J. exp. Biol. 152, 405-423 (1990)
Printed in Great Britain © The Company of Biologists Limited 1990
405
CONTROL OF LOCOMOTION IN THE FRESHWATER SNAIL
PLANORBIS CORNEUS
H. DIFFERENTIAL CONTROL OF VARIOUS ZONES OF THE CILIATED
EPITHELIUM
BY T. G. DELIAGINA* AND G. N. ORLOVSKY
A. N. Belozersky Interfaculty Laboratory, Corpus A, Moscow State University,
Moscow 119899, USSR
Accepted 3 April 1990
Summary
In the freshwater snail Planorbis corneus, the neuronal mechanisms of the pedal
ganglia that control ciliary locomotion were studied. The foot was attached to the
bottom of a recording chamber with the ciliated epithelium facing upwards. To
record the total motor effect produced by ciliary beating, a small disk with its edge
lying on the sole of the foot was used. The ciliary beating forced the disk to rotate.
In the pedal ganglia, efferent locomotor neurones (ELNs) were found, which
control the locomotor activity of the ciliated epithelium. This locomotor activity
increased with excitation of an ELN, and decreased with its inhibition. Axons of
the ELNs, controlling the anterior, middle and posterior zones, traverse the
corresponding pedal nerves. For the anterior zone, two ELNs were found. For the
middle and posterior zones, only one ELN per zone was found. The activity of
ELNs correlated with the intensity of ciliary beating during the following central
and reflex influences upon the locomotor mechanisms: (1) spontaneous fluctuations of the locomotor activity, (2) changes of temperature, (3) transections of
central connections (interganglionic connectives), (4) defensive reactions evoked
by tactile stimuli or switching off the light, and (5) activation of feeding behaviour
by natural stimuli. The data strongly suggest that ELNs are responsible for the
differential control of locomotor activity in various zones of the ciliated epithelium
during different behavioural acts.
Introduction
The freshwater snail Planorbis corneus (Gastropoda, Pulmonata) uses the
ciliated epithelium covering the sole of the foot for locomotion (Jones, 1975;
Miller, 1974; Trueman, 1983). In the preceding paper (Deliagina and Orlovsky,
1990), a general description of locomotion of Planorbis was given, and the
relationships between locomotion and various types of behaviour were con* Present address: Karolinska Institute, The Nobel Institute for Neurophysiology, Box 60400,
S-10401 Stockholm, Sweden.
I
zy words: Gastropoda, locomotion, ciliary beating, pedal efferent neurones.
406
T. G. DELIAGINA AND G. N .
ORLOVSKY
sidered. Planorbis can crawl both on the solid substratum (bottom of the pond or
river, algae, etc.) and below the water surface film, at a speed of up to 1.5 mm s" 1
The speed and the direction of locomotion are controlled by the nervous system in
relation to a behavioural task. It was demonstrated that the nervous system can
regulate the locomotor activity of the ciliated epithelium simultaneously over the
whole of the sole (when it is necessary to start or to stop locomotion, and to vary its
speed). But the nervous system can also affect the locomotor activity of the ciliated
epithelium in restricted zones of the sole. Thus, during feeding on the water
surface, the contact of a snail with food particles results in the inhibition of ciliary
beating in the posterior part of the sole.
There are numerous works devoted to the nervous control of ciliary beating in
various metazoans (for references, see Aiello, 1974; Arkett, 1987). In gastropods,
this nervous control of ciliary action has been studied both in veligers and in adult
animals. In veligers it was demonstrated that the frequency of ciliary beating
depends on the membrane potential of a ciliated cell: depolarization of a cell
results in the inhibition of beating. Under the influence of the central nervous
system (CNS), the ciliated cells can generate both postsynaptic potentials and
spikes. Serotonin excites ciliary beating (Arkett et al. 1987; Mackie etal. 1976).
An excitatory action of serotonin upon the locomotor cilia on the sole has also
been demonstrated in adult Tritonia diomedea (Audesirk et al. 1979) and Lymnaea
stagnate (Syed etal. 1988).
Central neurones controlling the pedal locomotor cilia were found in Tritonia
(Audesirk, 1977, 1978). A pair of neurones (Pd21) was identified in the pedal
ganglia, whose excitation initiated ciliary beating. These neurones participated in
the control of ciliary beating in various types of animal behaviour. In this paper,
the neurones controlling the pedal locomotor cilia of Planorbis corneus are
described. In many respects these neurones seem to be analogous to the Pd21
neurones of Tritonia.
A brief summary of this study has been published (Deliagina, 1988).
Materials and methods
Experiments were carried out on freshwater snails Planorbis corneus between
November and June. Adult snails (shell diameter of about 3 cm) were collected in
the autumn and kept in a refrigerator (5°C) in water-filled jars. Ten days prior to
the experiments, the snails were taken out of the refrigerator, put into water at
room temperature and fed with lettuce. They were subjected to an artificial
12h/l2h light/dark cycle. Surgical operations were carried out in the physiological saline for Planorbis (Kostyuk, 1968). The following preparations were used in
experiments. (1) Posterior part of foot+pedal ganglia (PeG) (Fig. 1A); (2)
Posterior part of foot+CNS (in this preparation the buccal ganglia were removed).
