VOL. IV, No. i SEPTEMBER 1926

ON THE NERVOUS CONTROL OF THE VELARCILIA OF THE NUDIBRANCH VELIGER

BY G. S. CARTER,Lecturer in Zoology in the University of Glasgow.

(With eleven Text-figures and Plates I—III.)

{Received nth January 1926.)

CONTENTS.PAGE

Introduction 1Material 5Structure:

(1) The velar cilia and their cells 5(2) Nerve-apparatus of the velum:

(a) Living material 7(6) Golgi preparations 7(c) Gold chloride preparations 8

(3) Summary of observations on the structure of the velum 9The beat of the velar cilia:

(1) Normal behaviour 10(2) Behaviour of isolated cells 11(3) Experiments on the effects of changes in the environment on the intermissions

of the ciliary beat:(a) General 12(b) Changes in the reaction of the medium:

1. Acids 132. Alkalis 14

(c) Narcotics and drugs:1. Some typical narcotics 162. Potassium chloride 193. Strychnine 204. Atropine 2.15. Pilocarpine . . . 216. Atropine and pilocarpine 217. Veratrine 218. Curare 22

(d) Conclusions . 22Summary 24Bibliography 24Explanation of plates 2S

INTRODUCTION.Two types of behaviour are found among the numerous and varied ciliated organswhich occur throughout the animal kingdom. The first is found in the greaternumber of these organs, and is characteristic of those which function continuouslyduring the lifetime of the animal. In these the beat is uninterrupted and apparently

Ktomatic, and, in many organs of the type, independent of the presence of the othersues of the body. Thus, the ciliated cells of the epithelia of the gills of themussel will continue to beat for days or even weeks after the tissues have been

2 G. S. C A R T E R

broken down into small masses of cells by being shaken in a small quantity ofwater (Gray (9)).

The second type of behaviour is that with which this paper is concerned. It isless common but occurs widely in the animal kingdom. In organs of this type thebeat is not continuous but intermittent or even occasional. Most commonly thistype of behaviour occurs among organs which are used to produce the movementsof the animal and in these it can often be observed that the variations of the beatproduce the reactions by which the animal responds to stimulation. When thisis so, the belief is forced upon the observer that the beat is somehow under thecontrol of the organism and, if the animal possesses a nervous system, it is naturalto suppose that the control is exerted over the cilia as it is over the muscles of thebody through the medium of the nerves. This supposition is rendered more prob-able as the similarity between the processes which underlie contraction in muscleand in the ciliated cell is made more clear by the results of physiological research(Gray do-13)).

Observation of the behaviour of animals which possess ciliated organs of thistype early suggested to biologists that the beat of the cilia was under control, andsince that time the existence and nature of this control has been discussed. Asummary of the many observations upon which a belief in its existence is foundedhas been given by Vignon ((39), pp. 633 ff.) and will not be repeated here.

Apart from these observational results, much work has been done with the objectof showing that the control is exerted through the nervous system and not otherwise.To demonstrate this two points must be established. It must first be shown thatnerves reach the ciliated tissues and there end in organs which are not obviouslysensory; and, secondly, proof must be put forward that these endings are in factregulatory and that it is through them that the beat of the cilia is controlled. Indiscussing the previous work on the subject these two problems will be dealt withconsecutively.

Numerous references could be given to authors who have described nerve-endings in ciliated cells. Most of these are clearly sensory but the distinctionbetween sensory and regulatory nerve-endings is not always easy. Sensory nerve-endings among ciliated cells may take the form of conical or rod-shaped bodiessimilar to those which occur elsewhere in the body. The sensory nature of these isbeyond dispute. Other types occur. Thus Meyer (as) described nerve-fibres endingin a layer of ganglion cells below the epithelium of the ciliated pits of the polychaetePolyophthalmus. The organ is sensory and in the absence of further evidence itmust be presumed that this is a sensory innervation. Similarly the fibres whichwere seen by Eisig (7) to run into the protoplasm of the ciliated cells of the sensorypits of the Capitellidae, or by Burger (3) following a similar course in the sensoryciliated organs of the Nemertines, cannot be said to be other than sensory.

In some animals, however, ciliated cells which appear to be under controlform innervated organs which are motor rather than receptor. This has ^ Bdescribed by Hatschek(i4) and Kleinenberg(i6) in the trochospheres ofchaetes where nerves pass directly into the protoplasm of the prototroch cells,

On Nervous Control of Velar Cilia of Nudibranch Veliger 3

d by Prouho (33-33) and Kupelwieserd9) in the ciliated cells of the pyriform andorgans of polyzoan larvae. In the polychaet larva the beat of the cilia of the

prototroch is under control. Kupelwieser brings forward evidence that the beatof the cilia to which he refers is also controlled.

By far the largest amount of the literature of the subject is concerned withthe possible nervous function of the internal fibres of the ciliated cell. That fibresreaching the basal granules of the cilia, first described by Rabl Riickhard(34),might be nervous in function was suggested by Engelmann (8), but he decided againstthis interpretation on a comparison of their nature with that of true nerve-fibrils.Apathy (2) revived the idea of the existence of nerve-fibrils within ciliated cellsand described a system of fibres in the epithelium of the intestine of Anodonta,reaching the basal granules and running through the cytoplasm parallel to thefibres of Engelmann. This new type of fibre he believed to be nervous, but hethought that the fibres of Engelmann had some other function. Only Metalni-koff (24) who confirmed Apathy's results, using tissues of Sipunculus, has describedcontinuity between these fibres and the nervous system of the animal. Laterauthors do not confirm the existence of this second type of fibre and conclude thatthe only "fibres" present are those of Engelmann, and that they are not nervousin function. Many believe that they are rather modifications of the alveolar networkthan true fibres (Vignon(39), PrenantteO, Kolacev(i7), Saguchios), etc.).

Finally, among the ciliate Protozoa cytoplasmic fibres have been describednear the bases of large cilia and in some forms {Diplodinium, Sharp (37); Euplotes,Yocum(42)) these fibres are connected with a central mass which has been calledthe "neuromotor organ." It has been shown experimentally by Taylor (38) thatin Euplotes damage to these fibres destroys the power of co-ordination of the move-ment of the cilia. It would seem that in these animals the mechanism by which thebeat of the cilia is controlled has been clearly demonstrated, but the relationbetween these intracellular fibres in the protozoan body and the nerve-fibrils of theMetazoa is by no means clear.

