With 2 plates and 12 text-figures Printed in Great Britain
A PREPARATION OF APLYSIA FASCI AT A
FOR INTRASOMATIC RECORDING AND STIMULATION OF
SINGLE NEURONES DURING LOCOMOTOR MOVEMENTS
BY R. DE G. WEEVERS
Department of Zoology, University of Bristol, and Institut de Biologie marine d'Arcachon, France
{Received 22 October 1970)
INTRODUCTION
By reason of their large size and ease of penetration with single or multiple micro-electrodes, the central neurones of opisthobranch and pulmonate gastropods have contributed a substantial proportion of current knowledge of invertebrate synaptic mechanisms, integrative processes and neurone geometry (Tauc, 1966). By contrast, far less is known of the behavioural roles of these same neurones, and in consequence it is difficult to assess the significance of the various postulated integrative mechanisms (Frazier et al. 1967).
The work of Willows (1968) and Willows & Hoyle (1968) was an important contri-bution in that for the first time behavioural acts were evoked from a gastropod by direct stimulation of certain specific single cells. The most dramatic of these acts was identical with the normal evasive activity of Tritonia when stimulated by the proximity of a starfish normally preying upon it. In addition to providing information about the functional significance of patterns of neural circuitry, the Tritonia preparation indi-cated the danger of assuming that isolated parts of nervous systems behave in the same way as when they are connected to their sensory inputs or to other ganglia. The majority of cells which would have exhibited pacemaking in isolated brains were silent unless stimulated when their peripheral connexions were intact (Willows, 1968). A similar phenomenon was reported more recently by Stinnakre & Tauc (1969) in Aplysia abdominal ganglia, where the well-known pacemaking cell R. 15 (terminology of Frazier et al. 1967) only becomes regularly autoactive once its connexions with the branchial ganglion and/or osphradium have been severed. Pacemaking in this case is normally inhibited by the action of hypo-osmotic sea water.
Two more recent studies (Kupferman & Kandel, 1969; Peretz, 1969) described various features of an abdominal ganglion preparation in Aplysia californica where connexions with the gill and organs of the mantle shelf were left intact. As in Tritonia, stimulation of certain motor cells evoked stereotyped and repeatable movements, whereas others evoked more complex effects indirectly via these 'motor neurones'. The behavioural functions of these movements were presumed to relate to gill venti-lation, expulsion of faeces and detritus or to protective withdrawal of the delicate respiratory exchange structures.
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R. DE G. WEEVERSactivities: retraction of branchial tufts, turning and swimming in Tritonia; gill with-drawal in Aplysia. It is perhaps not unexpected that this should be so, for the condition of most neurophysiological experiments must be fairly noxious from the point of view of the animal. Though there are undoubted technical advantages in working with escape-mechanisms (large size of cells involved, ease of evocation of behaviour), it is probable that study of these alone would yield an unduly simplified view of the neural mechanisms underlying behaviour. For instance, Dorsett, Willows & Hoyle (1969) reported that isolated Tritonia brains will generate patterns of neural activity in-distinguishable from evasive swimming when triggered by electrical stimulation of either the second or third cerebral nerve. Such independence from sensory feedback would not be expected in the less 'all-or-nothing' movements of normal locomotion in view of the known existence of proprioceptive reflexes in other gastropods (Herter, 1931a, b).
One of the large European species of tectibranch, Aplysia fasciata, is a powerful swimmer capable of covering considerable distances by active linear progression. Unlike the swimming of Tritonia, this is not an escape activity. The reaction of this species of Aplysia to noxious stimuli is to contract into a ball and to emit purple ink and mucous secretions. The three components of the response are recruited pro-gressively according to the intensity of the stimulus. Swimming, on the other hand, can reliably be evoked in the intact animal by suspending it free of the substratum. The present work was initiated to develop a preparation which would permit intraso-matic recording from central neurones during behavioural activities, especially the swimming referred to above.
Text-fig. 1. Swimming of intact Aplysia suspended in the experimental tank by means of a pair of hooks at the anterior and at the posterior end and a pair of hooks attached to each of the para-podia to limit the extent of their movement. The transducer was attached to the tail at 45° to the longitudinal axis so that both the vertical oscillations of swimming and changes in total body length were simultaneously displayed at equal amplification. The upper beam is displaced down-wards at the moment when the foot was allowed to grip an endless belt of Polythene. The resultant downward movement of the transducer trace indicates shortening of the foot. Cali-bration marks : 1 s, 1 cm.