In preparations 1 and 2 all the nerves were cut except for the pair of posterior
pedal nerves supplying the posterior half of the foot. (3) Foot+PeG (Fig. IB). (4j|
Foot+CNS. In preparations 3 and 4 the foot was split along the midline, and air
Control of ciliary locomotion in Planorbis
407
the nerves were cut except for three pairs of pedal nerves: anterior (APN), middle
(MPN) and posterior (PPN). (5) Semi-intact preparation. This consisted of the
foot (split along the midline)+CNS+lips, tentacles and eyes. All the nerves except
for pedal, tentacular and visual ones and those supplying the lips were cut.
The preparation was attached to the bottom of a Sylgard-lined chamber filled
with a physiological saline (Fig. 1A,B)- The foot was placed sole up and fixed by
means of several pins. The preparation with the posterior part of the foot was
additionally fixed by a small clamp located along the midline. The ganglia were
pinned by the ends of cut nerves. To record the locomotor activity of the ciliated
epithelium, a special transducer was used (see Fig. 1): a plastic disk (diameter
8 mm) with black lines drawn at equal distances at its periphery. The transducer
was placed (with a little play) upon a metal axis. The axis was embedded into
Sylgard by the side of the foot in such a manner that the peripheral part of the
under surface of the disk lay on the lateral area of the sole. The whole transducer
was submerged in physiological saline and did not touch the liquid surface. The
beating of cilia on the sole under the disk evoked its rotation, the linear velocity of
the periphery of the disk being a measure of the intensity of ciliary beating. This
velocity will be designated iocomotor speed'. The disk was brightly illuminated,
and its magnified image was projected by a lens onto a photocell in such a manner
,Tr(R)
PeG
II
0.5mnis
v/VAAAAA/WWV\A i 20s
Fig. 1. (A) Posterior part of foot+PeG preparation; (B) Foot+PeG preparation.
PeG, pedal ganglia; APN, MPN and PPN, anterior, middle and posterior pedal nerves;
F, foot positioned with the ciliated epithelium upwards; Tr(L) and Tr(R), transducers
placed on the left and right sides, respectively, of the foot; AZ, MZ and PZ, anterior,
middle and posterior zones innervated by the APN, MPN and PPN, respectively. The
continuous, interrupted and dotted circles in B show the positions of the transducer for
recording activity in AZ, MZ and PZ, respectively. (C) Two methods of recording the
locomotor speed. The lower curve is the output signal of the photocell. The length of
the vertical lines in the upper graph is inversely proportional to the distance between
neighbouring peaks in the lower curve.
408
T. G. DELIAGINA AND G. N .
ORLOVSKY
that the width of the image of a black line was equal to the photocell diameter.
Movement of the black lines during rotation of the disk resulted in periodic
oscillations of the photocell illumination and, consequently, in periodic changes of
its output signal (the lower curve in Fig. 1C). The higher the locomotor speed, the
shorter was the period of the oscillations. Such records of locomotor speed are
presented in Figs 2 and 8A. Since the distance between black lines at the periphery
of the disk was about 1.5 mm, the period of waves in the photocell output signal
(that is the distance between neighbouring maxima) corresponded to the 1.5 mm
displacement of the disk periphery. By dividing this value by the period of waves,
we measured the locomotor speed and presented it as a vertical line of
corresponding length (the upper graph in Fig. 1C). This type of representation of
the locomotor speed was used in Figs 5-7, 8B and 10. In some experiments, two
transducers located in different parts of the sole were used simultaneously.
The transducer described above, because of its dimensions, monitored the
locomotor activity on a rather large area of the ciliated epithelium. Thus, in
experiments where we examined exact boundaries between the zones of innervation of various pedal nerves in the ciliated epithelium, we used another method
for monitoring the locomotor activity of the epithelium. For this purpose small
black particles (dry China ink suspended in a physiological saline) were ejected
from a pipette near the anterior border of the foot. The passage of particles along
the foot sole, caused by ciliary beating, was observed through a stereomicroscope.
Neuronal activity in pedal ganglia was recorded intracellularly with glass
microelectrodes filled with 3moll" 1 KC1 (tip resistance 50-100MQ). To facilitate
the insertion of microelectrodes, the epineural sheath was softened with Pronase E
( 1 % solution). Pronase treatment was carried out locally: the aperture of the
micropipette (filled with pronase solution) was placed against the pedal ganglion
area from which neurones were to be recorded, and the solution was gradually (for
5-10 min) ejected from the micropipette.
To polarize the membrane of a neurone, current was injected into the neurone
through the same microelectrode that was used for recording. A bridge circuit was
used to reduce the artefact caused by this current. Although this circuit lowered
the artefact, it did not remove it. For this reason the records presented in this
paper do not demonstrate the true shifts of membrane potential that the current
caused in the polarized neurone.
To study the morphology of neurones, we made use of microelectrodes filled
with 5% Lucifer Yellow (Stewart, 1978). Cells were stained by passing a
hyperpolarizing current (10 nA) for 10-30 min. The ganglia were fixed in 4 %
formaldehyde, cleared in methyl salicylate and viewed in an ultraviolet microscope.
Neuronal activity was amplified with conventional electrophysiological apparatus. This activity, as well as the output signals of photocells monitoring the
locomotor activity of ciliated epithelium, was displayed on a pen recorder.