It appears that no evidence of the existence of nerve-endings of any form amongciliated cells is of much interest from the point of view of the problem of the controlof ciliary activity unless it is combined with observational evidence that the ciliaare under control. From this survey of the histological side of the subject it will beseen that the only animals, apart from the Protozoa, for which there is evidenceon both these points, are the larvae of some Polychaetes and Polyzoa.

This however does not form a complete solution of the problem. Evidencedealing with the second of the two points mentioned above must be added, namely,evidence that the cilia are in fact under the control of nerves. Such evidence mustfrom its nature be experimental but, when the literature of the subject is studied,it is found that experimental work on the control of cilia is not extensive and, untilvery recently, work which tended to show a definite control through the nervous

^ ^ t e m was non-existent.It was shown by Parker ((28,29), etc.) that a stimulus to contract might be

passed from one ciliated cell to the next without the intervention of the nervous

4 G. S. CARTER

system. This process, which he called "neuroid transmission," is responsible forthe metachronism of the beat in many forms and in sea-anemones (Metrid^^produced, as he believed, a reversal of the beat. The statement of Vignon ((39),pp. 659 ff.) that an intermittent beat may occur in the young veligers of Aplysiaat a stage of development at which no nerves can be demonstrated suggests that thecontrol may sometimes be of this type in the molluscan veliger. In the veligerswhich formed the subject of this paper true nerves were present but the inter-mittence occurs at a much earlier stage than that studied here and it is not impossiblethat it is at first due to influences which pass through the general cytoplasm of thebody and only later to impulses through definite nerve fibres.

The only available evidence of the direct control of cilia through the nervoussystem in the Metazoa is due to Mertonta). He observed that the cilia of theepithelium of the lips of a snail (Physa) are under control and found that theepithelium is supplied with nerves. He was able to cut out of the animal a pieceof the epithelium with the nerve that supplied it. Immediately after separation thecilia were at rest but they started to beat when the nerve was stimulated electrically,and continued to do so during the stimulation and for 2-3 seconds after it ceased.He drew the conclusion that stimuli conducted by the nerve were responsible forthe control of the beat. Owing to the smallness of the preparation the objectionmay be raised that the stimulation may have been caused by some other meansthan through the nerve, perhaps by leakage of the current through the water inwhich the epithelium lay. Merton rejects this suggestion on account of the carewhich was taken to keep the end of the nerve outside the water covering theepithelium and on account of the similarity between the results obtained by stimu-lation and the normal behaviour of the animal.

It will be seen from this discussion that, if we exclude the Protozoa, in no animalhas a demonstration of nerve-endings which may be regulatory among ciliated cellsbeen combined with experimental evidence that the cilia are under the control ofthe nervous system. The present paper is an attempt to do this in the Nudibranchveliger. When they escape from the egg-capsule in which the early part of thedevelopment is carried on, the movements of these veligers are caused mainly bythe beat of the single row of large cilia which surround the velum. The beat of thesecilia is intermittent and the means by which this intermittence is produced formedthe subject of these investigations. The intermissions occur frequently in theabsence of any recognisable stimulus but they are also a usual response to stimula-tion and this fact makes it clear that they are not due merely to some automaticresult of the continuance of the beat, such, for instance, as fatigue of the ciliarymechanism*. Rather it appears that the animal has some means whereby the beatof the cilia can be stopped and allowed again to recommence; that, in fact, the beatof the cilia is under control.

In view of its double object the paper falls naturally into two parts, thus:

* It will be shown below that isolated strips of these cells are able to beat as actively as innormal position in the body and continuously for many hours. This fact also excludes the possibilitythat the intermissions are due to fatigue of the ciliary mechanism.

On Nervous Control of Velar Cilia of Nudibranch Veliger 5

(1) A histological study of the cells and of the surrounding tissues, in whichit is claimed to show that they are innervated by nerve-fibrils with endingswhich resemble those of regulatory rather than of sensory nerves.

(2) An experimental investigation of the intermissions of the beat which leadsto the conclusion that these may be prevented

(a) by the isolation of the cells and the consequent destruction of thefibres reaching them, although the cilia beat actively after isolation;

(b) by the paralysis of the fibres by means of various narcotics.It is therefore concluded that these are regulatory nerve-endings and that, bymeans of them, the beat of the cilia is controlled.

The thanks of the author are due to Prof. J. Graham Kerr, F.R.S., Prof. D.Noel Paton, F.R.S. and Mr James Gray, M.A., who have given much help in thepreparation of the paper. The experiments were performed at the Millport MarineBiological Station and the author wishes to thank Mr R. Elmhirst, F.L.S., for muchinvaluable assistance during the course of the enquiry.

MATERIAL.The larvae of the Nudibranchs Archidoris tuberculata, Aeolis drummondi,

Aeolidia papillosa and Doto coronata were used. The eggs of these animals are laid,either singly or in groups of two to four, in capsules within a ribbon-shaped massof spawn. In these capsules the early development goes on and the larvae do notescape until they are fully formed veligers. The masses of spawn were collectedon the shore and kept in the aquarium until the larvae escaped. As soon as thishappened they were used in the experiments. The experiments were thereforealways performed on larvae of approximately the same age. The structure of thelarvae was studied in material preserved at the same stage.

STRUCTURE.The structure of the different veligers used in these investigations differs only

in details of the relative size and number of the cells and their parts. No separatedescription will be given of each.

(1) THE VELAR CILIA AND THEIR CELLS.

The general structure of the veliger at the stage at which it escapes from theegg-capsule is shown in PI. HI, fig. 14. The beat of the velar cilia is outwards and,the effective stroke of the beat being backwards, the animal is driven through thewater with the velum directed forwards. At rest the cilia lie in the position of thebeginning of the effective beat, directed forwards and somewhat over the disc ofthe velum. Like the cilia of the gills of Mytilus (Gray (9)), they are stiff in theeffective beat and limp in the recovery beat. As a consequence of this the formdiffers in the two halves of the beat. This is shown in PI. I l l , fig. 16, and it will beseen from this figure that the changes of shape during the beat are precisely similar|o those described by Gray for the cilia of Mytilus.