METHODS
controlled to 17 + 1 degC and aerated. Text-fig. 1 shows the response of a swimming undissected animal suspended in this way to objects touching the foot - very reminiscent of the flying insect's tarsal inhibitory reflex.
Unfortunately, it was found that as soon as the animal was opened to expose the
Padded clamp
Posterior parapodial nerve Mid-parapodial nerve
Posterior pedal nerve
Anterior parapodial nerve
Pedal ganglion Pleural ganglion Anterior aorta
Position of
ganglion table Anterior pedal nerve
Cerebral ganglia
Buccal mass and buccal ganglion
anterior ganglia, the greater proportion of the haemolymph rapidly escaped. In
Tri-tonia the condensed head ganglia allow the buccal mass to be used as an internal plug
to minimize this (Dorsett, personal communication). Such bled animals, though they will perform repeated parapodial folding and other movements for about 30 min, soon become inert except for single closure movements of the branchial chamber and para-podia every 2-4 min (the SGM of Peretz, 1969). Various methods were tried for success in retaining turgor, but this proved difficult because in order to obtain swimming it was evidently necessary that the foot should not be too strongly stimulated.
The method eventually adopted is shown in Text-fig. 2. First, the animal was sus-pended dorsal surface uppermost by the eight hooks attached as described under Text-fig. 1. A padded ring consisting of two semi-elliptical bands of Perspex hinged together at one side was then clipped around the 'neck' of the animal just anterior to theparapodia. The haemolymph in the posterior part of the body was retained by means of a latex balloon inserted into the haemocoele through a short slit in the body wall just anterior to the clamp; the balloon was inflated with air once it was in position so that it pressed the body wall outwards against the padded ring, displacing forward the contents of the crop which had previously occupied this position. The balloon consisted of a short length of latex tubing tied on to 3 mm O.D. thick-walled Polythene tubing bent into a right angle which gave the stiffness necessary for its insertion. This operation en-tailed little loss of haemolymph from the posterior part of the body. However, animals undergoing dissection always contracted the parapodia, thus forcing a considerable proportion of the haemolymph into the neck region; this escaped during subsequent dissection. In order to re-expand the posterior sinuses to their original volume, a small-diameter Polythene tube (shown at the top left in Text-fig. 2) was pushed down be-tween the balloon and the body wall. Sea water injected through this seemed to form an adequate substitute for haemolymph.
When this technique was first tried on an animal which had lost most of its haemo-lymph the effects were dramatic. As the posterior sinuses were expanded, the pre-viously inert animal started waving its parapodia, expanding and contracting its foot and crawling when a suitable substratum was presented. In addition to this seeming tonic excitatory effect of distension, such movements as were occurring became much easier to interpret. The action of the hydrostatic skeleton meant that contraction of, say, parapodial levator muscles resulted in the normal levation movement rather than local shortening which produced nothing more than enigmatic wrinkles.
The dorsal slit was then extended anteriorly, as shown in Text-fig. 2. A pair of hooks were used to open up the slit permitting further dissection. The crop was ligated posteriorly near to the clamp and removed. This allowed the buccal mass to be pulled forward and also exposed the circumoesophageal ring of ganglia, the pedal nerves and the pleurovisceral connectives. A further marked improvement in the activity of the animal was found to result from perfusing the anterior aorta with oxygenated sea water. This was done with a micromanipulated hypodermic needle inserted into the aorta. Animals often swam at this stage and had to be quieted by raising a platform to make contact with the foot (see Text-fig. 1). This platform consisted of a con-tinuous Polythene belt stretched over rollers, which apparently constituted a suitable surface for creeping.
