the relationshi of thp centrae l motor pattern to the feeding cycl oef lymnaea stagnalis · j. exp....

J. exp. Biol. (1979), 80, 137-163 137With 15 figures

Printed in Great Britain

THE RELATIONSHIP OF THE CENTRAL MOTOR PATTERNTO THE FEEDING CYCLE OF LYMNAEA STAGNALIS

BY R. M. ROSE AND P. R. BENJAMIN

School of Biological Sciences, The University of Sussex,Faimer, Brighton, Sussex BNi 9QG

(Received 31 July 1978)

SUMMARY

Electromyographic recordings from the buccal muscles of Lymnaea duringfeeding has shown that there are 4 component phases in the feeding cycle.Cinephotography of feeding cycles has confirmed that these correspond toprotraction, 2 phases of retraction, and an inactive phase. The 4 phases ofmuscle activity can also be related to the cycle of neural activity describedpreviously (Benjamin & Rose, 1979). Thus types 6, 4 group, and type 8 cellsare motoneurones involved in protraction and the two retraction phases,while the type 5 cell fires in the inactive period. The combination of physio-logical and anatomical approaches has led to the suggestion that the singleand double input cells described by Benjamin & Rose (1979) are involvedwith the control of buccal and oesophageal activity respectively.

INTRODUCTION

In the two preceding papers (Benjamin & Rose, 1979; Benjamin, Rose, Slade &Lacy, 1979) the electrical activity and distribution of axons of identified neurones inthe Lymnaea buccal ganglia were described. The object of this paper is to prove whichof the identified cells are motoneurones, and to show how their activity relates tomovements of the buccal mass.

From the point of view of the morphology, the Lymnaea buccal mass is an excellentsystem to study, since accounts of the musculature and feeding movements havealready been given by Carriker (1946), Hubendick (1957), and more recently byGoldschmeding & de Vlieger (1975). From the electrophysiological point of view thesystem also has the advantage that the muscles produce action potentials during con-traction. This means that muscle activity can be recorded extracellularly in feedingpreparations. In this paper much emphasis will be placed on neurone and musclerecording from the actively feeding preparation, 9ince many of the cell types can onlybe identified on the basis of their synaptic inputs during feeding cycles. In othermolluscs, such as Aplysia, the buccal muscles do not exhibit action potentials (Cohen,Weiss & Kupfermann, 1978), making a similar analysis more difficult. The Aplysiamuscle fibres are of larger diameter, however, so that intracellular recording is possible.At this stage we have not been able to record intracellularly from the small musclefibres of Lymnaea, but have tried to determine the functional role of identified cells inrelation to extracellular recording of muscular contraction. These recordings show

138 R. M. ROSE AND P. R. BENJAMIN

that in each feeding cycle there are three phases of muscle activity followed by aninactive phase. Motoneurones have been identified for each of the active phases. Inaddition a functional division occurs in that single input cells are involved in thecontrol of the buccal mass itself, while double input cells may control gut movementand secretion.

METHODS

All of the recordings in this paper were made on the semi-intact preparation. Thisconsisted of the buccal mass, oesophagus, and attached brain and buccal ganglia.Intracellular recordings from neurones were made as described in the earlier paper(Benjamin & Rose, 1979). Extracellular recordings were made from the buccalmuscles using glass suction electrodes in the conventional way. All recordings weremade from pairs of muscles or from neurones and muscles on the same side. In orderto record from neurones in the buccal ganglia during feeding movements, the buccalganglia were supported on a small metal table covered with a thin layer of Sylgard.The table was placed underneath the buccal ganglia, care being taken not to damageany of the bucca] nerves. This technique usually had the effect of stretching the buccalnerves slightly as the buccal ganglia were pulled away from the buccal mass. Theganglia were held on the table using only one or two micropins, a minimum amount ofstretch being applied to the ganglia themselves, since this may uncouple the inter-neurones which drive the feeding cycle.

In some cases the buccal mass was partially dissected. In order to explore the inner-vation of the large anterior jugalis muscle the buccal mass was cut in a dorsal longi-tudinal direction and the two halves of the anterior jugalis were spread out (seeBenjamin et al. 1979; fig. 2). To look at the innervation of the buccal sphincter, alateral longitudinal cut was made in the anterior jugalis near the point where thelateral buccal nerve enters this muscle (Fig. 1), and the anterior jugalis pulled asidewith fine forceps to expose the underlying muscles. Similarly the problem of theseparation of posterior jugalis and tensor innervation was investigated by making adorsal longitudinal cut across the top of the posterior jugalis, and folding this muscleback to expose the radula tensor muscles. In some cases the posterior jugalis wasisolated with its thin posterior jugalis nerve and attached buccal ganglia.

Cinephotography was carried out on the semi-intact preparation using a Bolex16 mm reflex camera at 25 frames/s. The feeding movements were investigated bothwith and without simultaneous extracellular recordings from the buccal muscles.Feeding movements were sometimes induced by dropping crystals of sucrose close tothe buccal mass. When simultaneous cinefilm and extracellular recordings were made,the two recordings were synchronized by noting the frame number as the camera wasswitched on and off. Operation of the camera switch was arranged to cause a steadyd.c. deflexion of the extracellular trace, and the times of 'on' and 'off' were measuredfrom the time base on the extracellular recording. This time interval was then checkedwith the time interval on the film, knowing the number of frames between ' on' and'off' and the frame speed. If these two measurements agreed satisfactorily, the recordwas subdivided and the frames corresponding to specific points in the feeding cyclewere projected and drawn. This method is accurate in the present case because thefeeding cycle itself is relatively slow, lasting 3-10 s, and the method is accurate towithin 2-3 frames (or o-i s).

Motor control of feeding 139

br

mlh

Fig. 1. Side view of the buccal mass of L. ttagnalit (based on Goldschmeding & de Vlieger,1975)> showing muscles from which extracellular recordings were made, bg, Buccal ganglion;igl, salivary glands; oet, oesophagus; mth, mouth; aju, upper part of anterior jugaJis; aji, lowerpart of anterior jugalis; dip, dorsolateral protractor; dlr, dorsolateral retractor; pel, preventrallevator; pvl, postventral levator; pvp, preventral protractor; pop, postventral protractor; pj/t,posterior jugalis/tensor. Triangle indicates position of fulcrum referred to in text.

RESULTS

Structure of the buccal mass

A comprehensive description of the structure of the buccal mass in Lymnaea stagnalishas been given by Carriker (1946). Using Carriker's account as a basis, Goldschmeding& de Vlieger (1975) regrouped the muscles into four concentric muscle systems. Froma physiological point of view our main problem has been to distinguish betweenprotractor and retractor muscles, and we have approached this problem by definingthe activity of each muscle rather than looking at the functional anatomy of thesystem. All we need to know initially is how the muscles are arranged externally, andhow the underlying muscles lie in relation to these. Fig. 1 should be consulted for thegeneral layout of the muscles of the buccal mass, and Fig. 6 shows the approximateposition of the odontophore during different phases of the feeding cycle.