To study the effects of temperature upon locomotion, two ways of cooling thei
preparation were used. When only the locomotor activity of the ciliated epi-"
Control of ciliary locomotion in Planorbis
409
thelium was recorded (without a parallel recording of neuronal activity), the
physiological saline in the recording chamber was gradually replaced with the
cooled one, the temperature in the chamber falling from 22 to 10°C. In
experiments with neuronal recording, the outer surface of the chamber was
cooled; in this case the temperature in the chamber fell to 13°C.
Results
Ninety-three experiments were carried out on various types of preparation. In
27 experiments, the general characteristics of locomotor activity of the ciliated
epithelium were studied, and in the remaining 66 the efferent neurones controlling
ciliary beating were investigated.
Effects of pedal nerve transection: projections of pedal nerves upon the ciliated
epithelium
Preparations consisting of a foot with pedal ganglia usually exhibited spontaneous locomotor activity: a transducer located in any area of the ciliated
epithelium rotated continuously. The study of the projections of various pedal
nerves upon the sole of the foot was carried out on this type of preparation. For
this purpose, the effects produced by nerve transection were observed. The first
experiments demonstrated that the effect of transection of a given nerve strongly
depended on the place of transection. Fig. 2A shows the locomotor activity on the
left side of the foot in a Posterior part of foot+PeG preparation (see Fig. 1A). At
the moment indicated by arrow 1, the left PPN was cut proximally, just at its exit
from the ganglion. After a short period of deceleration of the transducer
(apparently caused by contraction of the foot muscles), the locomotor speed
returned to its initial value. The same nerve was then cut distally, at the point of its
entrance into the foot (the moment of transection is indicated by arrow 2 in
Fig. 2A). After such a transection, locomotor activity ceased almost completely.
Transection of a nerve did not affect the speed of rotation of a transducer situated
on the opposite side of the foot.
Corresponding results were obtained with transections of APN and MPN. In
these experiments, the Foot+PeG preparation (Fig. IB) was used, and the
transducers were located on the anterior and posterior parts of the foot. Proximal
transection of both APN and MPN did not affect the speed of rotation of the
transducers. Distal transection of these nerves caused the anterior transducer to
decelerate, while the speed of rotation of the posterior one did not change. APN
transection was most effective when the transducer was located on the very
anterior part of the foot (this position is shown by the continuous circle in Fig. IB).
MPN transection was most effective when the transducer was shifted slightly
backwards (interrupted circle in Fig. IB).
The difference between the effects produced by distal and proximal transections
pedal nerves suggests that there exist, in the proximal part of the nerve,
'elements' (either efferent neurones or proximal parts of their axons capable of
410
T. G. DELIAGINA AND G. N. ORLOVSKY
B V(PL)
)\7%\AMAM/VMA/WVV^^
C V(PL)
\AAr^\^\X\y\A/\AA/^^A/\AAA/v-/
V(PR)
/XAAA/V/^AAftA/VVAAAA/VVA^V^NAAAVXAA/V^
D
V(PL)
E
V(AL)
^A
25s(A-C,E)
2min (D)
Fig. 2. (A,B) Effects of transection of the left PPN. The locomotor speed in the left
posterior zone, V(PL), was recorded (Posterior part of foot+PeG preparation). Arrow
1 indicates the moment of proximal transection of the left PPN; arrow 2, the moment of
distal transection of the same nerve. Recording B was performed 5 min after recording
A. V(PR), locomotor speed in the right posterior zone. (C-E) Examples of
spontaneous locomotor activity in various zones. (C,D) Locomotor speeds in the left
and right posterior zones, (Posterior part of foot+PeG preparation). C and D are from
different experiments. (E) Locomotor speeds in the left anterior and left posterior
zones (Foot+PeG preparation). In this and subsequent figures, L and R indicate left
and right sides of the foot.
spike generation) that activate the ciliated epithelium. However, we could not
detect any cell bodies in the proximal part of the pedal nerves.
To examine the boundaries between the zones innervated by various pedal
nerves, in four experiments (Foot+PeG preparation) the movement of dye
particles along the sole was observed (see Materials and methods). With the
innervation of a foot intact, particles that appeared at the anterior end of the foot
rapidly moved along the sole until they reached the tail. We then transected the
APN distally. After this transection, the speed of particles on the anterior part of
the foot sole sharply decreased, or the particles stopped, while the movement of
particles in the middle and posterior parts persisted at the initial speed. T h '
Control of ciliary locomotion in Planorbis
411
boundaries between the active and inactive epithelium could be seen rather
clearly. The zones innervated by the MPN and PPN were examined in the same
way. The area of the posterior zone (PZ) was about twice those of the anterior
zone (AZ) and the middle zone (MZ) (Fig. IB).
Correlation of locomotor activities in various zones
The Posterior part of foot+PeG preparation was used for studying the
correlation between locomotor activities in symmetrical areas (left and right
posterior zones) of the ciliated epithelium, and the Foot+PeG preparation was
used for studying the correlation between activities in the anterior, middle and
posterior zones of one side.