the ganglion table shown in Text-fig. 3 (b) through the circumoesophageal nerve ring ind under the left pedal and pleural ganglia, whose neurones were the subjects of the present investigation. The left side was chosen because the penis obscures the nerves on the right. Micropins through the outer sheaths of these ganglia immobilized and tensioned the surface of the sheath. In early experiments the sheath was slit with a sliver of razor blade; but in addition to damaging a proportion of cells, this procedure ruptured the blood sinuses around the brain so that perfusion of the ganglia became less effective. It may be also that the rather high perfusion pressure normally used
(a) (b)
Text-fig. 3. (a) Microelectrode mounted on a loudspeaker. The coil suspension is modified and consists of two thin copper diaphragms, each 25 /im thick. The inset diagram shows the form of the movement produced by a i-6 ms, 30 V rectangular pulse. Calibration: 50 fim, 1 ms. (6) Table used to support and immobilize ganglia. The prism and column were constructed from polished Perspex and together with the silvered undersurface of the ganglion table itself con-stituted a light guide. Ganglia were pinned out on to the wax contained in the cavity of the table. Scale mark for (a) and (b): 1 cm.
(60 cm sea water) distended the anterior foot sinuses with a beneficial effect com-parable to that of the balloon on the posterior sinuses, and that de-sheathing reduced the effective hydrostatic action on the foot.
It was found that specially stout microelectrodes mounted on the modified loud-speaker shown in Text-fig. 3 (a) would penetrate all but the thickest sheaths without slitting. The crucial features aiding penetration were the raised high-frequency response of the loudspeaker, the accurately axial movement conferred by the pair of copper diaphragms constituting the coil suspension and the stiffness of the microelectrodes themselves. These electrodes were pulled from thick-walled Pyrex capillary tubing with a modified Nastuk electrode puller which blew cooling jets of air on to the elec-trode tip at the time of the strong pull Chowdhury (1969). This resulted in elecelec-trodes
664 R. DE G. WEEVERS
which for the same tip size and shape had a much shorter and stouter shank. Penetration in most cases was best achieved with rectangular current pulses of 1-2 ms duration which produced a withdrawing movement of 30-60 /im amplitude followed by some undamped 'ringing'.
Amplifying, recording and electronic stimulating equipment was of the conventional type. A Tektronix 564 storage oscilloscope was found very useful because of the long time-scale of most behavioural activities. Behaviour and neurone discharge were viewed in 2 min episodes - longer than the longest persistence phosphor otherwise available. Even longer-term recordings were stored on magnetic tape using a four-channel Thermionics T3000 FM tape recorder, and relevant sections of such stored records were selected for filming later. Artists' paint brushes of various sizes were used to deliver tactile stimuli. Extracellular recordings from nerves and connectives were taken with suction electrodes using a multi-way switch to connect recording or stimulating leads to the desired channel. Movements were recorded with one or two light isotonic photoelectric transducers capable of linear registration of movements from 100 /im to 10 cm. These were routinely attached to the anterior rims of either or both parapodia with hooked micropins (see Plate 1). The loading could be adjusted from 10 to 100 mg according to the desired frequency response. The lightest load gave a high-frequency response of 1 s for the full 10 cm amplitude of movement. The tension in the foot muscles was monitored continuously with a Grass strain gauge whose compliance was varied as desired with springs attached in series or in parallel with the transducer.
RESULTS
Intracellular recording of responses to physiological stimulation Pedal ganglion
One disadvantage of working with pedal neurones is that, with the exception of about six large cells situated posteromedially on the dorsal surface of the ganglion, these are too numerous and too similar in size and pigmentation to be individually recognizable. Nevertheless it was thought important to investigate their behaviour as far as possible using only an approximate method of grid location and such physiologi-cal classifying criteria as presented themselves, since the large and identifiable cells hitherto studied in other ganglia may not be typical in all respects and certainly do not perform all possible integrative tasks. Moreover, the majority of the nerves to the foot and parapodia come from the pedal ganglia, and it is highly probable that the neurones of these large ganglia play an important part in co-ordinating locomotion and other behavioural activities. With these considerations in mind, all cells penetrated were routinely tested for receptive field, axon distribution, firing pattern during spon-taneous movements and motor consequences of stimulating the cell directly through the recording electrode.
Intrasomatic stimulation of single neurones
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(«)
S i
Ph o n R. tentacle
Text-fig. 4. Type responses from categories of pedal ganglion cells to various physiological stimuli, (a) Intense excitatory response to tactile stimulation of a small receptive field. (6) More or less prolonged inhibitory response to tactile stimulation almost anywhere on the body surface, (c), (d) Moderate excitation by tactile stimulation almost anywhere on body surface, (c) Without and (d) with post-excitatory inhibition, (e) Excitation by tactile stimulation within one receptive field, inhibition by tactile stimulation within (an) other(s). This was the commonest type. Stimuli marked by second beam or a bar below the trace. Time calibration at the end of each record: 1 s.
|sOmV
1 s Text-fig. 5. A cell of the type shown in Text-fig. 4(0). Starting at the top, the three consecutive records show respectively the response to tactile stimulation of the sole of the foot 1 cm anterior to, level with and 1 cm posterior to the rhinophores. Calibration: 50 mV and 1 8.