The muscles of the buccal mass are organized around the radula and its supportingcartilage, the odontophore. The odontophore consists of two symmetrical spoon-shaped structures, united ventrally, and the radula is stretched over its surface, lyingin the U-shaped groove between the two cartilages and running posteriorly into thebulbous radula sac (Fig. 6). To understand the muscular arrangement it is best toimagine the odontophore as a large oval-shaped structure seen in side view with itsventral base acting as a fulcrum about which it is rotated during the feeding cycle.Many of the buccal muscles are inserted at this fulcrum point. The unpaired posteriorand anterior jugalis muscles are inserted at this point on each side and pass over thetop of the buccal mass, the anterior jugalis enclosing the anterior two-thirds and theposterior jugalis the posterior one-third of the buccal mass. It is important to note thatwhile the anterior jugalis is a large thick muscle, the posterior jugalis is only a thin


muscle sheet, which besides covering the posterior end of the buccal mass also projectsanteriorly beneath the anterior jugalis, suggesting that it is a protractor muscle(Carriker, 1946). Also inserted at the fulcrum are the three paired tensor muscleswhich wrap around the posterior edges of the odontophore to be inserted on thesubradular membrane. Contraction of these muscles stretches the radula over theodontophore during rasping. Of these tensor muscles the largest is the supralateralradula tensor which forms the two large posterior-lateral bulges behind each fulcrum.It is these tensor muscles which are covered by the sheetlike posterior jugalis.

If the anterior jugalis is dissected away, the buccal sphincter can be seen under-neath. This muscle encircles the buccal cavity, as does the mandibular approximatorwhich lies just in front of it. There are two important muscles which lie under thebuccal sphincter - the dorsal and ventral odontophoral flexors, which insert on theupper and lower (fulcrum point) parts of the cartilage (Goldschmeding & de Vlieger,1975). We have been unable to record from these muscles, because they form widethin bands which are difficult to dissect from the thick overlying anterior jugalis, butit is interesting to note that Carriker (1946) assigns to the flexor muscles the functionof protraction, and according to his description protraction is brought about by thecombined contraction of the posterior jugalis and flexor muscles.

A small group of strand-like muscles are inserted ventrally below the fulcrum, andproject anteriorly to insert under the mouth. These include the pre- and postventralprotractors and the postventral levators (Fig. 1). Although the postventral levatorsserve to raise the posterior end of the buccal mass on the forward stroke of the odonto-phore, it seems that the pre- and postvential protractors are actually retractor musclesin that they pull the ventrally placed fulcrum point forwards during retraction, thushelping in the backward rotation of the odontophore. The buccal retractor musclesthemselves are also inserted laterally at each fulcrum point (Fig. i), and are extrinsicmuscles retracting the buccal mass as a whole.

It can be seen therefore that all the larger muscles concerned with the movementsof the odontophore are inserted at or near the fulcrum point. Of the remaining musclesthe dorso-lateral protractor is the largest of four small muscle pairs involved in turningthe buccal mass in the anterior direction, this muscle arising near the oesophagus andrunning across the sides of the buccal mass to insert in the mouth region (Fig. 1). Theposterior end of the radula continues into the bulbous radula sac which is supporteddorsally by a group of four small suspensory muscles (Goldschmeding & de Vlieger,1975; Carriker, 1946). Not included in this description are four muscles which openand close the mouth, and appear to be supplied by the labial nerves (Carriker, 1946),and also such minor muscles as the intra-cartilage tensors and the tensor of thecollostylar hood.

From a physiological point of view it is convenient that the most important musclesinvolved in the feeding cycles can be recorded externally without the need for dis-section. However, the buccal sphincter and flexor muscles can only be recorded bydissecting away the anterior jugalis. Although we have attempted to record from thesemuscles we are not satisfied that we have isolated them from muscle strands of theanterior jugalis, and will not therefore include any information on these muscles in thephysiological account. We have also grouped together muscles of similar functionssuch as the 'tensor' (3 tensor muscles), 'suspensors' (4 suspensory muscles) although


a more complete account would have to differentiate between different muscles inthese groups.

Electromyographic recording

In simple terms the feeding cycle consists of protraction and retraction of the radula.In reality it will be shown below that there are two phases of retraction and that thereis usually a period of inactivity between the end of retraction of one cycle and the startof protraction of the next. In Lymnaea the feeding cycle therefore consists of fourseparate phases; protraction (P), retraction (phases Ri and R2), and inactivity (I).

Electromyographic recordings were made with glass suction electrodes placed on theappropriate muscles. Recordings were always made from pairs of muscles on the sameside of the buccal mass. The tip diameter of the electrodes was up to 100 fim, so thatthe activity of large numbers of muscle fibres were being recorded. In relation to thesurface area of the more important muscles, this tip diameter is still relatively small.There are certainly variations in the activity of different parts of some muscles (e.g.anterior jugalis, see below) and our electrode size was a compromise between notlosing detail of the overall muscle activity and not having too much overlappingactivity from a number of motor units. It is nevertheless true to say that we are notabsolutely clear at this stage why some muscles have unitary potentials over a largearea (e.g. upper anterior jugalis), whereas others produce potentials of a compoundshape (e.g. tensors). It appears that the unitary potentials may be produced by singleneurones branching over a large area (a single motor unit), and the compound poten-tials by simultaneous activity in many muscle fibres innervated by different neurones(several motor units). An alternative explanation might be that the unitary potentialsresult from highly synchronized activity in several neurones supplying the same area.

The anterior Jugalis, posterior Jugalis and tensor muscles

The overall configuration of the feeding cycle can be distinguished by recordingfrom the anterior and posterior jugalis muscles simultaneously (Fig. 2). A point ofconfusion immediately arises when we refer to recordings from the posterior jugalismuscle. This is because the posterior jugalis is a thin muscle sheet covering the largetensor muscles, and the suction electrode unavoidably sucks up both the posteriorjugalis and some of the underlying tensor muscles. It is difficult to prove which part ofthe recorded activity is coming from which muscle, although we are now fairly certainof the distinction (see later). For the moment, we will refer to a recording made abovethe posterior jugalis as a 'posterior jugalis/tensor' recording.

The cycle begins with a burst in the posterior jugalis/tensor which we later show tobe the first (or protraction, P) phase of the feeding cycle. Muscle potentials occur athigh frequency during this burst and there may be several overlapping units. Retrac-tion in the feeding cycle consists of two phases, beginning with a burst in the lower partof the anterior jugalis (phase Ri; Fig. 2 b), followed by a burst of similar duration in theupper part of the anterior jugalis (phase R2; Fig. 2 a). Muscle potentials of the lowerpart of the anterior jugalis have a compound shape and appear to be made up of thesummed activity of a number of motor units. By contrast the burst in the upper part of


Fig. 2. Simultaneous extracellular recordings from the anterior jugalis and posterior jugalis/tensor (a and 6), and from different parts of the anterior jugalis (c). In this and all subsequentrecordings, simultaneous activity has been recorded from muscles (Figs. 2-4) or neurones andmuscles (Figs. 9—13) on the same side. Abbreviations: the component bursts of the posteriorjugalis/tensor are distinguished as pj and t; P, protraction phase; Ri, first phase of retraction;R2, second phase of retraction; /, inactive phase, (a) and (6) show that the burst in the lowerpart of the anterior jugalis follows the posterior jugalis burst in phase Ri (6), and that theupper part of the anterior jugalis fires even later in phase R2 («). (c) shows a pair of recordingsmade in a latero-medial position on the anterior jugalis, in which there is a mixture of activityderived from motor units supplying the upper and lower parts of the muscle.

the anterior jugalis has large unitary potentials occurring at very high frequency at theonset of the burst, suggesting the recording of a single motor unit.

