSON AND P. R.
BENJAMIN
of the body did cause some EPSPs and spikes in the left cerebral A cluster
motoneurone. Therefore, it would seem that there is some asymmetry in the
sensory convergence of tactile input, with the predominant response being
produced on the ipsilateral side of the body.
Effects of photic (light off) stimuli
When the light incident on semi-intact preparations was switched off (to mimic
the effect of passing a shadow over the animal) synchronous excitatory synaptic
input was evoked in all motoneurones (Fig. 5). In each case the input consisted of
a fast-rising compound EPSP which rapidly waned and often led to spikes
(Fig. 5A) or had lower-amplitude fast potentials superimposed on the depolarizing waveform. These latter potentials could have been either blocked spikes or
electrotonic EPSPs caused by spike activity in other, more active, motoneurones.
Muscle activity occurred after light off, even when the motoneurone being
recorded did not show full-sized action potentials (Fig. 5B), suggesting that some
other cells of the motoneurone population were, indeed, active. Cells in different
ganglia of the CNS received the sensory input synchronously (Fig. 5C). In
different preparations the latency between light off and the onset of the
depolarizing wave was consistently about 400 ms, the resultant compound EPSP
lasted for about 2 s and had an amplitude between 10 and 25 mV. Light off
stimulation was always followed by extracellular muscle activity (Fig. 5B) and
longitudinal shortening of the body similar to that occurring in the intact animal.
The eyes were not responsible for mediating the sensory input after light off, as
cutting the optic nerves had no effect on the input received by CM and DLM
motoneurones (Fig. 6A). However, the CNS had to be connected to the skin by
peripheral nerves for the input to be received (Fig. 6B). No EPSP input was
observed following the presentation of light off stimuli to isolated brain preparations, demonstrating that the response to light off was not merely due to the
direct activation of central neurones. These results suggest that dermal receptors
are involved in the reception of light off stimuli and are consistent with previous
findings (Stoll, 1972, 1973, 1976; Stoll and Bijlsma, 1973) of dermal light off
responses from the skin nerves but no activity within the optic nerve of Lymnaea in
response to either light off or shadow stimuli.
To identify the peripheral afferent pathways sending light off sensory information to the CNS, nerves connecting the skin to the CNS were cut. These
experiments were complicated by the finding that the amplitude of the EPSP
evoked by light off varied in different cells (see Fig. 5) and by the technical
difficulty of maintaining the penetration of a given cell whilst cutting nerves. In
general, cutting a single nerve made little difference to the amplitude of the EPSP
evoked in the motoneurone or to the activity in the DLM. This can be seen in
Fig. 7A (same preparation, left and right parietal nerves, Fig. 7Aii, and left and
right medial lip nerves, Fig. 7Aiii, were lesioned in turn and light off stimuli were
presented to the animal 5min after each lesion). The main exception to this
general rule followed cutting of the left and right tentacle nerves. This produced a,
Sensory activation of snail withdrawal
107
70 mV
Right cerebral A cluster
DLM motoneurone
Left DLM
Right DLM
Left cerebral A cluster
DLM motoneurone
20 mV
Left cerebral A cluster
DLM motoneurone
Light off
3s
Left CM
Left cerebral A cluster.
DLM motoneurone
A40 mV
Light off
Left parietal
DLM motoneurone
4s
Light off
Fig. 5. Excitatory effects of photic (light off) stimulation. After light off, excitatory
input was received by CM and DLM motoneurones. This input often caused cells to
fire action potentials (A). In cells that did not fire action potentials there were usually
electrotonic EPSPs in the depolarizing waveform (B) which were presumably due to
activity in coupled cells. The input was received synchronously by DLM motoneurones
in the same (B) and different (C) ganglia and was accompanied by a burst of EJPs in
the CM and DLM (B) and a longitudinal contraction of the foot.