In structure also these cilia resemble those of Mytilus very closely. Duringlife each easily breaks up into a bundle of fibres (PI. I, fig. 5) and it almost always

6 G. S. CARTER

does so when fixed (PI. I, figs, i , 2, 3). By means of the microdissection neediestcan be shown that the cilia consist (as do those of Mytilus w) of a series of trianguHplates set one behind the other in the plane of the beat of the cilium. When thecilium dies, these plates break up and a double row of fibres formed by their edges isleft. These are the external fibres of the fixed cilium. The plates are shorter on the sideof the cilium which is directed forwards in the effective beat. Together they form ablade-shaped cilium with the flat side of the blade in the plane of the beat of the cilium.The cilia vary from 20 to 40\L in length and from 1 to 2^ in thickness at the base.

The cilia are carried on a single row of cells surrounding the 8-shaped outlineof the disc of the velum. From two to four or occasionally more cilia are carriedon each cell. The cells vary in size from 25 x 20 x 5 /x in the large veligers ofArchidoris t o i o x i o x 4 / u , i n the much smaller veligers of Doto.

In PI. I, fig. 1, is shown a transverse section of the velum of Archidoris passingthrough a single frayed-out cilium on each side. A row of basal granules (b.g.)can be seen below the cilium. These correspond in number to the row of externalfibres (e.f.) on one side of the cilium and thus to the plates of which the cilium iscomposed (PI. I l l , fig. 15 a). From a study of sections cut in a plane perpendicularto that of this section and tangential to the velum {i.e. similar in position to that ofPI. I, fig. 3), there can be seen to be a double row of basal granules below eachcilium, i.e. one below each side of the blade. Thus each of the two external edgesof the triangular plate lies above a single basal granule.

Internal to the basal granules is the area of the internal fibres of the cilium.These did not appear in the sections as distinct fibres but the area in which theyare said to lie could be seen to be composed of protoplasm different in appearancefrom that of the rest of the cell. No definite conical arrangement could be seen.To regard this area as one of modified protoplasm rather than of fibres is in agree-ment with the more recent work on the internal fibres. (Saguchi, etc.)

Surrounding the basal granules is an area which stains darkly in sectionssomewhat overstained with haematoxylin (PI. I, figs. 2, 3, x). In PI. I, fig. 3,this area is stained in the cells of the right-hand part of the figure but not in thoseof the left. Its significance is unknown.

The nuclei of these cells are large and oval and lie in the centre of the cells.The rest of the cell is filled with protoplasm which shows no differentiation inthese sections.

It will be seen that the internal as well as the external structure of the ciliaryapparatus of these cells is closely comparable with that of the cells of Mytilus. Itis only more complex in that more than one cilium is borne on each cell.

The body of the animal around the velum and the disc of the velum itself arecovered by an epithelium of much smaller cells which bear short cilia. These ciliaare not shown in the figures. Between this epithelium and the underlying tissuesin the region of the disc are to be seen broad fibres (PI. I, fig. 1, m.f.) attacheddistally near the base of the cells which bear the velar cilia. These are about 2™in breadth and stain deeply with haematoxylin. They are the muscle fibres by whichthe contractions of the velum are brought about.

On Nervous Control of Velar Cilia of Nudibranch Veliger 7

(2) NERVE-APPARATUS OF THE VELUM.

(a) Living material.

A living veliger can often be fixed with the velum extended under a coverslip.The cells bearing the velar cilia then have the appearance shown in PI. I, fig. 4.Immediately within the outer surface of the cell and just below the bases of thecilia, there is a clear area representing the position of the basal granules and thepart of the cell surrounding them. Within this area the cell is full of small vacuoleswhich extend through the whole of the rest of the cell and surround the large ovalnucleus. These vacuoles are larger in the part of the cell internal to the nucleus.

By means of microdissecting needles the cells can be held apart and one ortwo separated from the remainder in the manner shown in PI. I, fig. 4. When thisis done, fibres («./.), which are much finer than the muscle-fibres, can be seenattached to the pointed ends of the ciliated cells and passing down into the tissuesof the velum. It was not possible in the unstained material to trace the course ofthese fibres towards the edge of the velum beyond the bases of the cells.

It is sometimes possible to obtain a side view of the ciliated cells in these prepara-tions. The appearance of one such in which the cilia have broken up into theirconstituent fibres (e.f.) is shown in PI. I, fig. 5. A fibre («•/.) is shown attachedto the pointed base of the cell.

The veligers can be stained intra vitam with methylene blue. The cells bearingthe velar cilia as they are seen in a preparation of this kind are represented inPI. II, fig. 6. The fine fibres («./.) which were seen in the unstained preparationsat the base of the cells take up the stain and can be seen to pass up between the cellsto the region just outside the nucleus. Here they break up into finer fibrils («./'.)lying apparently in the intercellular substance. Within the cell at the level of thesefibrils is a layer of granules (m.b.g.) lying among the vacuoles and staining darklywith methylene blue. These appear to occupy the inner part of the area which wasseen in the preparations stained with haematoxylin to be that of the internal fibresof the cilium.

In their property of staining with methylene blue these fine fibres resemblenerve-fibrils. Further evidence that they are true nerve-fibrils was obtained by theuse of specific nerve stains.

(b) Golgi preparations.

Sections were prepared by Golgi's bichromate of potash, silver nitrate method(Bolles Lee(21), pp. 458 ff.). One of these sections cut transversely to the plane ofthe beat of the cilia is shown in PI. II, fig. 7. The basal granules are visible as afaint line within the surface of the cells (b.g.). The nucleus and the area of theinternal fibres can be seen but are not darkly stained. The fibres («./.) take up the

tin and can be seen in about the same detail as in the methylene blue prepara-ns. Where they pass near the surface of the nucleus they stain for some reasonmore darkly than elsewhere and thus appear thicker in this region. They can some-

8 G. S. CARTER

times be seen to branch at their outer ends into the finer fibrils («./.') which wereseen in the methylene blue preparations but these fibrils do not stain darkly bdthis method.

Quite occasionally granules within the cells at the level of the fibrils take ona faint coloration in these preparations. It seems clear from the position andarrangement of these granules that they are the same as those which stain withmethylene blue. A figure of a preparation in which these granules have stained isgiven in PI. II, fig. 8.