In Fig. 2(c) two suction electrodes were positioned close together near the middle ofthe anterior jugalis, and it can be seen that at this recording site some of the musclepotentials of the lower part are transmitted to the other electrode which is primarilyrecording the anterior jugalis upper burst. As the electrode is moved more anteriorlyonly the second high frequency burst is recorded. This suggests that near the middleof the anterior jugalis there is some overlap of motor units supplying the upper andlower parts of the muscle. It seems unlikely that the upper burst could be coming fromthe underlying sphincter muscle because of the thickness of the anterior jugalis.

A burst of variable intensity occurs in the posterior jugalis/tensor simultaneous with;the lower anterior jugalis burst (Fig. 2b). This second phase of posterior jugalis/tensor

Motor control of feeding

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Fig. 3. Activity in other muscles, each being recorded with one of the muscles shown in Fig. 1.The muscles fire in the following phases of the feeding cycle: (a) dorsolateral protractor duringthe protraction phase (P); (b) buccal retractor during R2 phase; (c) postventral protractorduring Ri phase; (d) suspensor muscles during the protraction phase. Recording («) confirmsthe timing of the postventral protractor by its relationship to the burst in the upper anteriorjugalis. Each recording was made from a different preparation, except (c) and (e).

firing is usually much larger in amplitude than the first burst (Fig. 2b), although therelative magnitudes of the two bursts depend on the position of the electrode, thesecond burst tending to be larger the nearer the electrode is to the supramedianradular tensor. Evidence which will be given later suggests that the first burst is due tothe contraction of the posterior jugalis muscle, and the second large amplitude burstto the contraction of the large tensor muscles. The variation in amplitude of these twocomponent bursts with the position of the recording electrode is illustrated in Fig.4(c), where the first burst alone is clearly recorded by the upper channel electrode.This first burst is partially recorded by the other electrode which was placed over thesupramedian radular tensor, but in this position the predominant feature is the largeamplitude second burst. The explanation therefore appears to be that the tensor burstfollows the posterior jugalis burst, and since it terminates before the onset of the burstin the upper part of the anterior jugalis (Fig. \b), we may conclude that it is syn-chronous with the first retraction burst (Ri) in the lower half of the anterior jugalis.

Each protraction-retraction cycle is separated by a period of virtual inactivity in thejugalis muscles (the I phase). It will be shown later that several identified neurones


I cycle10s

Fig. 4. Variations in the duration of the feeding cycle, (a) slow cycle, (6) fast cycle, (c) decayingcycles of activity elicited in the semi-intact preparation by application of sucrose (arrow). Thefeeding cycle is defined as the time from the onset of protraction of one cycle to the beginningof protraction in the next, and can only be defined in this way for (6) and (c), since a protractionburst is absent in (a). The recording in (6) is a particularly clear example which shows repre-sentative bursts in three phases of the cycle (P, Ri and R2), the fourth inactive phase (/) beingof very short duration. In (c) the electrodes were placed in two different positions on theposterior jugalis/tensor and show pure posterior jugalis activity on the upper channel, andpredominantly tensor activity on the lower channel.

discharge during this quiescent period, and that some of these project to the oeso-phagus. It seems likely that this period is associated with peristaltic activity in theoesophagus following the feeding cycle of the buccal mass.

Activity in other muscles

Further detail of the feeding cycle is provided by determining the relationship ofactivity in other muscles to the protraction-retraction cycle of the jugalis muscles. Thepostventral protractor muscle discharges at the same time as the tensor burst (Fig. 3 c),and is therefore a retractor muscle, as Kater has also pointed out in Helisoma (Kater,1974). Futher confirmation of the timing of this burst is given by recording it with theupper anterior jugalis burst (Fig. 3 c), which it precedes. The dorso-lateral protractorand the suspensor muscles of the radula sac fire bursts which are in phase with theprotraction burst of the posterior jugalis (Fig. 3 a, d), the suspensor muscles dis-charging at similar frequency to the posterior jugalis burst, while the dorso-lateralprotractor burst is composed of low frequency and large amplitude potentials. Finally,one or two large amplitude potentials occur in the buccal retractor muscle during thesecond phase of retraction (R2), as is shown by recording this muscle with the anteriorjugalis upper burst (Fig. 3 b).


Variation of cycle period

The cycle period is defined as the time interval between the onset of one phase ofprotraction (posterior jugalis burst), and the next. There are considerable variationsin the cycle period, some extreme examples being shown in Fig. 4. Typically the cycleis 10 s total duration, but in extreme cases the cycle period may be as short as 5 s(Fig. 46). The most variable component is the duration of the inactive period (I),which is of the order of 1 s in Fig. 4(6), compared with as long as 20 s in other cases(Fig. 2 a). The next most variable component is the protraction phase, although this isnot particularly evident in the extracellular recordings shown here. When we come tolook at the protraction-retraction cycle in more detail it will be seen that the protrac-tion phase is sometimes very short (Fig. 11 c), and at other times long (Fig. 12 a). Suchvariations are not seen in the set of paired extracellular recordings (Figs. 2-4) becausethe short-duration protraction phases are associated with poorly patterning preparationswhich have been rejected here. The retraction phases (Ri and R2) are relativelyconstant.

Frequently the cyclical bursting behaviour occurs for periods of a minute or so,punctuated by periods of inactivity or rather random firing typical of the non-feedingpreparation. It is possible to initiate feeding cycles by dropping crystals of sucroseclose to the buccal mass (Goldschmeding & Jaeger, 1973), and an example of activityinitiated in this way is shown in Fig. 4(c). After application of sucrose (arrow) thereis intense burst activity in the posterior jugalis and tensor muscles, which slowlydeclines over 8 or 9 cycles. Such initiated sequences should be compared with theexponentially decaying sequences initiated by food extract in Aplysia depilans (Rose,1976) and Archidoris (Rose, 1971).

Summary of muscle activity

The sequence of muscle potentials is summarized in Fig. 5 for muscles which areaccessible to recording without further dissection of the buccal mass. It was founddifficult for instance to separate the odontophoral flexor and buccal sphincter musclesfrom the overlying anterior jugalis muscle and still retain an actively feeding prepara-tion.

During the first (or protraction, P) phase of the cycle the most important featureis the burst in the posterior jugalis. The suspensor muscles of the radula sac, and thedorsolateral protractors are also active during this phase. It is also probable that thedorsal odontophoral flexor (not recorded) acts in conjunction with the posteriorjugalis in everting the radula to the mouth (Carriker, 1946).

Retraction is divided into two phases; Ri and R2. During Ri the tensor muscles,anterior jugalis lower, and postventral protractor muscles are active. During R2 theupper anterior jugalis and buccal retractor fire. Between the end of R2 and the begin-ning of the next cycle is the inactive phase (I) in which no buccal muscles participate.Variations in the cycle period are associated with differences in the durations of theinactive and protraction periods. These variations will be shown later to be associatedwith variations in the durations of the underlying postsynaptic potentials of themotoneurones.


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5sFig. 5. Summary of timing of activity in different muscles based on recordings shown in Figs.2-4. The burst activity has been aligned with phases of the feeding cycle using evidence givenin Fig. 8.