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perhaps because the pacemaker region was electrically remote from the soma. Direct stimulation of such cells evoked no motor effects and their behaviour was what wouldi be expected of first-order sensory interneurones. These 'type a' cells resemble the 'type i ' cerebral neurones of Anderson (1967) and were the only ones in the present study to show any suggestion of systematic localization in the ganglion with respect to either sensory or motor field or adequate sensory modality. A large proportion of them had receptive fields in the anterior foot or head regions and formed a loosely defined group near to the root of the anterior pedal nerve (see Text-fig. 2).
A rather small proportion of cells showed only inhibitory responses. Text-fig. 4 (a) is an example of this class which constituted only 10% of the total sampled. Receptive fields were mostly large.
Excitatory responses with or without after-inhibition were commoner - 33 % of the total (see Text-fig. 4c, d). These were quite distinct from type a in always exhibiting a tonic discharge before stimulation, in their sensitivity to depolarizing current which always evoked a tonic discharge, and most of all in the possession of large and often ill-defined receptive fields; excitation was usually only moderate in degree. Adequate stimuli for types c and d were also often non-specific with respect to modality. The neurone illustrated in Text-fig. 4(c) responded moderately or weakly to a variety of tactile, proprioceptive-like, vibrational, water-current and diffuse photic stimuli. It is possible that such neurones are only driven strongly by certain precise stimulus con-figurations which were not hit upon in this survey.
Numerically the largest class (41 % of penetrations) were of the type shown in Text-fig. 4(e) - mutually antagonistic receptive fields in different parts of the body. The antagonistic fields tended to be anterior and posterior rather than in bilaterally symmetrical positions. Only 4 % of cells failed to respond to any inputs tested.
The size of receptive field in all the above classes varied continuously in extent, but those of type a cells were always smaller than the smallest field cells of other classes, and type a cells never had demonstrable peripheral axons. The criteria separating the other types are less objective and the classification there may only be one of descriptive convenience.
Pleural ganglion
cated qualitative discrimination of different types of object touching the visceral bump - the receptive field of this neurone. The occurrence of different sizes of completely invading 'A spike' suggests the existence of spatially separated and in-dependently pace-making integrative loci, possibly on different branched axons as suggested by Tauc & Hughes (1963). This cell had two ipsilateral peripheral axons (Text-fig. 6 b). Several pedal type a cells (Text-figs. 4, 5) converging on this pleura! cell would be a simple arrangement capable of mediating such spatial summation.
Text-fig. 6. The responses of a pleural cell to tactile stimulation of successively larger areas of the visceral hump. The areas stimulated were (a) i mm2, (6) 25 mm8, (c) 6 cm8. The transducer recorded movements of the ipsilateral parapodium (up = levation). Spikes retouched. Vertical calibrations for (a)—(c): 50 mV, 1 cm. Time calibration: 1 s. (d) Orthodromic spikes in the two peripheral axon branches — in the ipsilateral posterior and anterior parapodial nerves. Sweep triggered from the soma spike, axon spikes are superimposed during zo sweeps without change in latency; vertical calibration: 50 mV upper, 100 fiV lower trace. The third frame shows an antidromic soma spike, evoked by electrical stimulation of the posterior parapodial nerve and progressively blocked by hyperpolarizing the soma. Vertical calibration, iomV; time cali-bration for all three frames, 50 ms.
Pedal ganglion ' motor cells'
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R. DE G. WEEVERSfurther peripheral modification must be slight since the evoked movement bears a close and consistent relation to the frequency at which the cell discharges (see graph Text-fig. 7).