noticeable decrease in both the amplitude of the EPSP evoked in the DLM
motoneurone and the muscle activity in the DLM after the light off stimulus
(compare Fig. 7Bi, before, with Fig. 7Bii, light off stimulus presented 5 min after
bilateral tentacle nerve cuts). These findings suggest that there are many afferent
pathways by which photic light off information is fed into the CNS from the skin,
but that the tentacle nerves are particularly important routes for carrying this
sensory information. This is compatible with the ultrastructural studies of Zylstra
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G. P. FERGUSON AND P. R. BENJAMIN
Ai
30 mV
Left cerebral A cluster
DLM motoneurone
Light off
Bi
Light off
Bii
10 mV
Left cerebral A cluster
DLM motoneurone
4
Light off
Light off
Fig. 6. Reception of light off stimuli is mediated by the skin, not the eyes. (A) A
similar excitatory input was received by cerebral A cluster DLM motoneurones before
(Ai) and after (Aii) both optic nerves from the eyes had been cut (i and ii are from
different cells). (B) Isolating the CNS from the skin, by cutting all nerves, abolished
the excitatory synaptic input (Bii) normally received by the DLM motoneurones (Bi).
(1972), who found that ciliated receptor cells (which may be photosensitive) are
located throughout the epidermis of the head-foot and are most concentrated in
the tentacles.
Although the responses of CM and DLM motoneurones to tactile and photic
sensory stimuli have been described separately, all motoneurones tested received
sensory input of both modalities. An example of this is given in Fig. 8. The same
left cerebral A cluster DLM motoneurone was stimulated first with light off
(Fig. 8A), and then with two tactile stimuli delivered to the left lip (Fig. 8B). In
response to both types of stimuli the motoneurone fired action potentials,
electrical activity was recorded from the left CM, and the semi-intact preparation
underwent a longitudinal contraction.
Sensory inputs received by other identified neurones
In addition to the CM and DLM motoneurones, some other previously
identified Lymnaea neurones also received input after sensory stimulation of the
body wall of the animal (for the positions of these cells see Benjamin et al. 1985).
Three examples of this are given in Fig. 9. In the first of these (Fig. 9A) the giant
cell RPD1 (identified by Benjamin and Winlow, 1981) was recorded together with
a left cerebral A cluster DLM motoneurone. Tactile stimulation of the left anterior
foot caused simultaneous excitation of both the DLM motoneurone and RPD1,
Sensory activation of snail withdrawal
109
Ai
Left DLM
30mV
Left cerebral A cluster
DLM motoneurone
t
Light off
Bi
Left cerebral A cluster
DLM motoneurone
Left DLM
Fig. 7
3s
Fig. 7. Effect of cutting nerves on the amplitude of excitatory synaptic input received by
left cerebral A cluster DLM motoneurones in response to light off stimuli. (Ai-iii) Same
preparation, suction electrode maintained in constant position on left DLM (top trace).
Different cerebral A cluster cells recorded in Ai, Aii and Aiii. (Ai) Light off, all nerves
intact. (Aii) Light off 5min after cutting left and right parietal nerves. (Aiii) Light off
5 min after also cutting left and right medial lip nerves. Note slight reduction in amplitude
of compound EPSP in Aiii. EJPs in left DLM are presumably due to activity of other
motoneurones. (B) Different preparation. (Bi) AH nerves intact, light off evokes a spike
within the left cerebral A cluster DLM motoneurone (top trace) and EJPs in the DLM
(bottom trace). (Bii) Light off 5 min after cutting both tentacle nerves. No spike in
motoneurone (different cells in Bi and Bii) and reduced activity in the DLM.
110
G. P. FERGUSON AND P. R. BENJAMIN
60 mV
Left cerebral A cluster
DLM motoneurone
Left CM
4
Light off
4s
I
Touch left lip
Fig. 8. CM and DLM motoneurones receive both modalities of sensory input. Light
off (A) and touching the left lip (B) evoked action potentials in a left cerebral A cluster
DLM motoneurone and EJPs in the left CM.
Similarly, tactile stimulation of the left anterior foot caused simultaneous
activation of pleural D group cells (Haydon and Winlow, 1982), a cerebral A
cluster DLM motoneurone and the left DLM and CM (Fig. 9B). Like the CM and
DLM motoneurones, these cells also received sensory input after light off. This is
shown for a pleural D group neurone and a right cerebral A cluster DLM
motoneurone in Fig. 9C.