Cinephotography of the feeding cycle

The feeding cycle in most molluscs involves two main types of movement, namelyrotation of the buccal mass relative to the body (using extrinsic muscles), and move-ment of the radula relative to the buccal mass (using intrinsic muscles). In laboratory-reared animals, whose body wall is semi-transparent, the rotation of the buccal masswithin the body cavity can be clearly seen. However, we have only filmed movements


r.sac

mth

10

Fig. 6. Sequence of events in a spontaneous feeding cycle at 400 msec intervals. Inset showsdiagrammatically how the radula (rad) lies over the odontophore (od) and is continued poster-iorly into the radula sac (r.sac). The approximate shape and orientation (bar line) of the odonto-phore has been indicated in each frame to aid understanding of the shape changes of the buccalmass. Protraction involves contraction of the posterior jugalis (pj: shaded area of frames 1—4),and forward rotation of the odontophore towards the mouth (mth). Retraction begins at frame5, and the radula sac gradually emerges as the odontophore rotates backwards (frames 6-9).The odontophore is rotated past the vertical position between frames 8 and Q, and the con-traction of the anterior jugalis seen as a narrowing of the anterior end of the buccal mass inframe 9, forces the radula towards the oesophagus. Finally the radula returns to the restingposition (frame 10).


of the isolated buccal mass, in which the extrinsic muscles which suspend the buccalmass and the muscles round the mouth have been cut. This description is thereforeprimarily concerned with radula movements within the buccal mass. A further con-straint is that because the muscles round the mouth have been cut, the radula does notemerge properly from the mouth, and details of the rasping movements cannot bedistinguished. Nevertheless, the filmed sequences of movements are useful becausethey define the relationship between muscle activity and the different phases of pro-traction and retraction.

A complete feeding cycle is shown in Fig. 6. In the resting state the postero-ventraledges of the two halves of the odontophore bulge out ventrally, and the posteriorjugalis can be clearly distinguished (shaded region). The approximate orientation of theodontophore within the buccal mass is indicated by a bar line, and the oval-shapedodontophore cartilage has been drawn schematically on each diagram to help explainthe external shape changes. During the slow protraction phase (frames 1-4) the buccalmass changes to an ovoid shape as the radula is pushed towards the mouth, and thearea occupied by the posterior jugalis is progressively diminished. In frames 3-4 theleading edge of the radula can be seen pressing against the dorsal wall of the buccalmass anteriorly at the end of the protraction phase/start of the rasp movement. Itappears that the two halves of the odontophore become fairly closely approximatedat this stage, explaining the ovoid shape of the buccal mass. It will be shown later onthat the diminishing area of the posterior jugalis is associated with posterior jugalismuscle activity. It is also worth noting that during this protraction phase there isevidence of a forward thrusting of the whole buccal mass which corresponds to theforward rotation of the buccal mass seen in the intact animal.

Retraction (Fig. 6, frames 5-9) is a continuous process, in which the radula is movedbackwards and downwards, and its leading edge is rotated towards the oesophagus.As the radula is moved backwards and rotated, the radula sac appears ventro-posteriorly(frame 6). The radula sac enlarges and swings forwards as retraction continues. Thisresults in the radula assuming a vertical orientation (frame 8) compared with thenear-horizontal position at the start of retraction (frame 5). The radula is then rotatedbackwards beyond the vertical position, and its leading edge can be seen pushingup against the roof of the buccal mass at the point where it joins the oesophagus(frame 9). Finally the radula returns to the resting position (frame 10).

Although the sequence shown in Fig. 6 illustrates the main features of the cycle, itdoes not enable us to distinguish between the two phases of retraction (Ri and R2).By filming the dorsal surface of the buccal mass it is possible to watch the backwardmovement of the radula by the indentation that its leading edge makes in the dorsalwall of the buccal mass (Fig. 7). A distinct V-shaped line can be seen as the radulamoves backwards. This backward movement occurs as two separate phases, whichcorrespond to phases Ri and R2 of the muscle recordings (see below). The first phaseinvolves the rotation of the radula to the vertical position (Fig. 7, frames 3-5). Thisslow phase Ri is followed by R2, a powerful and very rapid continuation of the back-ward rotation of the radula (frames 6-8). It has been necessary to space the framescloser together to detect this rapid movement. This second phase of retraction (R2) isaccompanied by contraction of the upper part of the anterior jugalis which forces thqradula towards the oesophagus (frame 8), completing the cycle.


Fig. 7. Another spontaneous feeding cycle filmed from a more dorsal position to show thespeed of backward movement of the radula. Frames were selected at the times shown, and arespaced closer together during more rapid movements. The approximate orientation of theradula is indicated by a dotted line, which is thickened at the leading edge of the radula. As theradula moves backwards its leading edge makes an indentation in the roof of the buccal mass.Frames 1 and 2 cover the protraction phase (P). Frames 3-5 show the radula moving slowlybackwards during the first phase of retraction (Ri ~ 1-7 s), and frames 6-8 illustrate the rapidmovement which characterizes the second phase of retraction (R2~o-$ s).

As a final confirmation of our interpretation, six cycles were filmed and the activityof pairs of muscles recorded simultaneously. A typical example is shown in Fig. 8with recordings from the posterior jugalis/tensor and anterior jugalis upper. Asexpected, the initial posterior jugalis burst discharges during protraction (Fig. 8,frames 1, 2). The activity which follows is associated with the tensor burst (phase Ri,frames 3, 4), and involves the beginning of retraction with the emergence of the radulasac. The muscle potentials from the tensor muscle are much smaller than usual in thisrecording. The radula is in a near-vertical position at the end of the Ri phase. How-ever, the most dramatic event is the powerful contraction of the anterior jugalis, whoseelectrical activity corresponds with a marked narrowing of the anterior half of thei>uccal mass. This is the rapid movement referred to previously, and its rapidity is"reflected in the high frequency of the anterior jugalis upper burst (phase R2). It is this

R. M. ROSE AND P. R. BENJAMIN

Fig. 8. Simultaneous recording of buccal mass movements and electrical activity in theposterior jugalis/tensor und upper anterior jugalis muscles. Black circles indicate the positionsof the recording electrodes, and times at which frames occur are indicated next to each drawing.A small amplitude burst occurs in the posterior jugalis/tensor during phases P and Ri of thefeeding cycle. Typically the tensor component would be of larger amplitude during phase Ri.The large-amplitude burst in the upper anterior jugalis is simultaneous with contraction of theanterior jugalis (phase R2) in the cinefilm, this contraction forcing the vertically positionedradula towards the oesophagus.


rapid movement which forces the vertically positioned radula backwards, deliveringfood to the oesophagus. Recordings of this type therefore prove that the two phases ofretraction (Ri and R2) correspond to a slow Ri followed by a fast R2 backwardrotation of the radula.