Posterior part of foot+PeG preparations exhibited spontaneous locomotor
activity, at speeds of 0.1-0.5 mm s" 1 , which were considerably lower than those of
the intact animal (about l m m s " 1 ) . The speeds on the left and right sides of the
foot varied somewhat with time. Usually, we did not observe any strict correlation
between the speeds (as in the example shown in Fig. 2B). In about 30% of
experiments, a periodic inhibition could sometimes be observed of one or both
locomotor speeds, with a period of 30-60 s. If both speeds underwent these
periodic changes, they varied in phase. An example of such in-phase coordination
in the left and right posterior zones is presented in Fig. 2C. Besides the in-phase
coordination, we also observed (but rarely, in five experiments only) an out-ofphase (reciprocal) coordination. As is shown in Fig. 2D, when the left zone was
active, the right zone was inhibited: alternations between the two sides occurred
irregularly, at intervals of 1-10min or more. In the course of an experiment, the
preparation could successively exhibit in-phase coordination, reciprocal coordination and independent activities of the two sides of the foot.
Fig. 2E shows a simultaneous recording of the locomotor speeds in the anterior
and posterior zones (Foot+PeG preparation). The speed in the anterior zone, in
various experiments, was 0.5-1.5 mm s" 1 (corresponding to the speed of an intact
animal), while the speed in the posterior zone was 2-3 times slower. Fluctuations
of the two speeds were not usually linked with one another, but in some
experiments one could observe in-phase fluctuations (with a period of 30-60 s,
Fig. 2E) corresponding to those described for the symmetrical areas of the foot
(Fig.2C).
Serotonin activates the ciliated epithelium
In Tritonia diomedea and Lymnaea stagnalis, serotonin excites ciliary beating
(Audesirk et al. 1979; Syed et al. 1988). The same result was obtained in Planorbis
corneus. The effects of serotonin (5-HT) and its precursor (5-hydroxytryptophan,
5-HTP) were studied in a preparation of the isolated foot with the pedal nerves
transected distally, in which spontaneous locomotor activity was absent. Serotonin
was gradually added to the physiological saline in the recording chamber. Its
|effects are shown in Fig. 3A. Locomotor activity appeared at a threshold
'concentration between 10~7 and lO^moll" 1 . At higher concentrations, the speed
412
T. G. DELIAGINA AND G. N. ORLOVSKY
1.0-
0.5-
0J
0.5-
10" 7
10"6 10" 10"
[5-HT](moir')
10"
2
5-HTP(l(T4moir1
fy
3
Wash
Time (h)
Fig. 3. Excitation of ciliary beating by serotonin (5-HT) and its precursor
5-hydroxytryptophan (5-HTP) (Posterior part of foot+PeG preparation). (A) An
increase in 5-HT concentration results in an increase of locomotor speed. (B) Effect of
5-HTP.
increased until it reached a plateau at 10 4 mol 1 1. The maximal value (1 mm s"1)
was close to the maximal speed observed in intact animals (1.5 mm s" 1 ). After the
preparation had been washed in clean physiological saline, locomotor activity
ceased.
The serotonin precursor also activated the locomotor epithelium. One can see in
Fig. 3B that 5-HTP (10~ 4 moir 1 ) evoked locomotion. The speed increased
gradually and in 2 h reached 0.7 mm s" J . After the preparation had been washed in
clean physiological saline, the locomotor activity persisted unchanged.
Efferent locomotor neurones
Efferent locomotor neurones (ELNs), controlling the anterior, middle and
posterior zones of the ciliated epithelium, were found in the pedal ganglia.
Neurones of the anterior zone
These were studied in the Foot+PeG preparation. The transducer was placed
on the anterior zone of the foot (continuous circle in Fig. IB). On the anterior pole
of each pedal ganglion there were two neurones, ELN(Al) and ELN(A2) which
affected the speed of locomotion in the anterior zone. Fig. 4A shows the typical
position of these neurones. We could not identify these neurones visually
(according to their size and position) with certainty; for their identification it was
necessary to know their effect on locomotion. Staining with Lucifer Yellow
showed that these neurones had a soma diameter of about 50/xm. Each of the
neurones had one axon going to the ipsilateral PPN. The background discharge of
these neurones had a frequency of 1-2 Hz. Sometimes in the background
discharge one could observe a short regular inhibition repeated at intervals of
15-60 s (as shown for the neurones of middle and posterior zones in Figs 6A and
8C). The ELN(Al) and ELN(A2) from one ganglion were found to be weakly
electrically connected, but this coupling was revealed only when passing a very
strong current (see Fig. 5D). This connection was so weak that neither spikes nor
Control of ciliary locomotion in Planorbis
413
PPN
CP
200/on
Fig. 4. (A,B) Location of efferent locomotor neurones (ELNs) in the pedal ganglion.
In A the ganglion is shown with the anterior pole up; in B, with the posterior pole up.
CPC, cerebropedal connective; CN, collumellar nerve; St, statocyst. (C) Morphology
of ELN(P) stained with Lucifer Yellow. Other abbreviations as in Fig. 1.
postsynaptic potentials arising in one of the neurones were reflected in the
membrane potential of the other neurone (Fig. 5E). The effects of ELN(Al) and
ELN(A2) on locomotion were different. One can see in Fig. 5A that when a
current of -0.5 nA was injected into the ELN(Al) (the minimal current
suppressing the spike activity), the speed of locomotion was about O.lmms" 1 .
When the current was switched off the neurone generated a high-frequency burst
and then, at the resting potential, fired at a frequency of 1.5 Hz. When ELN(Al)
was excited, the speed of locomotion increased over 3-5 s and reached 1.3 mm s" 1 .