Text-fig. 8 illustrates the properties of a parapodial levator motor cell. Again the movement evoked was smooth and its minimum latency of 400 ms is very long. Assuming that no drop in conduction velocity occurs peripherally due to tapering of the axon, conduction time only accounts for 100 ms. But unlike the gill preparation of Kupferman & Kandel (1969), the transducer in the present experiments could not be attached to the contracting structure and the interposition of extremely compliant visco-elastic tissues in series could well account for the rest of the delay and would also smooth and 'blunt' individual twitches if present.
50 mV I
1s
1 cm
3P
SOmVI
.§.2
10 20
Frequency (s"')
30
Text-fig. 7. Spike activity of a pedal' motor cell' during (a) spontaneous movements of the ipsi-lateral parapodium and (6) during movements evoked by lightly stroking the siphon; the cell was slightly depolarized in (6) to show the powerful inhibition accompanying levation movements (parapodial closure), signalled by a downward movement of the middle trace, (c) Movements evoked by depolarizing the cell, causing it to fire bursts of spikes. The ventral surface of the parapodium contracted in the direction of the arrow on the inset diagram. The graph shows the relation between discharge frequency and the maximum extent of contraction. (The parapodia were held slightly raised above the horizontal, so depression of the parapodia in (a) and (b) produced an outward swinging movement of the margin. The contraction in (c) was too local for proper action of the hydrostatic skeleton and the margin was pulled in towards the midline.
(d) The axon of this cell was in the anterior parapodial nerve. The three frames show, in order:
Behavioural activities and patterns of neuronal activity Creeping
The consistent correlation between spontaneous discharge pattern of such motor cells and behavioural activities occurring at the time is particularly clear in Text-fig. 9. All four of the cells illustrated fired a burst of impulses at a particular phase of each step, a slightly different phase being characteristic of each cell. Their motor fields were all in the sole of the foot and depolarizing current evoked local longitudinal contraction, detectable when observed visually from beneath. Cells a and b evoked contraction about level with the siphon, a slightly more posterior than b. Cell c had the most anterior motor field, slightly anterior to the haemolymph-retaining clamp. The field of cell d
Text-fig. 8. Apedal motor cell which fired during the relaxation phase of 'stepping', (a) Shows the unstimulated discharge of the cell on the top trace (cal. 10 mV); the second trace recorded tension changes in the foot (cal. 20 g, tension increasing downwards); the third and bottom traces recorded vertical movements of the contralateral and ipsilateral parapodia respectively (cal. 1 cm), levation being signalled by an upward deflexion of the trace. The same convention with respect to the direction of increasing tension and levation movements is followed in subsequent figures. Time calibration: 5 s. (6) Forced discharge of the same cell and the contraction evoked proximally in the upper surface of a strip in the middle of the ipsilateral parapodium (cal. 10 mV, 1 mm, 500 ms). (e) Oscilloscope triggered from spikes evoked by depolarizing the cell, as in (6). The lower trace shows a time-locked orthodromic spike in the axon of the cell recorded extracellularly from the ipsilateral anterior parapodial nerve. Calibration: upper beam, 10 mV; lower beam, 100 /tV; time calibration, 5 ms.
670 R. DE G. WEEVERS
but this could be a non-specific effect of deformation where the posterior hooks were inserted into the muscles of the tail. Text-fig. g(d) shows an apparent decrease in regu, larity of stepping when this one motor cell was hyperpolarized to prevent firing, but this effect was not consistently observed and would in any case hardly be expected as a direct effect since at a conservative estimate there must be at least 150 such longi-tudinal motor units in the whole foot (see Discussion). If there were recurrent axon branches from the motor cells, these might influence pre-motor interneurones with
Text-fig. 9. Four different motor cells in the pedal ganglion firing at various phases of 'step-ping'. In (a), (6) and (c) calibration for the upper beam is 40 mV, and for the lower beam 20 g tension, increasing downwards, recorded by an auxotonic transducer on the tail. Time calibra-tions for all four frames are 20 s. In (6) the tension on the foot was increased experimentally by 15 g after the first step. In {d) the two added bottom traces show movements of the parapodia (up = levation); trace 3 contralateral, trace 4 ipsilateral, cal. 1 cm. After four steps the cell was hyperpolarized so that spiking was blocked and only e.p.s.p. remained.
sufficiently large fields to produce such an effect; Dorsett (1968) did show the existence of numbers of neurones in the pedal ganglia of Aplysia punctata with ipsilateral peri-pheral axons (which could therefore have been motor cells) and having crossed axon branches ending in the contralateral pedal ganglion, so such an effect is at least possible.