Relationship of neurones to muscles

Having established the pattern of muscle activity, the next problem is to relateactivity in identified neurones to muscle recordings. This subject will be introduced bygiving evidence for 1:1 nerve and muscle activity to the anterior jugalis and posteriorjugalis/tensor muscles in the non-feeding preparation. The finding of a 1:1 relation-ship between an identified neurone and muscle does not necessarily prove that theneurone is a motoneurone. It is possible, for instance, that the neurone is electro-tonically coupled to another cell which is the motoneurone for that muscle. While wehave observed electrotonic coupling as a common organizational feature in the buccalganglion we have only rarely observed such 1:1 following of action potentials betweenelectrotonically coupled cells. Usually the coupling is not sufficiently strong for suchfollowing to occur. Even if such coupling did occur, it could at least be stated thatsome elements of a given set of synchronously firing neurones were motoneurones. Themain problem is that of relating the activity of the identified groups of neurones(Benjamin & Rose, 1979) to identified muscles. In this respect it is important todemonstrate the relationship between nerve and muscle activity in the actively feedingsystem. The non-feeding preparation can give information on visually identified cellssuch as the 4-group cells, but it is necessary to have an actively feeding preparation tobe able to identify other cell types on the basis of their synaptic inputs (see Benjamin& Rose, 1979). In this analysis we demonstrate this relationship for buccal cell types3-8 as identified in the previous paper (Benjamin & Rose, 1979), cell types 1 and 2 notbeing included, since the anatomical work has shown that their axons go exclusively tothe salivary glands and oesophagus respectively and not to the buccal musculature(Benjamin et al. 1979). Where possible some comments will be made on the ability ofthe muscle to follow at different frequencies, but generally emphasis has been placedon determining the overall nerve-muscle relationship rather than discussing specificdetails. In all recordings (Figs. 9-13) muscle activity was recorded on the same sideas the neurone.

Identification of motoneurones in the non-feeding preparation

In the non-feeding preparation there is usually a low level of nerve and muscleactivity, and sometimes many of the neurones are silent. Consequently it is easier toshow 1:1 firing since the muscle potentials are not obscured by background spikeactivity. The limitation is that it is only really possible to identify the 3 cell and 4-group cells with certainty, since these cells are usually visually identifiable, and oftenhave a characteristic inhibitory input followed by rebound excitation. Even in non-feeding preparations the inhibitory inputs to the 4-group cells are often of sufficientamplitude to give rise to a short post-inhibitory burst.


(a) 80 mi

4,

40 mV

BR4

10s 200 miFig. 9. Examples of the i : i relationship between (a) 4 cluster, (6) 8 cell, and (c) 4-cell spikes,and muscle action potentials. In (a) three different 4-cluster cells were each penetrated with amicroelectrode and the surface of the anterior jugalis was explored with a suction electrode tofind the area innervated. Each 4 cluster cell (4^ 41 or 4a) is associated with activity in the areasindicated: 4i-»-®, 4,->-#, 4,->-0. The illustrated recordings are each from a recording sitedistinguished in the figure by a ' + '. (6) 8-cell activity associated with 1:1 firing of the upperanterior jugalis (from the same preparation as Fig. xzd). (c) 4 cell activity associated with 1:1firing of the lower anterior jugalis. The gain for the muscle recording in (6) is x I that in theother recordings, which gives an indication of the amplitude of the upper anterior jugalis spikes.The recording in (6) shows spontaneous activity, whereas bursts of action potentials wereelicited in the remaining recordings by a depolarizing current pulse.

Anterior jugalis motoneurones

The anterior jugalis muscle is innervated by 4-group snd 8 cells. Of these cells themost striking results were obtained from the main 4 cell, which was observed manytimes to have a 1:1 relationship of nerve to muscle action potentials (Fig. 9 c). Anumber of 4 cluster cells also innervate the same muscle (Fig. 9 a). By exploring thesurface of the anterior jugalis with a suction electrode it was shown that there was noconsistency in the areas innervated by specific cells from animal to animal. In particularthe main 4 cell sometimes covered a wide area, while in other preparations it wasfairly restricted. A representative example showing the spread of the innervation fromthree 4 cluster cells is shown in Fig. 9 (a). The only generalization which we can makeis that the main 4 cell usually supplies a much larger area of this muscle than any ofthe 4 cluster cells. During feeding the anterior jugalis undergoes quite complex move-ments, and it is interesting that 4-group cell bursts have different times of onset(Benjamin & Rose, 1979). This will presumably lead to differential contractions ofdifferent parts of the muscle. However, a given pattern of spatial innervation cannot badistinguished presumably because there are variations in the depths at which the"fibres run, the recordings being made from the muscle surface.

Motor control of feeding 153(b)

Pill *W

BL4

(c)

Pill. l l l l l U l l l l l l l l .

(rf)

pih

BL4CL

40 mV

10 >

Fig. 10. Examples of the 1:1 relationship between action potentials in (a) 4 cell, (6) unidentifiedsmall cell (sc), (c) 4 cluster cell, (


(a)

pilt •

BR3

mI cycle

BR4

rm

BR4CL

3 s

40 mV

Fig. n . Recordings of (a) 3 cell, (6) 4 cell, (c) 4 cluster cell with the posterior jugalis/tensorduring feeding cycles. In all three cells burst activity occurs at the same time as the tensor burst,and in (6) and {c) the posterior jugalis burst occurs at the same time as the period of inhibitionpreceding bursting in 4 and 4 cluster cells. In (a) the 3 cell activity has no 1:1 relationship tothe compound potentials in the tensor muscles.

jugalis. It could be argued that the ventral buccal nerve might also innervate theposterior jugalis, and that fine branches to this muscle had been cut in the dissectionprocedure. It is clearly very difficult technically to be absolutely certain that there is noinnervation of the posterior jugalis from 4-group cells. Later we will show that 6 cellsalso innervate the posterior jugalis/tensor. In this case it seems likely that the 6 cellsconstitute the small cells which project down the posterior jugalis nerve to the posteriorjugalis muscle. One way to demonstrate this in the non-feeding preparation would be toisolate the posterior jugalis and attached posterior jugalis nerve and buccal ganglion,and to record from the 6 cells to show their 1:1 innervation of this muscle. We havenot concentrated on this experiment because of the difficulty of finding and identi-fying 6 cells, but it might be technically feasible.

Identification of motoneurones in the feeding preparation

The easiest cells to record from in the feeding preparation are the large 4-group and3 cells. A common feature in proteased preparations is that the burst recorded overthe supra-median radular tensor (plus posterior jugalis) often has a large amplitudespike-like deflexion at the onset, followed by a high-frequency burst of musclepotentials. The 4-group and 3 cells discharge at the same time as this burst, but anexamination of the 3 cell recorded at high speed (Fig. 11 a) reveals that there is no1:1 relationship between the high-frequency 3-cell burst and the complex musclepotentials of the tensor muscle. Recordings from a 4 cell (Fig. 11 b) and a 4 cluster cell(Fig. 11c) together with the posterior jugalis/tensor, not only show that these cells,fire at the same time as the high-frequency tensor bursts, but also show that t h^inhibitory wave occurs at the same time as the first burst in the posterior jugalis. It is

Motor control of feeding 155(a)

pili

pi 1

i /' RUR21 /

BR5

I cycle

40 mV

40 mV

B1.7

Fig. 12. Relationship of double input cells to muscle activity, (a) In the 5 cell the first phase ofinhibition is simultaneous with the posterior jugalis burst, the second phase with the tensorburst, and firing is restricted to the inactive (/) period. The reduced amplitude spikes occurringin 5 cells during the first phase of inhibition have been remarked on previously (Benjamin &Rose, 1979). (b) In the 8 cell the first and second phases of inhibition are also simultaneouswith the posterior jugalis and tensor bursts respectively, but unlike the 5 cell, the 8 cell dis-charges at high frequency during the Rz phase as a result of rebound from inhibition, (c) The7 cell receives excitation during the protraction phase and inhibition during Ri simultaneouswith the tensor burst. This is in agreement with the alignment of 5 and 8 cells shown in (a) and(6) if the cycle is as given by Benjamin & Rose (1979, fig. 14). There is no 1:: relationship of7-cell action potentials to the muscle potentials in the posterior jugalis tensor, (rf) The 8 cellcauses 1:1 following of muscle potentials in the upper anterior jugalis. In the two cycles shownthere is only a small first phase of inhibition (arrow). The gain of the muscle recording is x }that shown in (a), indicating the large amplitude of the upper anterior jugalis burst.

nevertheless difficult to base the alignment of the neural sequence with the musclesequence on information of this kind, because i.p.s.p.s in 4-group cells often occurwith little change in membrane potential (Benjamin & Rose, 1979).