These data demonstrate that tactile and photic input, in addition to being widely
distributed to the DLM and CM motoneurones (see Fig. 4), is also received by
other cell types. At present, the function of these cells is unknown, and although
both RPDl and pleural D group neurones have axons in the nerves innervating the
CM and DLM (Ferguson, 1985), they were not motoneurones for these muscles
and were not involved in longitudinal shortening of the body. However,
considering how similar their activity is to that of the CM and DLM motoneurones, it is possible that they play some other role during whole-body
withdrawal.
Discussion
The role of the CM and DLM motoneurones
The results presented in this paper support the hypothesis that the previously
Sensory activation of snail withdrawal
111
|-\J
Left cerebral A cluster.
DLM motoneurone
60mV
RPD16s
Touch left anterior foot
60mV
Left DLM
Right cerebral A cluster
DLM motoneurone
Left pleural D group
neurone
Left CM
30mV
Left cerebral A cluster
DLM motoneurone
20 mV
Left CM
Left pleural D group
neurone
3s
I
4s
Light off
I
Touch left anterior foot
Fig. 9. Reception of sensory inputs by cells that are not CM and DLM motoneurones.
Tactile stimulation of the left anterior foot evoked synchronous EPSPs in left cerebral
A cluster DLM motoneurones, the giant cell RPDl (A) and a pleural D group cell (B).
This usually led to simultaneous spike activity. The pleural D group cell was also
excited, synchronously with a DLM motoneurone, after light off (C).
112
G. P. FERGUSON AND P. R.
BENJAMIN
identified CM and DLM motoneurones of Lymnaea (Ferguson and Benjamin,
1991) are involved in the whole-body withdrawal response. Contraction of these
muscles and subsequent shortening of the body in the semi-intact preparation are
always accompanied by spike activity in neurones of the CM and DLM motor
pools. This can be induced by spontaneous excitatory synaptic inputs to the
motoneurones (relatively rare) or by sensory inputs from the skin, which are
known to initiate whole-body withdrawal responses in the intact snail. The
behavioural role of the 'spontaneous' activity in the CM and DLM motoneurones
is unclear, but it must be centrally generated as it occurs in isolated, as well as
semi-intact, preparations. It may underlie the spontaneous withdrawal responses
that are sometimes seen in the intact snail or play a role in locomotory activity that
involves shell movements generated by endogenous central activity (Haydon and
Winlow, 1986). In the absence of sensory input from the skin, the high resting
potentials of the CM and DLM motoneurones ensure that they are silent. This is
presumably important for the snail because strong withdrawal of the snail into its
shell prevents other types of behaviour occurring.
Whether the centrally located motoneurones of Lymnaea are entirely responsible for the whole-body withdrawal response is unclear because the large number
of neurones involved (see Fig. 4 of the previous paper) made it impossible to
assess the contribution of particular neurones to the total response. Peripheral
motoneurones are known to play a role in other defensive withdrawal responses
(e.g. the gill withdrawal response of Aplysia californica, Peretz, 1970; Peretz et al.
1976), but normally these are localized responses occurring in one part of the
body. The whole-body withdrawal response of Lymnaea requires the coordinated
response of two muscles covering most of the body surface and it seems unlikely
that this could be achieved by a peripheral neural network. This conclusion is
supported by the work of Cook (1975), who showed that cutting the columellar
and cervical nerves to the periphery, or the central pleuro-pedal connectives (all of
which contain many of the axons of the CM and DLM motoneurones; Ferguson
and Benjamin, 1991), abolishes most of the withdrawal response in Lymnaea.
Sensory input to motoneurones
Further evidence supporting the role of the motoneurones in the whole-body
withdrawal response is that the sensory modalities that normally cause whole-body
withdrawal also activate the CM and DLM motoneurones. In the semi-intact
preparation, both photic (light off) and tactile stimulation of the skin were
effective in causing activation of the CM and DLM motoneurones, strong EJP
activity in the CM and DLM and a resultant longitudinal contraction of these
muscles. It is the reception of these common inputs and the electrotonic coupling
of the CM and DLM motoneurones (Ferguson and Benjamin, 1991) that are
responsible for coordinating the activity of the motoneurones involved in the
whole-body withdrawal response.