(c) Gold chloride preparations.

By far the best results were obtained by this method. The method used wasthat of Lowit (Bolles Lee (21), p. 204), except that 25 per cent, formic acid was usedin place of the full strength both before and after the impregnation with the goldsalt (Paton and Pembrey(3o), p. 43).

A figure of a section in which the velum has been cut in the plane of the discand the cells bearing the velar cilia somewhat obliquely is given in PI. II, fig. 9.It will be seen that the outer region of the cell is stained darkly and that betweenthis and the nucleus is a region much less darkly stained. The nucleus and theinternal tissues of the velum are quite unstained. The fibres («•/.) can be seen darklystained between the cells. In the region just external to the nuclei they break upinto fibrils («./.') which can be traced almost to the outer edges of the cells. At thebase of the cells they pass out of the plane of the section.

In PI. II, fig. 10, is shown a section perpendicular to the plane of the disccutting the cells of the velar cilia longitudinally. The various parts of the cell stainin the same manner as in the section shown in fig. 9. The basal granules which arenot in the plane of section of fig. 9, can be seen to be stained more darkly than thesurrounding protoplasm. Outside them a small area of the cell is not stained. Thefibres have stained more darkly than in the section of fig. 9 and therefore appearthicker. They can be traced a short distance below the cells.

PI. II, fig. 11, is a semi-diagrammatic reproduction of part of a similar sectionsomewhat nearer the centre of the disc of the velum. At the side of the section thecells lie somewhat over one another and the fibres appear closer together. The figureis given because in it the fibres can be traced for some distance below the cells. Thefibres of several cells unite to form a single fibre which passes deeper into the tissues.In other respects the section is similar to that of fig. 10.

PI. I l l , fig. 12, was drawn from a section passing transversely through the velumand similar in position to that of PI. I, fig. 1. It was taken from a preparationof a larva of Doto coronata in which the size and number of the cells are both muchsmaller than in the larva of Archidoris tuberculata. The cells bearing the velar ciliaare cut longitudinally in the plane of the beat of the cilium. The region which stainsdarkly with gold chloride can be seen to occupy approximately the area of theinternal fibres of the cilium. Many other parts of the cell also stain but less dar

The fibres («./.) can be seen passing up the external side of the cell and endinin fibrils {n.f.') midway between the level of the nucleus and that of the basal

On Nervous Control of Velar Cilia of Nudibranch Veliger 9

granules. They lie in a slightly different plane of focus from that of the nucleus. TheI^A of a second fibre serving a neighbouring cell can be seen on the left of thefigure. Below the cell, fibres can be traced radiating from a ganglion just within thebase of the foot. A study of a series of sections showed this ganglion to be thecerebral ganglion of the larva. These fibres pass to various tissues of the front partof the body and some to the neighbourhood of the cells bearing the velar cilia. Itwas not possible to trace a single fibre throughout its whole length between theganglion and the ciliated cell but the fibres below the cells follow the same generalline as those leaving the ganglion and their arrangement leaves little doubt that theyare continuous with them. A study of a large number of similar sections confirmedthis conclusion. The fibres leaving the cerebral ganglion are clearly nerve-fibresand it seems therefore clear, apart from their staining properties, that the fibresbetween the cells of the velar cilia are also nervous.

It was possible in these preparations to make out further details of the structureof the end-fibrils of these fibres. It was noticeable that the end-fibrils appearedmuch darker than the inner parts of the fibres. They did not do so in preparationsmade by other methods. Fig. 13 is a semi-diagrammatic representation of theirappearance in higher magnification. It will be seen that the nerve-fibril splits upinto two or more thin fibrils which pass almost to the outer edge of the cell («./')•Among the fibrils are present short thick rods (w.e.). They are either thicker thanthe fibrils or appear to be so because they stain more darkly. Under lower magnifi-cation, by which the fibrils and rods cannot separately be distinguished, the wholegives the appearance of more darkly stained fibrils. It was not possible to makeout whether these rods form part of the fibrils or are closely applied to them. Itseems more probable from the preparations that they are modified parts of thefibrils. If this is so, the parallel is close between them and the nerve-endings ina muscle. Both consist of branching fibrils and of swellings along the course of thefinest branches.

(3) SUMMARY OF OBSERVATIONS ON THE STRUCTURE OF THE VELUM.

A diagram of the structure of the cells of the velar cilia and of their nerve-supplyis given in PI. I l l , fig. 15. The cell and the ciliary apparatus are shown in side-viewin PI. I l l , fig. 15 a, and in end-view in fig. 15 b.

The structure consists of the following parts:

(1) Ciliary apparatus.(a) The cilium consists of a series of triangular plates set one behind the

other in the plane of the beat of the cilium. Each plate is associated with a pairof basal granules within the cell, one below each of its two external sides. The platesincrease in length towards the side of the cilium which is hindmost in the effectivebeat. Two, four or sometimes more cilia are borne on each cell.

(b) Ciliary apparatus within the cell.1. A double row of basal granules below each cilium. Each contains about

15 basal granules.

io G. S. CARTER

2. An area around the basal granules which stains with haematoxylin but less,darkly than the granules themselves.

3. An area of differentiated protoplasm lying between the basal granules andthe nucleus. This represents the area of the internal fibres of the cilium describedby many authors.

4. An area of protoplasm containing granules which stain with tnethylene blue,situated within the area of the internal fibres and on a level with the nerve-endings.

(2) Nervous apparatus.

(a) Fibres which pass up between the cells from their bases to the level of thegranules which stain with methylene blue.

(b) Finer fibrils into which these fibres break up at this level. There are two orsometimes more of these fibrils between each pair of cells and they pass almost tothe outer edge of the cell.

(c) The rod-like organs which occur on the course of these fibrils and are eitherthickened parts of the fibrils or lie very close to them.

From a comparison of PI. Il l , figs. 15 a and b, it will be seen that both the fibresand their end-organs are out of the plane of both figures. They are therefore showndotted.