Double input cells

Much more information is given by recording muscles with the double input 5,f and 8 cells, since there are more clearly denned transition points in a cycle.

The recording of Fig. 12 (a) from the 5 cell and the posterior jugalis/tensor is


particularly illustrative because the first and second bursts of the posterior jugalis/tensor are of large amplitude. The first point is that the 5-cell burst occurs during theinactive (I) phase. The first-phase inhibition on the 5 cell is simultaneous with thefirst (protraction) burst in the posterior jugalis/tensor, and the second phase ofinhibition occurs during the first phase of retraction (Ri) simultaneous with the secondor 'tensor' burst of the posterior jugalis/tensor. The 5 cell recovers from inhibitionduring phase R2 and fires once more during the I phase. The 5-cell burst itself is nottherefore synchronized with any activity in the buccal musculature. This is in agree-ment with the anatomical findings (Benjamin et al. 1979), which showed that the 5cell sends a single axon to the dorsobuccal nerve. It seems likely that the 5 cell, like the2 cell, is concerned with oesophageal movements following the transfer of food to theoesophagus at the end of the feeding cycle.

The results for the 5 cell are compatible with a similar recording from the 8 cell(Fig. 12b). Here the first (protraction burst) is of small amplitude because of theposition of the electrode. This first burst (phase P) occurs at the same time as the firstphase of inhibition on the 8 cell. The second inhibitory wave-form is synchronizedwith the 'tensor' burst (phase Ri) as in the 5 cell. Although the p.s.p.s are verysimilar in the 5 and 8 cells, the 8 cell shows rebound excitation immediately followingphase Ri, and fires during phase R2. This suggests that the 8 cell is involved in thecontraction of the upper part of the anterior jugalis. The 8 cell also has an axon in thelateral buccal nerve, which supplies this muscle (Benjamin et al. 1979). Confirmationof this prediction is given by the recording shown in Fig. iz{d) from the 8 cell and theupper anterior jugalis in a feeding preparation. Although it can be clearly seen that the8 cell causes large amplitude muscle potentials in this muscle following a period ofinhibition, the recording is confusing because the first phase of inhibition on the 8 cellis very short in duration (Fig. 12 d, arrow). We confirmed that there was a very shortfirst phase of inhibition by recording the 8 cell with a 4 cluster cell, which has only thefirst inhibitory input. The recording of Fig. I2{d) therefore appears as a succession ofsecond inhibitory phases, followed by rebound excitation. The 1:1 relationship of8-cell activity to muscle potentials in the upper anterior jugalis is shown in more detailin Fig. 9(6).

The 7-cell activity also ties in with that of the 4, 5 and 8 cells (Fig. 12 c). Thetypical acceleration of the 7-cell burst occurs during the protraction phase, the 7 cellreceiving excitation at the same time as the 5 and 8 cells receive the first phase ofinhibition. Although it is possible that the 7 cell could generate part of the first (pro-traction) burst in the posterior jugalis, this seems unlikely, since there was no 1:1correspondence of nerve and muscle potentials in Fig. i2(c) even when this cell wasfiring at low frequency. The excitation on the 7 cell is followed by a second phase ofinhibition during phase Ri of the muscle cycle, as found for 5 and 8 cells. Whilerecording from a 7 cell the whole surface of the buccal mass has been explored with asuction electrode, including the dissected buccal sphincter muscle. Since no inner-vation to any muscle was found we suggest that the 7 cell may supply the oesophagus.Goldschmeding, Bruins & Everts (1977) show a cell '/?' next to the 3 cell which pro-jects down the dorsobuccal nerve only, and from its size and position it is possible th?*this is the 7 cell.


at,

BR4

40 mV

1 cycle 10s

Fig. 13. Relationship of single input 4 and 6 cells to muscle activity, (a) 1:1 firing of actionpotentials in the 6 cell and muscle potentials in the posterior jugalis/tensor. (6) Later in thesame preparation as (a), the 6 cell fires bursts during spontaneous feeding cycles. The 6-cellburst occurs during the protraction phase of the cycle, although the 1:1 relationship to musclepotentials is no longer apparent because of the high frequency of 6-cell discharge, (c) 1:1firing of action potentials in the 4 cell and muscle potentials in the lower anterior jugalis. (d)Later in the same preparation as (c) the 4 cell receives inhibition during the protraction phase,and fires a burst during the retraction phase of several spontaneous feeding cycles. The 1:1relationship to muscle potentials is obscured by the high frequency of firing and the activity ofother motoneurones to the lower anterior jugalis. In this particular recording the 4 cellreceives a second phase of inhibitory input (arrow) which delays the onset of bursting.

Single input cells

Having established the timing of the neural and muscle sequences in terms ofdouble input cells, we can re-examine the single input cells. Since the 6 cells receiveexcitation simultaneous with the first phase of inhibition of 3, 4, 5 and 8 cells, and arenot 8ynaptically driven at any other part of the cycle, it seemed likely that that the 6cells could cause the first phase protraction burst in the posterior jugalis. That this isthe case is proved by the recordings of Fig. 13(0), (b). In Fig. 13(0) the 6 cell fires atlow frequency in a 1:1 correspondence to small muscle potentials in the posteriorjugalis/tensor. When this preparation is showing patterned feeding cycles (Fig. 136)the 6 cell receives strong excitation during the protraction phase of the cycle. Unfor-tunately, because the 6 cell discharges at such a high frequency, individual muscleaction potentials fuse together to form a poorly distinguishable burst of small ampli-tude, and it is no longer possible to distinguish the 1:1 correspondence. It is clear,however, that the 6-cell burst and the first burst in the posterior jugalis correspond.The 1:1 firing and the fact that the cell fires in exactly the phase which we wouldpredict for the 6 cell is convincing evidence that 6 cells are involved in the protractionphase.

EXB SO


A final detail may be added in regard to the main 4 cell. This cell fires in 1:1correspondence with the lower anterior jugalis at low frequencies (Fig. 13 c). Duringfeeding cycles (Fig. 13d) the inhibition on the 4 cell corresponds with the protractionphase. In this particular preparation the 4 cell receives a second phase of synapticinhibition (Fig. 13 d, arrow) like the 8 cells. This leads to delayed firing of the 4 cellrelative to the 4 cluster cells, and has been remarked on previously (Benjamin & Rose,1979). The delay is such that the 4 cell may contribute partially to contraction of theanterior jugalis during phase R2, in conjunction with the 8 cell. Comparison of thesimilar muscle recordings in Fig. 13 (A) and (d) from two different preparations showsthat the 4 cell receives inhibition as the 6 cell receives excitation during the pro-traction phase of the cycle, entirely in accord with previous findings (Benjamin &Rose, 1979).