Then the neurone was hyperpolarized again and the speed of locomotion
decreased over 10-15s to O.lmms" 1 . Thus, when the frequency of ELN(Al)
discharge was changed from 0 to 1.5 Hz, the speed of locomotion increased 13fold. Hyperpolarization of ELN(Al) with a current of —0.5nA produced no
noticeable effect upon ELN(A2) activity.
ELN(A2), in contrast to ELN(Al), affected the locomotor speed only slightly.
When it was hyperpolarized (—0.5 nA), its discharge terminated and the speed of
locomotion decreased by 10-20% (Fig. 5B). Only with much stronger current
injected into ELN(A2) could a pronounced inhibition of locomotor activity be
observed. As shown in Fig. 5C, when a current of —10 nA was injected into
ELN(A2), the speed of locomotion decreased from 1 to 0.6mms" 1 . Since the
resistance of ELNs for hyperpolarizing currents was about 20 MQ (measured
several times when inserting two microelectrodes into a neurone), with a
hyperpolarizing current of - l O n A , the membrane potential of the ELN(A2)
reached about 200 mV, i.e. it far exceeded the normal physiological range. As seen
in Fig. 5C, when such a current was injected, ELN(Al) was noticeably inhibited
owing to the electrical connection with ELN(A2). Since ELN(Al) had a very
kstrong influence on locomotion, one cannot rule out the possibility that a
significant part of the effect of the ELN(A2) polarization shown in Fig. 5C is
414
T. G. DELIAGINA AND G. N. ORLOVSKY
A
B
V(A)
ll I ,
-0.5 nA
-0.5 n A
H
lmms
50 mV
50mV
20 mV
Fig. 5. Characteristics of the ELNs controlling the anterior zone. The activities of
ELN(Al) and ELN(A2), as well as the locomotor speed in the anterior zone, are
presented (all recordings from the same pair of cells). The period of current injection is
marked by a solid line, the polarity and current strength being indicated near the line.
ELN(Al) affects the locomotor speed much more strongly than ELN(A2) (compare A
and B). To obtain a more pronounced effect, ELN(A2) was hyperpolarized by a very
strong current. With such a current ELN(Al) was also affected (C) owing to the
electrical coupling demonstrated in D. This coupling is too weak to synchronize the
cells (E). Scales shown in C also apply to A and B.
mediated by ELN(Al). It seems likely that, besides ELN(Al) and ELN(A2),
there are no other neurones in the pedal ganglia electrically connected with them
and affecting locomotor activity in the anterior zone. The following experiment
confirmed this hypothesis. When ELN(Al) and ELN(A2) were hyperpolarized by
a current of -0.5 nA (the minimal current needed to suppress their spike activity),
the speed of locomotion decreased from 1 to 0.1 mm s" 1 , but a further increase of
the current (to - 1 0 nA) produced no additional decrease in speed. If ELN(A1)|
and ELN(A2) had electrically connected partners affecting locomotion, such a
Control of ciliary locomotion in Planorbis
415
strong current would have inhibited them and produced an additional decrease in
speed.
ELN(Al) and ELN(A2) affected only the anterior zone of the ciliated
epithelium. When we excited or inhibited these cells with polarizing current, we
could affect the speed of rotation of the transducer in the anterior zone of the foot,
but the locomotor speeds in the posterior zone, and in the contralateral anterior
and posterior zones, did not change.
Neurones of the posterior zone
These were studied in the Posterior part of foot+PeG preparation. On the
ventral side of each pedal ganglion we found one efferent neurone, ELN(P),
controlling locomotor activity in the posterior zone. Fig. 4B shows the typical
position of this neurone. ELN(P) had a soma diameter of about 65 /xm and one
axon in the PPN (Fig. 4C). Its background discharge had a frequency of 1-2 F£z. In
almost all experiments, there were short periodic pauses in the activity of ELN(P)
repeated at intervals of 15-60 s (Fig. 6A). Each pause was followed (with some
delay) by a slowing of locomotion.
An example of the influence of ELN(P) upon locomotor activity is shown in
Fig. 6B. Initially, the neurone was hyperpolarized by a current of — lOnA. At this
current the speed of locomotion in the ipsilateral posterior zone was about
0.25mms" 1 . When the current was switched off, the neurone became active and
V(PR)
0.5mms
50 mV
ELN(PR)
0.5 mm s
50 mV
-lOnA
Fig. 6. Characteristics of the ELN controlling the posterior zone. (A) Spontaneous
locomotor activity in the posterior zone and background activity of the corresponding
ELN. (B) Effects produced by hyperpolarization of ELN(PL) on the locomotor speeds
in the ipsi- and contralateral posterior zones. Time scale in B applied also to A.
416
T. G. DELIAGINA AND G. N . ORLOVSKY
the speed of locomotion increased gradually (over 5-7 s) to 0.8 mm s" 1 , i.e. about
threefold. When the current was switched on again, locomotion gradually
decelerated over 15-20 s. The switching off of the hyperpolarizing current was
repeated once more (in the middle of the recording) with the same result. At the
end of the recording, the hyperpolarizing current was not switched off but
decreased to - 2 n A . In this case, ELN(P) did not spike, but the speed of
locomotion almost doubled. This may indicate that ELN(P) has electrically
connected partners. Unfortunately, they have not been found yet. As shown in
Fig. 6B, changes of ELN(P) activity did not affect the locomotor speed in the
contralateral posterior zone.