The course of the nerve-fibres within the body of the larva is shown in PI. I l l ,fig. 16, which was drawn from a reconstruction made from a series of sections of alarva. Above the very large otocysts (ot.) lie the cerebral ganglia (e.g.). From thesethe nerves supplying the cells of the velar cilia arise. Three or four nerves leave theganglion on each side and these by repeated branching give rise to the fibrils endingbetween each two adjacent cells. This is the interpretation given in the previouspages and is derived from an examination of a large number of these sections butit was not possible to trace the course of a single fibre from the ganglion to the cells.

THE BEAT OF THE VELAR CILIA.

(1) NORMAL BEHAVIOUR.

When the veliger is swimming actively through the water, the greater part ofthe animal is outside the shell and the velum is fully expanded in the positionshown in PI. I l l , fig. 14. The velar cilia then beat actively and metachronically,the rhythm passing down the whole line. The smaller cilia covering the body alsobeat actively. The movement of the animal through the water is directive and quitedistinct from the circus movements which often occur when the animal has lostcontrol of its cilia under experimental conditions.

Even in an animal which receives no perceptible stimulation, active movementthrough the water is never continuous. It is rare to find a perfectly healthy animalin normal conditions swimming continuously for more than 30-60 seconds andusually the period is much less (10—20 seconds). In every case a sudden stoppajfl6f the beat can be observed if the animal is kept within the field of the microscopefor a very few minutes.

On Nervous Control of Velar Cilia of Nudibranch Veliger 11

The reaction to stimulation varies with the strength of the stimulus. After^ stimuli, such as contact with an obstacle or another animal, the usual responseis a sudden stoppage of the beat of the velar cilia. This is indistinguishable from thestoppage which occurs without perceptible stimulation. The cilia covering the restof the body are not affected and continue to beat. After a slightly stronger stimuluscontraction of the velum may occur at the moment at which the beat stops and,if the stimulus is still stronger, the whole body may be contracted within the shell.All these contractions are sudden. If the stimulus is very strong and is repeatedor continuous, a series of slight impulsive contractions within the shell may occur.This is observed most often during strong chemical stimulation under experimentalconditions.

If no further stimulation occurs after the animal has contracted, it slowlyexpands. The velum as it passes out of the shell gradually opens and, at a stageat which its expansion is almost complete, the velar cilia again begin to beat.

The intermission of the beat is always easily recognisable. The cilia stop beatingsuddenly and simultaneously and are held in the position of the beginning of theeffective beat, i.e. directed forwards and somewhat arched over the disc of the velum(PI. I l l , fig. 16). Stoppages of other kinds, such as a gradual slowing or shorteningof the beat, occur in unhealthy animals but these are quite distinct from the normalintermission. The period during which the cilia are at rest after an intermissionis variable but, in the absence of further stimulation, is fairly constant for any oneveliger. From 2 to 5 seconds is a very usual time.

The recommencement of the beat after an intermission occurs in quite adifferent way from the stoppage of the beat which preceded it. In active veligersthe whole of the cilia may appear to start beating simultaneously but, if the beat isslow, it may be seen that some cilia start before the rest. It seems probable that thestart of the beat of the separate cilia is always independent. The stoppage of thebeat, on the other hand, is simultaneous in all the cilia and this difference betweenthe behaviour at the beginning and end of the intermission suggests that the re-commencement of the beat is due rather to the passing away of the impulse whichcaused the intermission than to a new impulse.

The metachronism of the beat is not regained until the beat becomes general.Often the first few strokes of the cilia after an intermission are shorter than thenormal.

This description of the normal behaviour is true for all the veligers used inthe experiments. It is also true for veligers which are still enclosed within the egg-capsule. The velar cilia beat for some days before the veligers escape and typicalintermissions occur.

(2) BEHAVIOUR OF ISOLATED CELLS.

In veligers which have become unhealthy, the cells bearing the velar cilia

f luently break free and can be seen driven about through the water by the beat ofeir cilia. This also occurs in veligers which have become unhealthy before escapingfrom their capsules. The interior of the capsule may become filled completely

12 G. S. C A R T E R

with free cells which have the shape shown in PI. I l l , fig. 15 a. Their beat maycontinue for many hours and is quite continuous until it is stopped by the death Mthecell. Atypical intermission was never observed. It will be noted below that certaindrugs, especially pilocarpine, cause a similar breakdown of the tissues of the velum.

By means of microdissecting needles strips consisting of several cells bearingvelar cilia can be torn off the edge of the velum. The cells lie in their naturalposition alongside one another in a single row. The cilia continue to beat afterseparation and in these cells also the beat is continuous and may continue for manyhours. These cells were in a quite healthy condition before isolation and the beatbecomes continuous immediately after isolation. The absence of intermissionscannot therefore be due to previous unhealthiness which might perhaps be the causeof the continuous beat of the cilia of cells which break free in the unhealthy veliger.The beat remains active and apparently normal and it is therefore clear that nodamage has been done to the ciliary mechanism. It must be concluded that theabsence of intermissions is due to some effect produced by the isolation apart fromthis mechanism. The results of these experiments are therefore in agreement withthe experiments to be described later which indicate that the intermission is due toan impulse along the nerves which reach these cells.

(3) EXPERIMENTS ON THE EFFECTS OF CHANGES IN THE ENVIRONMENT

ON THE INTERMISSIONS OF THE CILIARY BEAT.

(a) General.

If it be true that the intermission of the beat is caused by a nerve-impulsealong the fibres whose endings among the ciliated cells have been described, itshould be possible to prevent these impulses from reaching the cells by treatingthe animal with narcotics. These substances destroy the power of conduction innerves in lower concentration than that in which they affect the other living pro-cesses of the body. All, however, are toxic in high concentration, and will destroyall living activity if they are allowed to act upon the tissues in these concentrations.In high concentration, therefore, they will stop the beat of the cilia but, if theintermission is due to a nervous impulse, it should be inhibited and the beatrendered continuous in the presence of a lower concentration of the narcotic thanthat which affects the ciliary beat itself. During continued treatment with anarcotic substance the concentration of the substance within the tissues willgradually rise as more and more penetrates. In animals thus treated the intermissionsshould stop before the beat is affected. It is therefore clear that, if the intermissionsare nervous in origin, it should be possible to find (1) concentrations of the narcoticat which the intermissions are inhibited sooner or later but the beat never affected,and (2) others in which the intermissions are inhibited before the beat is affected.