Swimming: pedal neurones and the left pleural giant cell (LGC)
pre--parations episodes of flapping could be evoked for a period of 6 h following the start if recording. The present observations are thus from a small number of preparations, though caution is clearly necessary at this stage, they are included because among other things they do suggest one possible behavioural role for the left pleural giant cell (LGC). The almost uniquely extensive peripheral branching of the axons of this cell (at least nine major branches - Hughes, 1967) render its behavioural role of particular interest.
The swimming obtained from dissected preparations is shown in the flash photo-graph of Plate 1, fig. 2. The parapodia are turgid and fully extended, the gill is fully exposed and the foot long and narrow with the creeping surface folded in on itself.
Text-fig. 10. A motor cell in the pedal ganglion which discharged irregularly during stepping and tonically at a high frequency during swimming. Calibration: top trace, 25 mV; second trace, 1 cm right parapodium, bottom trace, 1 cm left parapodium; time calibration 20 s. The spike height recovered after the long burst during swimming and declined similarly in amplitude during subsequent bursts.
Text-fig. 10 shows the tonic activity of one of the motor cells with a transversely oriented motor field which fold the foot in this way during swimming. By contrast, during creeping the same animal was shorter and had a turgid foot with a wide creep-ing surface, the gill was covered and the parapodia were closed as far over the turgid visceral hump as the restraining hooks would allow (Plate 1, fig. 1). The only apparent differences in creeping and swimming between intact animals and dissected animals were that in the latter the periodicity of both kinds of locomotor movements was about two-thirds what would have been expected from an animal of this size, and that the tentacles were not extended forwards in front of the mouth in the characteristic posture during swimming, almost certainly because of the absence of a hydrostatic skeleton anterior to the haemolymph-retaining clamp.
the LGC during (a) creeping and (b) swimming. During creeping both neurones dis-charged irregularly, but during swimming the pedal cell fired a burst corresponding to each beat, whereas the LGC discharged erratically with high-frequency bursts ai intervals between episodes of regular swimming. Both cells were slightly hyper-polarized in (a) to show the changes in discharge frequency more clearly. It is of interest that, even when slightly hyperpolarized, the LGC in these preparations showed high-frequency synaptically evoked spike activity; in isolated nervous systems or under less good experimental conditions, it is a silent cell responding only phasically to de-polarization. The beats of the parapodia were usually rather irregular for a while at
Text-fig. 11. (a) and (6) are two consecutive frames of recordings from (1) a large cell towards the posterior median corner of the pedal ganglion, and (2) the left pleural giant cell (LGC). The third trace shows movements of the right parapodium and the bottom trace of the left parapodium. Calibration: 50 mV, 1 cm, zo s for both (a) and (6). The first third of (a) shows stepping which is followed by incipient swimming movements. Towards the end of (6) the beats became fairly regular. Slightly hyperpolarized in (a), current off at the start of (6) - the baseline drift is an artifact resulting from this.
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could be evoked by allowing the animal to 'rest' with the foot in contact with the Polythene belt and then releasing this by passing a flat strip of Perspex between roller md foot so that the platform dropped. Sometimes the animal spontaneously released the platform, which fell under its own weight as swimming started. It was found that if the previously hyperpolarized LGC was depolarized, causing it to fire steadily at a fairly high frequency, such episodes of swimming were terminated within 10-20 s (Text -fig. 12). In one preparation this was repeated six times within a period of 2 h. Each time, after a variable latency, the sole of the foot became wide and flat and shortened markedly, and very frequently swinging movements of the foot would follow (see the slow levation of the right and depression of the left parapodium with accompanying longitudinal contraction of the foot shown in Text-fig. 12).
Text-fig. 12. An episode of swimming apparently terminated by depolarizing the LGC, causing it to discharge at an average frequency of 5 imp./s for 80 s (individual spikes are fused at this sweep speed; the period of depolarization is marked by a line above the top trace). Top trace: LGC, capacity-coupled, cal. 10mV. Second trace: tail tension, increasing downwards, cal. 20g. Third trace right, bottom trace left parapodium movement, cal. 1 cm. Time calibration: 20 s.