Neurones of the middle zone
These were studied in the Foot+PeG preparation. The transducer was located
in the middle zone of the foot (the interrupted circle in Fig. IB). In each pedal
ganglion we found one efferent neurone, ELN(M). This neurone was located on
the ventral side of the ganglion, in the same region as ELN(P), but somewhat
more medially (Fig. 4B). The axon of ELN(M) entered the MPN; and the soma
diameter was about 50 pan. The background activity of ELN(M) was similar to that
of ELN(P). The effect of ELN(M) on locomotor activity is shown in Fig. 7. When
the neurone discharge was suppressed by a hyperpolarizing current, the locomotor
activity in the middle zone disappeared almost completely, while the speed in the
posterior zone did not change. It seems likely that ELN(M) also has electrically
coupled partners. This is supported by the observation that variations in a strong
hyperpolarizing current (sufficient to inhibit the spike discharges of the neurone)
injected into ELN(M) could still affect the locomotor speed, as was shown for
ELN(P) (Fig. 6B).
V(P)
I
V(M)
I
I
I
I
i l l
0.5 mm s
50mV
ELN(M)
Fig. 7. Characteristics of the ELN controlling the middle zone. Effects produced by
hyperpolarization of ELN(M) on the locomotor speed in the middle and posterior
zones.
Control of ciliary locomotion in Planorbis
417
ELNs mediate different influences upon the locomotor mechanism
The inhibitory influence of cerebral ganglia
The speed of locomotion in Foot+CNS preparations was usually slower than
that of Foot+PeG preparations. The locomotor activity in the middle and
posterior zones was particularly low, and in some experiments activity in these
zones was completely absent. This inhibitory influence came from the cerebral
ganglia. In Fig. 8A (the beginning of recording) the locomotor activity in the
posterior zones (left and right) in the Foot+CNS preparations is shown. One can
see that this activity was very low. The right cerebropedal and cerebropleural
connectives were then cut (the moment of transection is marked by arrow 1). After
transection, locomotor activity on the right side increased sharply, but activity on
the left side remained low. When the left cerebropedal and cerebropleural
connectives were cut (arrow 2) the left side was also inactivated.
Recordings of ELN(M) and ELN(P) in the Foot+CNS preparation demonstrated that their activity was low. In Fig. 8B the activity of ELN(M) and the
locomotor speed in the middle zone in such a preparation are shown. The mean
frequency of the discharge during recording was 0.5 Hz, and the locomotor speed
was about 0.07 mm s" 1 . After the ipsilateral cerebropedal and cerebropleural
connectives had been cut (Fig. 8C), the frequency increased to 3 Hz (i.e. sixfold)
and the speed of locomotion increased to 0.5 mm s" 1 (i.e. sevenfold). The
rhythmic modulation of the neuronal discharge and of the speed of locomotion,
with a period of 30 s, is clear. Thus, the cerebral ganglia exert an inhibitory effect
upon the ELNs of the middle and posterior zones. It seems Likely that it was
disinhibition of these neurones (after cutting the cerebropedal and cerebropleural
connectives) that resulted in the increase of locomotor activity in these zones.
V(PL)
2min
0.5 mm s
V(M)
,
|ELN(M
1111
I
50 mV
Fig. 8. Effects of transection of the cerebropedal and cerebropleural connectives.
(A) Transection of the right connectives (arrow 1) and the left connectives (arrow 2)
resulted in excitation of the ciliary beating on the corresponding side of the foot.
(B) The speed in the left middle zone and activity of the corresponding efferent
neurone (Foot+CNS preparation). (C) As for B, but 1 min after transection of the left
cerebropedal and cerebropleural connectives.
418
T. G. DELIAGINA AND G. N. ORLOVSKY
ELN(Al) and ELN(A2), which control the anterior zone, were as active in the
Foot+CNS preparation as in the Foot+PeG preparation. Correspondingly,
locomotor activity in the anterior zone was high in both preparations.
Effects of temperature
The speed of locomotion in gastropod molluscs is known to decrease with
cooling. We found, however, that there are important differences in the
temperature curves for the Isolated foot and Foot+PeG preparations. The
dependence of locomotor speed on temperature in the Foot+PeG preparation is
shown in Fig. 9A. With a change in temperature from 10 to 15°C the speed
increased from 0.1 to 0.5 mm s" 1 . With a further increase in temperature (to 22°C)
the speed did not change. The pedal nerves were then cut proximally, resulting in a
dramatic change in the temperature curve: the speed of locomotion now depended
linearly on temperature over its whole range. Thus, the pedal ganglia ensure that
the snail's locomotor speed is independent of temperature within the range
15-22°C.
We carried out simultaneous recordings of the locomotor speed and of the
activity of ELN(M) when changing temperature. Since we used another method of
cooling (see Materials and methods) the range of temperatures in these experiments was narrower (13-21 °C). The temperature curves for the discharge
frequency of ELN(M) and for the speed of locomotion are shown in Fig. 9B.