A reaction to stimulation is produced by an impulse along the whole courseof the reflex arc. A substance which paralyses any part of this arc will prevent t Himpulse from reaching the cell. Intermissions due to stimulation should thereforebe prevented by all narcotics wherever the seat of their action on the reflex arc

On Nervous Control of Velar Cilia of Nudibranch Veliger 13

may be and not only by those which paralyse effector nerves and their end-organs,result of the experiments was to show that apparently all intermissions are

ented by any one of the narcotics employed and it seems necessary to concludethat even those intermissions which follow no obvious stimulus are due to somestimulus too small to be observed, either within or outside of the body.

As wide a selection of narcotics as possible was used in order to establishdefinitely that their effects were due to their narcotic property and not to anyaccidental property of the substances themselves. The action of some other drugswas also observed for comparison with that of the typical narcotics. The additionof these substances to the sea-water sometimes causes slight changes in the reactionof the water. It was therefore necessary to observe the effects of changes in thereaction both in the alkaline and in the acid direction.

The animals were placed in a solid watch-glass filled with the prepared medium.As it was desired to observe the effect of the drug during at least an hour, someprecaution had to be taken against evaporation of the drug. With this object thewatch-glass was covered with a plate of glass so as completely to exclude air. Itwas found that the effect of even the most volatile of the substances remainedconstant under these conditions for a much longer period than one hour and it wastherefore assumed that no appreciable evaporation occurred. It was also observedthat veligers would live and behave normally in sea-water in these watch-glassesfor many hours.

All the experiments were performed on veligers which had just left the egg-capsules. If it had been desired to use as young veligers as possible, they mighthave been carried out on veligers still within the capsule, but the power of thedifferent drugs to penetrate the membrane was unknown and complications wouldhave arisen from this cause.

(b) Changes in the reaction of the medium.1. Acids.

It was found by Gray (9) that the cilia of the gills of Mytilus beat more slowlyin slightly acid sea-water and more rapidly in slightly alkaline sea-water. In morestrongly acid or alkaline sea-water the cilia are brought to rest. The addition ofany acid to the water produces this effect but a much longer treatment is requiredwith the mineral acids than with those which more easily penetrate the cell-membrane, such as the fatty acids.

Similar results were obtained with the cilia studied in these experiments. Bothmineral and fatty acids were used. It was observed that in acid media the inter-missions were not prevented before the beat of the cilia was stopped. They wereoften longer than normal and the cilia were slow to start beating again after anintermission. Often a few cilia on the velum would start beating some time beforethe rest. There was a tendency under these conditions for the intermissions to

only some of the cilia on the velum, the remainder continuing to beat,he following table gives the results of typical experiments with the veligers of

Aeolis drummondi.

G. S. CARTER

Table I. Hydrochloric acid added to sea-water.

ioo c.c. sea-water+.. . c.c.

N/io HC1

pHBeat after a period of

45 60 min.

20

2-53040

6-4

3'731

«——Normal >•—Rather slow—

-Very slowStopped

-Rather slow *•-Very slow > Stopped< Stopped >

It will be seen that the full effect of the additions of hydrochloric acid was notproduced in all cases until the veligers had been at least one hour in the acid medium.After corresponding additions of butyric acid the full effect was produced in atreatment of 5 minutes. The results of experiments with butyric acid on the sametype of veligers is given in the next table.

Table II. Butyric acid added to sea-water.

100 c.c. sea-water+ ... c.c.

N/10 but. ac.

i-oi-52-O2-25

pH

6-8645-8S-4

Beat after a treatmentof 5 min.

NormalRather slowVery slowStopped

2. Alkalis.The increase in the rapidity of the beat observed by Gray in alkaline media

was also observed in these experiments. This occurs of pH. 8-4-10-0. In morealkaline media the ciliary mechanism becomes affected and the beat becomes shortand inactive and finally stops.

The following tables give the results of experiments with sodium hydrate andammonia. The veligers of Aeolis drummondi were used.

Table III. Sodium hydrate added to sea-water.

i ooc.c.sea-water+ ... c.c.

JV/io NaOH

S

•75

i-o

i-5

2-O

4 0

pH

8 S

8 7

8-9

c. 9 4

C. IOO

above n o

Beat after a period of

IS 3° 45 60 min.

Normal rather rapid beat throughoutY + + + +

Normal but distinctly rapid beath + + +(rare) +(rare)—Beat very rapid > < Beat less active

+ + (rare) + (incomplete)—> •* Beat less active0 0 o—> -e-Only a few cilia beating0 0 o—Beat very inactive and short

-< ^Beat very rapid+ f

< Beat very rapid-+ o

Beat active Beat short

:)1+ = Intermissions present. 0.= Intermissions absent.

On Nervous Control of Velar Cilia of Nudibranch Veliger

Table IV. Ammonia added to sea-water.

w-ioo ex.sea-water

+ ... ex.N/io NH.OH

5

•75

i -o

2"O

8-6

8-8

90

c. io-o

Beat after a period of

5 30

+ 0 0

•< Very rapid beat+ (at2min.) 0 0

•

0

X

Less active0

X

Beat v0

X

Beat v

60 min.

0

X

Beat less active0

X

beat >•0

X

ery inactive0

X

ery inactive

x = Cells becoming free from the velum.

It will be seen from these tables that the intermissions are prevented in alkalinesea-water some time before the beat begins to show signs of stopping. These resultsare in contrast with those of the experiments with acid sea-water. In the experi-ments with the more alkaline solutions of ammonia the velum began to breakdown after a time and the ciliated cells to become free by their own movement.When this occurs, it is natural, in view of the observations on isolated cells describedabove, that the intermissions should not occur. It will be seen, however, that theyceased to occur some time before there was any sign of the cells becoming separated.It seems that, if the intermission is caused by a nervous impulse, the nervous tissueis more sensitive to alkalinity than the ciliary mechanism.