This coordination of motoneurone activity is important because the CM and
DLM motoneurones are located in at least seven ganglia of the CNS ( F e r g u s o ^
Sensory activation of snail withdrawal
113
and Benjamin, 1991). During whole-body withdrawal these cells must be
synchronously activated; the CM and DLM must contract together to pull the shell
forward and shorten the ventral area of the head-foot. Much of this coordination
of the response can be explained by the fact that once the sensory inputs reach the
CNS they are widely distributed to the CM and DLM motoneurones. Thus,
sensory inputs converge onto cells located in widely separated ganglia of the CNS,
and they receive synchronous EPSP input from both photic (light off) and tactile
stimulation from all parts of the skin of the head-foot. It should be noted that the
sensory inputs received by CM and DLM motoneurones and by RPD1 and pleural
D group neurones were not received by all neurones within the CNS. A neurone
located in the right pedal F cluster (identified by Slade et al. 1981) and causing
contraction of the circular muscle of the head-foot is one example of a cell that
received no input after presentation of either modality of sensory stimulus
(Ferguson, 1985).
The receptors responsible for both types of sensory activation of the withdrawal
response are presumably located in the skin (Zylstra, 1972; Zaitseva and
Bocharova, 1981). Direct evidence from nerve cutting showed that the eyes are
not involved in the light off response and that this is probably mediated by light off
receptors in the skin, as previously proposed by Stoll (1972, 1973, 1976). The
degree of sensory convergence could not be tested directly with the photic stimulus
used here (switching off the main source of light to the preparation) as the whole
body surface was stimulated, but cutting particular nerves did not prevent the
excitatory response in CM and DLM motoneurones. Given that the innervation of
the skin from different nerves is discrete (although different nerves do have
innervation areas that overlap to a limited extent; Janse, 1974), receptors
responsible for the response presumably project along separate peripheral nerve
pathways from different parts of the body surface. Thus, the convergence of light
off stimuli onto many different motoneurones must mean that the sensory input is
widely distributed once it reaches the CNS from particular nerve roots. This is also
true for the tactile inputs to the CNS, as different areas of the body wall relay
information about tactile stimulation to the CNS through particular nerves (Janse,
1974). Again, the tactile inputs initiated by touching particular parts of the body
wall affected motoneurones in many different ganglia and thus the tactile input
must be widely distributed once it reaches the CNS. With tactile stimulation,
sensory convergence could be shown directly by probing different parts of the
surface of the body and recording similar inputs to a particular cell. Some of this
convergence may be facilitated by the presence of several afferent pathways to the
CNS from a particular part of the body. For instance, whole-body withdrawal can
be evoked by tactile stimulation of the lips. The sensory information is conveyed to
the cerebral ganglia of the CNS via the lip nerves and to the pedal ganglia via the
superior cervical nerves (Janse, 1974). However, the motoneurones causing
contraction of the CM are located in the cerebral and pedal ganglia, and the
motoneurones that cause contraction of the DLM are located in the cerebral,
jiedal, pleural and left parietal ganglia (Ferguson and Benjamin, 1991). For whole-
114
G. P. FERGUSON AND P. R.
BENJAMIN
body withdrawal to be complete and coordinated, motoneurones in different
ganglia must be excited synchronously by sensory input, thus requiring that the
sensory information relayed via nerves to the cerebral and pedal ganglia also
converges onto cells in other ganglia of the CNS.
Recording from the peripheral nerves of Lymnaea, Janse (1974,1976) and Janse
and Van Swigchem (1975) distinguished two major categories of touch-sensitive
neurones that had cells bodies within the CNS: primary and higher-order cells.
Primary sensory cells responded to touch or stretch and had receptive areas
varying in size from small to large (up to hah0 the body surface); higher-order
sensory neurones responded to touch, pressure and stroke, or to stretch, and had
large receptive fields. The type of stimuli applied during the present investigation
probably activated the touch, pressure and stretch receptors described by Janse
(1974, 1976) and Janse and Van Swigchem (1975). Therefore, more selective
presentation of tactile stimuli to the animal is required to determine exactly which
type of tactile sensory neurones normally evokes whole-body withdrawal behaviour. Similarly, further experiments are required to localise sensory neurone cell
bodies within the CNS and to determine whether the connections between sensory
cells and the CM and DLM motoneurones are mono- or polysynaptic.
We thank Dr E. M. Sommerville for the gift of cat's whiskers. G.P.F. was
supported by an SERC studentship.
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