674 R- DE G. WEEVERS
DISCUSSION
The respective roles of motor cells and the peripheral plexus
Observations from the early literature on the role of the different gastropod ganglia (reviewed in Bullock & Horridge, 1965) make it clear that normal muscular function is impossible without the pedal ganglia, the pleural being less essential. In both Aplysia and Helix denervation of the pedal ganglia leads to progressive shortening of the sole of the foot, ending in death. Together with the observation that the shortening is delayed by reducing the amount of peripheral stimulation, this suggests an excitatory action of any peripheral motor cells which would normally be subject to inhibition by the pedal ganglia. Were it not for the rather enigmatic peripheral plexus, the present observations of single apparently motor cells in the pedal ganglion might be taken to indicate a direct route of excitation to muscle fibres of the kind described by Ramsay (1940) for Helix buccal retractor muscles. Willows (1968) described the same kind of directly evoked movements in Tritonia (unidentified cells near to cells 3, 4, 33, 34). He suggested that perhaps the peripheral plexus took care of relatively local and reflex responses. In the present work 15 % of sampled cells behaved like motor neurones. A conservative estimate of the number of cells in the pedal ganglion would be 2000-3000 on each side. Assuming no sampling errors this would therefore include 600-900 motor cells. A fully expanded 400 g Aplysia has a surface area of approximately 600 cm2, which would allow considerable overlap between motor fields 2 cm2 in area (about the average observed here) arranged in two overlapping sets oriented at right angles to each other. Such a complement would probably suffice for swimming movements, but would constitute a rather crude system, probably insufficient to co-ordinate the finer movements during creeping. Either the peripheral plexus must have a degree of autonomy in co-ordinating waves in the foot, or some of the cells found in the pedal ganglion having peripheral axons which yet evoke no apparent muscular contraction must be small field motor cells. The progressive shortening of the foot which results from denervation imposes a further degree of complexity on this picture and may imply peripheral inhibitory or 'relaxing' innervation, either of muscle or of other motor cells in the plexus.
The role of the pleural ganglia
Ten Cate (1928) believed the pleural ganglia of Aplysia to be without significance in the defensive movements of a parapodium which he evoked reflexly by contralateral stimulation. The present observation that discharging the LGC discourages swimming and promotes creeping suggests that this ganglion may be involved in activities of a more complex kind than these protective reflexes. There are also certain preliminary indications that other large pleural neurones may act as command cells.
as well as their follower populations, have axons that pass to the periphery in the nerve fcrunks appropriate to the various functions. It is hard to conceive that the information content of a series of bursts in one or a few command cells could be sufficient to co-ordinate directly a complex activity such as swimming. In any case, the pedal motor cells seem to be active at the same time, so any built-in co-ordinating circuitry of the pedal ganglion is likely to be activated in a manner subordinate to driving by appro-priately connected command cells. Perhaps the peripheral axons of the command cells have an effect on the peripheral plexus. It may be no coincidence that both are features peculiar to gastropods.
SUMMARY
1. Methods are described for suspending and clamping Aplysia fasciata so as to permit intrasomatic recording from neurones of the head ganglia during locomotor and other behavioural activities.
2. Sensory responses of neurones in the pedal ganglion are classified into four main types, all being distinct from those of pleural ganglion cells.
3. The pedal ganglion may well contain 'motor cells' for the greater part of the somatic musculature.
4. Preliminary results suggest that the pleural LGC may be involved in promoting a change from swimming to creeping behaviour.
Valuable technical advice and many useful discussions with Professor G. M. Hughes are gratefully acknowledged. I am indebted to the University of Bordeaux for excellent laboratory facilities and accommodation at the Arcachon marine station, to the S.R.C. for financial support through a grant to Professor G. M. Hughes and to Bristol University for travelling expenses.
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EXPLANATION OF PLATES
Fig. 1. Dorsal view of the body and parapodia of Aplysia during creeping. The inset frame shows the activity of the LGC at the time. This was depolarized to fire steadily at 3-5 imp./s. The recording system was capacity-coupled. The second trace shows the fluctuations in tail tension. The double-ended arrows point to three consecutive steps; cal. 20 g. The bottom trace shows movements of the right parapodium; cal. 1 cm. See photograph for the point of attachment of the transducer. The flash artifact two-thirds of the way along the bottom trace shows the instant of the photograph (thick arrow). Time calibration:
10 s.