Summary of nerve-muscle relationships

It is now possible to describe how the burst activity of cell types 1-8 are correlatedwith the activity of the muscles described earlier. The neurones which have beenproved to have a 1:1 relationship to buccal muscles are shown in Fig. 14, while thealignment of the remaining neurones with the feeding cycle is given in Fig. 15. Only afew representative muscles are shown in Fig. 14, and only the phases of the cycle itselfin Fig. 15.

The cycle begins with a burst in the 6 cells (Fig. 14), which causes a burst in theposterior jugalis (first burst in posterior jugalis/tensor), and protraction of the radula.Undoubtedly there are more neurones involved in protraction since the suspensor,flexor and dorso-lateral protractor muscles also contribute to this phase. Although wehave found other candidate neurones which burst in this phase their identification asmotoneurones is complicated by the fact that interneurones also discharge during thisperiod, and we have not been able to demonstrate the 1: r relationship as done for the6 cells.

The retraction movement is divided into two phases (Ri and R2). In phase Ri the4-group cells fire, causing the contraction of the lower part of the anterior jugalis andthe tensor muscles. These muscles cause the rasp movement, the anterior jugaliscontracting differentially to produce a backward movement, and the tensor musclesstretching the radula over the odontophore. The postventral protractor muscles alsocontract, moving the fulcrum towards the mouth and thus helping to swing theodontophore to a vertical position.

Phase R2 is primarily caused by the burst in the 8 cell, which causes contraction ofthe upper part of the anterior jugalis. This forces the odontophore beyond the verticalposition so that food can be released into the oesophagus. The 4 cell may also con-tribute to this movement. The buccal retractor also pulls the whole buccal mass back-wards during R2. Finally the odontophore returns to the resting position, and staysthere until the start of the next protraction phase.

In Fig. 15 the remaining cell types are aligned with the feeding cycle. The physio-logical and anatomical (Benjamin et al. 1979) evidence suggests that none of these cellsare involved with the control of buccal muscles. The 1 cells are probably involved withsalivary gland activity as in Helisoma (Kater, 1974), and the 3 cells may also supplyglandular cells in the walls of the buccal mass and oesophagus (Benjamin et al. 1979).Anatomically, the 2, 3, 5 and probably also the 7 cells, supply the gut (Benjamin et al.


* gp

5sFig, 14. Summary of the relationship of activity in identified motoneurones to the cycle ofmuscle activity. The relationship* are as follows: 6 cell-*posterior jugalis (Fig. 13a, A), 4 groupcell-f-anterior jugalis lower (Figs. 13c, d, gd), 8 cell-»-anterior jugalis upper (Figs, ixd, gc).Cycles of muscle activity are separated by an inactive phase (I).

1979), The alignment of neural activity to the feeding cycle shown in Fig. 15 is baaedon recordings of 5 cell (Fig. 12a), 3 cell (Fig. 11 a), and 7 cell (Fig. 12c) activity, andthe remaining 1 and 2 cells are shown in their usual temporal positions relative to 3, 5and 7 cells (Benjamin & Rose, 1979). Bursting in the 5 cell is clearly restricted to theInactive period (I), and this activity may be combined with short bursts in the 2 cell(Benjamin & Rose, 1979) to produce oesophageal contractions.

6-2

5sFig. 15. Summary of the relationship of remaining identified cells to the feeding cycle, basedon recordings of the 3 cell (Fig. 11 a), 5 cell (Fig. 12 a) and 7 cell (Fig. 12 c), and the neuralcycle described by Benjamin & Rose (1979). With the exception of the 1 cell, all cells are doubleinput cells and may control gut function.

DISCUSSION

Relationship of neural activity to movements

The object of this paper was to relate the pattern of neural activity of identifiedneurones in the buccal ganglion to the sequence of muscular contractions duringfeeding. It would be very difficult to give a complete account of this relationshipbecause the buccal mass has 46 separate muscles (Carriker, 1946). However, most ofthese muscles are arranged as symmetrical pairs, many occur in groups having similarfunctions (e.g. 4 suspensor muscles, 3 paired tensor muscles), and this number alsoincludes small muscles which play an insignificant part in the feeding cycle. A furthersimplification arises when it is realized that most of these muscles discharge in separate


phases of a 4-phase feeding cycle (Fig. 5). The main result of this paper is that activityin 6, 4 group and 8 cells are components of the protraction, Ri and R2 phases respec-tively, and activity in the 5 cell occurs in the inactive phase (I). By obtaining examplesof burst activity in these cells during feeding cycles, it has been possible to align theneural cycle with the muscle activity cycle (Figs. 14, 15). It is also interesting that thedistinction between single and double input cells discussed in the first paper (Benjamin& Rose, 1979) seems to have a functional significance. Thus the single input 6 and 4groups cells are involved in controlling buccal mass movements, whereas the doubleinput 2, 3, 5 and 7 cells send axons to the gut (Benjamin et al. 1979). The exception tothis scheme is that the 8 cells are double input cells which supply the upper part of theanterior jugalis causing contraction during phase R2. However, the contraction ofthe upper part of the anterior jugalis is essentially the initiating phase of peristalsisin the oesophagus, and in this sense the 8 cell is also a double input cell whoseactivity relates to gut function.

If the single and double input cells serve different functions, can they be active asindependent groups ? The hypothesis put forward in the first paper was that the twophases of input might result from activity in two oscillatory networks of interneurones,with the first network being capable of entraining the second. In isolated ganglia wehave often observed double input cells which go into long periods of firing in whichonly the second input is present, suggesting that the proposed second oscillator iscapable of independent activity. It is possible that a series of feeding cycles could befollowed by activity in the gut alone, and that this could result from independentbursting of the second oscillator which drives the 2, 3, 5, 7 and 8 cells only.

As we have remarked in the introduction, Lymnaea differs from Aplysia in that themuscles appear to generate action potentials. In certain buccal muscles in Aplysiacontractions are produced by summation and facilitation of excitatory junctionpotentials (Orkand & Orkand, 1975; Cohen et al. 1978) and the muscles contract in agraded fashion proportional to the size of these junctional potentials. By contrast thebuccal muscles in Helisoma produce action potentials, this being confirmed by intra-cellular recording from the posterior jugalis muscle (Kater, Heyer & Hegmann, 1971).In relation to the innervarion pattern we have found that several motoneurones caninnervate the same muscle (e.g. anterior jugalis), and that certain motoneurones caninnervate more than one muscle (e.g. 4 cell to anterior jugalis and tensor muscles (seealso Goldschmeding, 1977)). In Helisoma different motoneurones also innervatedifferent parts of the anterior jugalis (Kater, 1974), while in Aplysia the lower extrinsicprotractor (Orkand & Orkand, 1975) and accessory radula closer (Cohen et al. 1978)also receive more than one excitatory axon.