A parallel for this "narcotic" action of alkaline media may perhaps be foundin the experiments of Adrian (O on the recovery process in nerve. He found thatthe supernormal period in the recovery process did not occur in media that weremore alkaline than normal but did occur in those that were more acid and increasedwith increasing acidity. He believed that the nerve impulse produced a negativecharge on some membrane within the nerve, that the insensitive period followingan impulse was due to the presence of this charge and that the nerve did not againbecome sensitive until the charge was removed. In acid media the membranepassed through a neutral phase in the recovery process when it was hypersensitiveto a positive phase in full recovery when this hypersensitivity was lost. In alkalinemedia the neutral phase was not reached and the nerve never became hyper-sensitive. The supernormal phase was therefore absent. If this interpretation isthe true one, the membrane should take on a permanently negative charge in asufficiently alkaline medium and the nerve be permanently insensitive. It wouldseem, therefore, that the results of the experiments recorded above might beexpected to occur on the hypothesis put forward by Adrian.

It is, however, possible that the effect is due to other causes than the increaseconcentration of the OH~ ions. Mansfeld(22) found that the movements

of preparations of the intestine of the dog and cat and of nerve-muscle preparationsof the gastrocnemius of the frog were inhibited by the presence of alkalis in the

16 G. S. CARTER

Ringer's solution with which they were perfused. He ascribed these resultchemical binding of the CO2 in the medium by the alkalis. Zottermann(43) repelhis experiments on the gastrocnemius but failed to confirm his results except whenammonia was added to the medium. He believed that this effect was due to aspecific action of the NH'4 ions. It is noteworthy that by far the most evidentresults in the experiments with veligers were obtained when ammonia was added.Whatever the mechanism of its action, it is clear that ammonia may produce a"narcotic" effect on nerves.

It is again possible that the cause of the lack of intermissions in alkaline medialies rather in the increase of the activity of the ciliary mechanism which occurs underthese conditions than in a narcotic effect of the alkalinity. As the cilium becamemore active, a larger and larger stimulus would be required to cause an intermissionand it is possible that a condition might arise in which the nerve impulse wasentirely unable to stop the beat.

(c) Narcotics and drugs.

i . Some typical narcotics.

The following drugs gave a typical "narcotic" effect:Alcohol. Chloral hydrate.Ether. Phenyl-urethane.Chloroform. Menthol.Morphine hydrochloride. Nicotine.

With the exception of curare none of the substances which were used and whichare known to be typical narcotics failed to produce this effect. The action of someother drugs which was not so typical will be described below.

The first effect of these substances in weak concentration is to cause an increasein the activity of the ciliary beat. This is parallel to their stimulating action on animaltissues when present in too weak concentration to cause narcosis. At this stageintermissions occur and may be much more frequent than normally, perhaps onaccount of stimulation of the receptor organs. If the substance is present insufficient concentration, the intermissions become rarer and rarer and finally ceaseto occur, the beat remaining active.

In greater concentration the substances do not produce the initial period ofactivity. The first effect is usually a prolonged stoppage of the beat, often accom-panied by a contraction, also perhaps due to stimulation of the receptor organs. Itlasts usually for 2-5 minutes but in the presence of some drugs, not typical nar-cotics {e.g. veratrine), it may be prolonged for a very much longer period. It isfollowed by a renewal of the beat. In the renewed beat intermissions do or do notoccur according as the substance is or is not in sufficient concentration to inhibitthem before the beat recommences. When the substance is a typical narcotic theyalways cease to occur sooner or later and some time before the beat of the ^jiashows signs of weakening.

As the intermissions become rarer and rarer, a larger and larger stimulus isrequired to cause them. Such stimuli are bound to be rare and in the experiments

On Nervous Control of Velar Cilia of Nudibranch Veliger 17

it was therefore not possible to define very accurately the time at which the inter-^•ssions finally became impossible. The times given in the figures for the various

drugs are those at which the last intermission was observed in a body of a con-siderable number of veligers. Before it was decided that intermissions had beenprevented by the treatment, the watch-glass was violently shaken. If no inter-mission occurred when this was done it was assumed that they had been in-hibited.

The beat of the cilia in the absence of intermissions was often apparently quitenormal and metachronic. Frequently, however, the animal, instead of swimmingforwards in a directive manner as it does in its normal life, was observed to swimround and round in circles without making any progress.

The metachronicity of the beat was not the main object of these experimentsand only occasional observations were made upon it. It appeared that in generalthe beat remained metachronic so long as all the cilia on the velum were beating.Occasionally a metachronic beat round only part of the velum was observed.

Text-fig. 1. Alcohol. Arcfndoris tuberculata. Text-fig. 2. Ether. Archidoris tuberculata.x = Intermissions cease. + =Beat begins to weaken. O = Cells begin to come free.

•Ol -OZ

Text-fig. 3. Chloroform. Aeolis drummondi. Text-fig. 4. Morphine HC1. Aeolidiapapillosa.x = Intermissions cease. + =Beat begins to weaken.

BJEB-IVl 2

i 8

70

60

so

AO

JO

20

10

MINS

6c

so

3o

20

(0

G. S. CARTER

niNS.

60

So

LO

30

zo

10

•Ol -o* •

On Nervous Control of Velar Cilia of Nudibranch Veliger 19

the intermissions and the beat. This difference is a measure of the "narcotic"• o n of the drug.

It will be seen that the separation between the curves is very different in extentwith the. various drugs. By far the most marked effect was obtained with nicotine.The other drugs all gave a distinct separation, differing in extent from one drug toanother but in a less degree.

The reaction of the sea-water was not altered to an appreciable extent by thepresence of these drugs in the highest concentrations used in the experiments, withthe exception of morphine hydrochloride. The reaction of sea-water to which•4 per cent, of this drug has been added is pH 7-4, that of the sea-water used at thetime being 8-3. It will be seen from the experiments on the occurrence of theintermissions in acid media that this difference could not have been the cause ofthe cessation of the intermissions.

Certain differences in behaviour in the presence of the different drugs wereobserved. In the presence of the higher concentrations of ether some cells brokefree from the velum but not for some time after the intermissions had ceased.With chloral hydrate the beat stopped almost immediately when the concentrationwas greater than -07—08 per cent. It was therefore not possible to observe the effecton the intermissions of higher concentrations. With phenyl-urethane contractionsof the muscles of the body were often observed after the intermissions had beeninhibited. This may have been due to slow penetration of the drug, the nerve-endings in the ciliated epithelium being paralysed before the deeper lying nervessupplying the muscles were reached.