Between 15 and 21 °C these curves are parallel and are only slightly dependent on
temperature. Therefore, we suggest that the shape of the temperature curve of
locomotor speed in the Foot+PeG preparation is determined by ELNs.
Activation of the locomotor system during feeding arousal
In intact Planorbis, presentation of food evokes rhythmic feeding movements of
the radula and of other parts of the buccal apparatus. If the animal had been
motionless up to this point, it would begin to creep in the direction of the source of
food (Deliagina and Orlovsky, 1990). Similar reactions were observed in the semi1.01
8.
0.5-
0.5BO
o
c
•c
0J
10
15
20
0
"
10
Temperature (°C)
c
15
20
Fig. 9. Dependence of locomotor speed and ELN(M) activity on temperature.
(A) The speed as a function of temperature in the Foot+PeG preparation before (•)
and after (O) proximal transection of all pedal nerves. (B) The locomotor speed in the
middle zone ( • ) and the firing rate of the corresponding ELN(M) (O) as a function of
temperature (Foot+PeG preparation).
Control of ciliary locomotion in Planorbis
419
V(M)
I
)
I
J
I
I
ELN(M)
20 s
B V(M)
I
I
I
I I I1
ELN(M)
ill
0.5mms
50 mV
Fig. 10. Reflex influences on locomotor speed and ELN activity (semi-intact preparation). (A) Touching the lip with a lettuce leaf (the period of touching is marked by a
solid line) evoked the feeding rhythm in a buccal ganglion neurone (BN), activated
ELN(M) and increased the locomotor speed in the middle zone. (B) Tactile
stimulation of the tentacle (marked by the arrow) decreased both the locomotor speed
and the neuronal activity. (C) A short break in the illumination (marked by a solid line)
evoked both inhibition of the neuronal discharge and a decrease in locomotor speed
(all recordings from the same preparation).
intact preparation. As shown in Fig. 10A, touching the ventral surface of the lip
with a crushed lettuce leaf (touching is marked by a solid line) evoked rhythmic
activity in the feeding system which was demonstrated by rhythmic bursting in one
of the neurones of the buccal ganglia (BN). At the same time, ELN(M) became
active, and the speed of locomotion increased considerably.
Inhibition of locomotor activity during defensive reactions
In intact Planorbis, tactile stimulation of various parts of the body and switching
off the light evoke defensive reactions, including inhibition of locomotion. The
420
T. G. DELIAGINA AND G. N .
ORLOVSKY
defensive reaction could also be observed in the semi-intact preparation
(Fig. 10B,C). Tactile stimulation of the tentacle or switching off the illumination
evoked inhibition of ELN(M), and locomotor speed decreased.
Thus, all the effects upon locomotion (both acceleratory and deceleratory)
observed by us were accompanied by corresponding changes of the activity of the
ELNs.
Discussion
The foot of Planorbis corneus is innervated by three pairs of pedal nerves.
Transections of these nerves demonstrated that each of them has a definite
projection in the ciliated epithelium of the foot sole: a distal cutting of the nerve
resulted in the cessation of locomotor activity in the corresponding zone.
According to these projections of pedal nerves, six zones could be distinguished
(Fig. IB). This result corresponds well to the morphological data obtained in the
nudibranch gastropod mollusc Hermissenda (Richards and Farley, 1987): by
staining the pedal nerves it was demonstrated that their projections in the sole do
not overlap.
In the pedal ganglia of Planorbis we found efferent locomotor neurones (ELNs)
controlling the locomotor activity of the ciliated epithelium. When these cells are
excited, locomotor activity increases, and when they are inhibited, it decreases.
Each ELN has only one axon, which runs in one of the pedal nerves and controls
the locomotor activity in the corresponding zone. Two neurones, ELN(Al) and
ELN(A2), were found for the anterior zone, ELN(Al) being much more
'influential' than ELN(A2): by changing its activity within the physiological range,
we could obtain almost the whole range of locomotor speeds (from 0.2 to
1.3 mms" 1 , Fig. 5A).
We found one efferent locomotor neurone for the middle zone [ELN(M)] and
one for the posterior zone [ELN(P)]. However, it seems likely that, for each of
these zones, there are some more influential neurones weakly electrically
connected with ELN(M) and ELN(P). This is supported by the observation that
we could change the locomotor speed by means of hyperpolarizing current
(injected into the ELN), even when spike activity in the ELN was suppressed
(Fig. 6B).
We think that the group of ELNs that we have found could provide the
operative control of locomotion. This is supported by the following observations.
(1) By changing activity in the ELNs we could change the speed of locomotion
over a wide range. (2) The ELNs are affected by the nervous system in all cases
when changes of locomotor speed are observed. We have found parallel changes in
ELN activity and in locomotor speed: (a) during feeding arousal (Fig. 10A); (b)
during defensive reactions (Fig. 10B,C); (c) during transection of the cerebropedal connectives (Fig. 8B,C); (d) during temperature changes (Fig. 9B); and (e)
during spontaneous fluctuations of locomotor speed (Figs 6A, 8C). (3) Exper-.
imental polarization of ELNs has shown that, after excitation of an ELN, the
Control of ciliary locomotion in Planorbis
421
speed of locomotion increased and reached a maximum in about 5 s. After
termination of the ELN activity, the reduction in locomotor speed lasted for about
15 s (Figs 5A, 6B, 7). Corresponding time dependences between the activity of the
ELNs and the speed of locomotion were found during spontaneous fluctuations of
both ELN activity and locomotor speed (Figs 6A, 8C).