Relation to other work

Another important result is that activity in the semi-intact preparation is verysimilar to that recorded from the isolated ganglion, implying that feeding cycles aregenerated centrally. But how is this activity initiated and maintained, and is it modifiedby proprioreceptive feedback ? It is certainly possible to initiate sequences in the semi-intact buccal mass using sucrose (Fig. 4 c), but at this stage we do not know whetherthis provides a phasic or slowly decaying stimulus to the pattern generator, or whetherthe chemosensory stimulus acts by way of higher order interneurones. The fact that


isolated buccal ganglia can produce periods of intense cyclical feeding suggests thatfeeding can also be initiated internally. In Helisoma, Granzow & Kater (1977) haveshown that feeding cycles can be initiated by passing current into certain inter-neurones in the cerebral ganglia. Gillette & Davis (1977) have also demonstratedfeedback from the buccal ganglia to cerebral neurones in Pleurobranchea. The relation-ship between the buccal and cerebral ganglia in Lymnaea is at present being investi-gated in this laboratory (McCrohan, unpublished observations). In relation toproprioreceptive input, Kater & Rowell (1973) have shown that stretching of mechano-receptors in the wall of the buccal mass of Helisoma evokes e.p.s.p.s in retractormotoneurones and i.p.s.p.s in protractor motoneurones, and suggest that the mechano-receptors cut off protractor activity before the inhibitory input to the protractors(Kater & Rowell, 1973; fig. 13). Kater (1974; fig. 23) seems to have modified this idea,in that he shows the mechanoreceptor input as being simultaneous with the inhibitoryinput to protractors from the interneurones. By comparison all that we can say withcertainty is that in the isolated buccal ganglion of Lymnaea the 4-group cells terminatesharply before the inhibitory input from interneurones, suggesting that mechano-receptors are not important in limiting the duration of 4-group bursts at high cyclefrequencies.

Two previous studies are directly relevant to the present work. Goldschmeding &de Vlieger (1975) have discussed the feeding cycle of Lymnaea stagnate based onanatomy and visual observation, while Kater (1974) has based his description of theclosely related pulmonate Helisoma trivolis on electromyographic and neuronerecording. The major difference between our work and that of Goldschmeding & deVlieger is that we put much greater emphasis on the rasping and retraction phases(Ri and R2) as active phases of the cycle, whereas the latter authors regard retractionas a passive relaxation of all muscles, with 19 of the 28 muscle types involved in pro-traction. This difference arises primarily because Goldschmeding & de Vlieger claimthat the anterior jugalis contracts during protraction and that the tensor muscles alsostretch the radula over the cartilage during this phase. By contrast we have shown thatthe anterior jugalis contracts strongly during the two retraction phases (Ri and R2)and that the tensor muscles contract during Ri. In other respects the two descriptionsare similar - for instance, during protraction we both agree that the dorso-lateralprotractor, suspensory muscles, and posterior jugalis are active, and during the returnof the radula to the resting position the posterior odontophoral protractor and buccalretractor contract. Goldschmeding & de Vlieger's interpretation was partly based onthe finding that the main 4 cell supplied both the anterior and posterior jugalis, whichshould therefore contract together. We claim that the posterior jugalis recording islargely tensor muscle activity, and that it is the tensor and anterior jugalis whichcontract together in retraction.

On comparison of our results with Kater's findings in Helisoma, there is somesimilarity as regards the activity patterns (Kater, 1974; fig. 8). We both find that theposterior jugalis and dorso-lateral protractor are protractor muscles, the tensor isinvolved in rasping (our phase Ri) and the buccal retractor and anterior jugalis inretraction. Kater does not find two phases of anterior jugalis activity, and has theposterior odontophoral protractor muscle firing in the later phase of retraction (our R2).What is very puzzling is that Kater's protractor motoneurone group have a marked


hyperpolarization preceding the burst of action potentials, the retractor motoneuronebursts being in antiphase to these protractor motoneurones. This is exactly the oppositeof the system in Lymnaea, where it is the retractor (4-group) motoneurones which havethe hyperpolarization, and the protractor (6 cells) are in antiphase. Our descriptionalso differs in that we add the activity of double input cells (2, 3, 5, 7) and show that2, s and 7 cells are active during the inter-feeding phase. It appears that the 1 cell issimilar in controlling the salivary glands.

We thank the M.R.C. for financial support, and Mike Land for help with thecinephotography.

REFERENCES

BENJAMIN, P. R. & ROSE, R. M. (1979). Central generation of bursting in the feeding system of thesnail, Lymnaea stagnalis. J. exp. Biol. 80, 93-118.

BENJAMIN, P. R., ROSE, R. M., SLADE, C. T. & LACY, M. G. (1979). Morphology of identified neuronesin the buccal ganglia of Lymnaea stagnalis. J. exp. Biol. 8o, 119-135.

CARRIKER, M. R. (1946). Morphology of the alimentary canal of the snail Lymnaea stagnalis apressa Say.Tram. Wise. Acad. 38, 1-88.

COHEN, J. L., WEISS, K. R. & KUPFBRMANN, I. (1978). Motor control of buccal muscles in Aplytia.J. Neurophysiol. 41, 157-180.

GILLETTE, R. & DAVIS, W. J. (1977). The role of the metacerebral giant neuron in the feeding behaviourof Pleurobranchea. J. comp. Plcysiol. 116, 129-159.

GOLDSCHMEDINO, J. T. (1977). Motor control of eating cycles in the freshwater snail Lymnaea stagnalis.Proc. K. Ned. Akad. Wet. C 80, 171-189.

GOLDSCHMHDING, J. T., BRUINS, H. J. & EVERTS, W. M. (1977). Topography of buccal and cerebralneurones involved in feeding in the freshwater snail Lymnaea stagnalis. Proc. K. Ned. Akad. Wet.C 80, 83-06.

GOLDSCHMEDINO, J. T. & DE VLIEGER, T. A. (1975). Functional anatomy and innervation of the buccalcomplex in the freshwater snail Lymnaea stagnalis. Proc. K. Ned. Akad. Wet. C 78, 468-476.

GOLDSCHMEDINO, J. T. & JAECBR, J. C. (1973). Feeding responses to sucrose in the pond snail Lymnaeastagnalis after nerve section and tentacle amputation. Neth.J. Zool. 33, 118-224.

GRANZOW, B. & KATER, S. B. (1977). Identified higher-order neurons controlling the feeding motorprogram of Helisoma. Neuroscience a, 1049—1063.

HUBENDICK, B. (1957). The eating function in Lymnaea stagnalis (L). Arkiv Zool. 10, 511-521.KATER, S. B. (1974). Feeding in Helisoma trivolvis: The morphological and physiological basis of a

fixed action pattern. Am. Zool. 14, 1017-1036.KATER, S. B. & ROWELL, C. H. F. (1973). Integration of sensory and centrally programmed components

in the generation of cyclical feeding activity of Helisoma trivolvis. J. Neurophysiol. 36, 142-155.KATBR, S. B., HEYER, C. & HEOMANN, J. P. (1971). Neuromuscular transmission in the gastropod

mollusc Helisoma trivolvis: Identification of motoneurones. Z. vergl. Physiol. 74, 127-139.ORKAND, P. M. & ORKAND, R. K. (1975). Neuromuscular junctions in the buccal mass of Aplysia: Fine

structure and electrophysiology of excitatory transmission. J. Neurobiol. 6, 531—548.ROSE, R. M. (1971). Patterned activity of the buccal ganglion of the nudibranch mollusc Archidoris

pseudoargus.J. exp. Biol. 55, 185-204.ROSE, R. M. (1976). Analysis of burst activity of the buccal ganglion of Aplysia depilans.J. exp. Biol.

64. 38s-4°4-

the relationshi of thp centrae l motor pattern to the feeding cycl oef lymnaea stagnalis · j. exp....

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