Nervous mechanisms controlling the ciliated epithelium of the sole of the foot
have also been studied in Tritonia diomedea (Audesirk, 1977, 1978). In the pedal
ganglia, two symmetrical efferent neurones (Pd21) were found to affect ciliary
beating in the posterior part of the sole. Each had two axons: one running in the
pedal nerve and another in the contralateral pedal ganglion. When these neurones
were excited, beating of the cilia in the posterior part of the sole intensified.
Activity of Pd 21 was correlated with the intensity of ciliary beating during various
reflex influences on locomotion. These neurones appear to be analogous to the
ELNs of Planorbis.
Because of the restricted projections of the ELNs, the nervous system can
differentially control various zones of the ciliated epithelium. Evidence for such
control was obtained in behavioural experiments: when the snail was fed on the
water surface, ciliary beating in the posterior part of the foot was inhibited
(Deliagina and Orlovsky, 1990). In experiments on the Foot+CNS preparation we
found that locomotor activity in the middle and posterior zones of the foot was
rather low. This was caused by a strong tonic inhibitory inflow from the cerebral
ganglia to ELN(M) and ELN(P): after the removal of the cerebral ganglia the
tonic activity of ELN(M) and ELN(P) as well as the speed of locomotion increased
considerably (Fig. 8).
Our experiments also demonstrated the existence of differential control of the
left and right sides of the foot: in some experiments reciprocal, alternating activity
of the two sides was observed (Fig. 2D). It seems likely that such a regime is used
by the snail during turns; for example, during 'looping' (Deliagina and Orlovsky,
1990). Unfortunately, the activity of ELNs during such a locomotor regime was
not recorded. However, it seems likely that it is the alternating activity of the
symmetrical ELNs that determines the locomotor regime described above.
However, in the majority of behavioural tasks, all zones of the ciliated
epithelium are controlled in parallel, i.e. excitation and inhibition of ciliary
beating occurs simultaneously over the whole sole. In experiments on the semiintact preparation, it was demonstrated that all the ELNs have common inputs.
All of them were activated during feeding arousal and inhibited during defensive
reactions. Another example of the parallel control of different ELNs is the 30-s
rhythm, i.e. a simultaneous decrease in the locomotor speed in all the zones
(Fig. 2C,E), the 'locomotor pauses' being preceded by pauses in ELN activity
(Figs 6A, 8C).
There is considerable evidence that serotonin plays an important role in the
excitation of ciliary beating in gastropods. Serotonin excites beating of locomotor
Cilia in veligers (Arkett etal. 1987; Korobzov and Sacharov, 1971; Mackie etal.
1976) as well as in adult molluscs {Tritonia diomedea, Audesirk etal. 1987;
422
T. G. DELIAGINA AND G. N . ORLOVSKY
Lymnaea stagnalis, Syed et al. 1988). Our experiments demonstrate that the
ciliated epithelium of the isolated foot of Planorbis can also be activated by
serotonin (Fig. 3A) and by its precursor 5-HTP (Fig. 3B). An increased amount of
serotonin was found in neurone Pd21, which excites ciliary beating in Tritonia
(Audesirk et al. 1979). A great number of nerve endings containing serotonin and
forming synapses on ciliated and mucus cells have been found in the foot of
Lymnaea (Syed et al. 1988). Finally, there are numerous pedal efferent neurones
containing serotonin and projecting to the foot in Lymnaea (McKenzie et al.
1987a,b; Slade et al. 1981). However, the question of whether the ELNs
controlling ciliary locomotion in Planorbis use serotonin as a mediator remains to
be answered.
Our experiments with transection of pedal nerves gave different results
depending on the level of transection: when the transection was performed
proximally, locomotor activity persisted, but with a distal transection, it ceased
(Fig. 2A,B). We could not detect any cell bodies in the proximal part of the pedal
nerves. In contrast, it was found that the ELNs are very important for exciting the
locomotor cilia. Thus, it seems very likely that the proximal part of the ELN axon
plays a crucial role in maintaining ELN activity. It is possible that the spiketriggering zone is located in the proximal part of the ELN axon. This is supported
by findings obtained in two other species of gastropod molluscs. In Hermissenda it
was demonstrated that, in pedal efferent neurones, the spike-triggering zone is
located in the proximal part of an axon (Richards and Farley, 1987). In Clione
limacina, the spike discharge in axons of efferent pedal neurones persisted after
removal of the soma (Arshavsky et al. 1986).
In the present study we found that the dependence of locomotor speed on
temperature differed in the Isolated foot and Foot+PeG preparations. In the
range 15-20°C, locomotor speed in the isolated foot almost doubled, whereas with
the pedal ganglia intact it was almost constant (Fig. 9). Thus, owing to the
influence of the pedal ganglia, the ciliary locomotor apparatus is independent of
temperature. In the pteropod mollusc Clione limacina, the frequency of wing
oscillations does not depend on temperature, provided that the cerebral ganglia
are intact. After removal of these ganglia, the frequency becomes dependent on
temperature (Suslova, 1985). The nervous mechanisms responsible for the
stabilization of ciliary beating under conditions of varying temperature need
further investigation.
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