2. Potassium chloride.

It was found that the addition of isotbnic potassium chloride to the sea-waterproduced similar results to those of the narcotic substances. The results of ex-periments on veligers of Aeolis drummondi are given in Text-fig. 9. It will be seen

MINS.

110

100

SO

60

40

10

5 35 /s ioyo n/i.KCL.

Text-fig. 9. Potassium chloride.Aeolis drummondi.

x = Intermissions cease. + =Beat begins to weaken.

20 G. S. CARTER

that there was a considerable interval after the cessation of the intermissions duringwhich the beat was active in media to which from 5 to 20 per cent, of the solu^Kof the salt had been added. Overton ((27), pp. 261-9) found that the presence ofa disproportionate amount of potassium in a salt solution in which a nerve-musclepreparation of a frog was lying paralysed the motor nerve-endings before themuscle itself was affected. Several references to work of other authors showing thatpotassium has a narcotic effect are given by Clark ((5), p. 444).

3. Strychnine.A saturated solution of strychnine in sea-water was prepared by stirring the

water for 24 hours by means of a strong current of air in the presence of more ofthe drug than would dissolve. There was no appreciable difference in reactionbetween the saturated and fresh sea-water.

The action of this drug is complex. In all strengths from 1 to 100 per cent, ofsaturation the intermissions are inhibited within the first five minutes. In a strengthof -i per cent, of saturation they continue for more than one hour. The beat of thecilia continues actively except in the fully saturated medium in which there is aslight loss of activity after an hour. So far the action of the drug is similar to that

MINS.

120

IO0

90

60

KO

10

INTER-MISSIONSREAPPEAR

MINS.

60

So

40

30

20

10

1%SAX PK..&I3

•Ol

2-201

S25

^Oi 01^ -05%

i-L ?5 8-7Text-fig. 10. Strychnine. Aeolis drummondi. Text-fig. 11. Atropine. Aeolis drummondi.

Logarithmic curve x = Intermissions cease. + =Beat begins to weaken.

of the typical narcotic substances. It was found, however, that when the animalswere treated with the drug in strengths of 1-10 per cent, saturated, the inter-jnissions reappeared at the end of 70 minutes. This did not happen in a higher orlower concentration of strychnine or with any of the other drugs used in theseexperiments. No evidence of rhythmic contractions similar to those which occurin vertebrates was obtained but it was observed that the animals frequently con-tracted within the shell for long periods and that the beat often continued acti^fcwithin the shell.

That strychnine should act as a narcotic might be expected. In the vertebrates

On Nervous Control of Velar Cilia of Nudibranch Veliger 21

its narcotic effect is masked by the much more obvious convulsions that it causes,^ i t it is also known to paralyse nerves. In the vertebrates this effect is not pro-nounced (Dixon(6), p. ioo). Overton(z6), however, observed that tadpoles whichhad been treated for a long time with a weak solution of strychnine behaved ' ' asif they had been poisoned with curare," i.e. were completely paralysed (p. 170).Laqueur(2o) found that frogs poisoned with strychnine were insensitive to acousticstimuli at a time at which the smallest stimulation of the skin by a touch or a currentof air set up convulsions. Here the acoustic organs were paralysed and not thetactile. It is surprising that in the veligers so marked an apparent narcotic effectshould be produced and that convulsions should be completely absent. That thenarcotic effect is not typical is shown by the fact that the intermissions laterreappear.

4. Atropine.The presence of this drug renders the sea-water more alkaline and the results

of the experiments with it were therefore inconclusive. A well-marked narcoticeffect was obtained in sea-water + -02 per cent, atropine (/>H 8-5). In higherconcentrations the complications arise that the velum begins to break down intoits component cells and that a prolonged stoppage of the beat follows the intro-duction of the veligers into the medium. It is therefore impossible to observewhether intermissions can occur at this stage. Later the beat recommencesand is continuous. This occurs some time before the velum begins to break down.It would appear, therefore, that atropine acts as a narcotic but that its action iscloaked by other effects.

5. Pilocarpine.The most striking effect of the presence of this drug is the rapid breakdown of

the velum which occurs. The effect is much more marked with pilocarpine than withany of the other drugs used. A similar effect occurs in other ciliated tissues, e.g.it was observed in the ciliated epithelium of the oesophagus of frogs into whichpilocarpine had been injected by Heidenhainos) and Schmidt(36). In the case ofthe velum the intermissions cease a short time before the cells begin to break free,but it was not possible to decide whether this was due to the initial stages of thefreeing of the cells or to a specific effect of the drug. The reaction of the water wasnot appreciably altered by the presence of pilocarpine in the strengths used.

6. Atropine and pilocarpine.The effect of these drugs in combination was tested in order to find out whether

any antagonistic action could be observed. No antagonism was found, the effectsof the two drugs together being a summation of their effects singly.

7. Veratrine.A solution of veratrine in sea-water was made by stirring the water for 24 hours

by strong aeration in the presence of the drug. The reaction of the water wasunchanged.

22 G. S. CARTER

The most obvious effect of this drug was to cause an immediate and prolongcontraction of the veliger within the shell. The cilia could be seen to be b e ^within the shell. The beat was active and metachronic. The effect is thereforedifferent from that of atropine which causes the cilia to stop beating without anymarked contraction. Intermissions occurred within the shell during the first20 minutes of the experiment. Thereafter they appeared to cease but doubtfulintermissions were seen at a later stage in a few experiments. About 40 minutesafter the beginning of the experiment the veligers began to expand and in another20 minutes were completely expanded. The beat remained active and metachronic.

The fact that intermissions occur in the contracted veliger in the early part ofthe experiment shows that their absence later is not due to the contraction or toabsence of stimulation. It would seem that an incomplete inhibition of the inter-missions occurs in the presence of veratrine.

This result recalls the evidence that has been brought forward that this drugacts on nerve- as well as on muscle-tissue (Kondo(i8), Vinogradoffui)). That itshould produce an effect similar to that of recognised narcotic drugs is thereforenot impossible.

8. Curare.

I was indebted for a sample of this drug to Prof. D. Noel Paton, F.R.S. Asaturated solution was prepared by aerating the water in the presence of the drug.Not quite all the drug dissolved. The reaction of the water was unaltered.

The drug was found to have very little effect on the veligers. The beat wasat first abnormally active and remained active for more than two hours. There wasa tendency for the cells of the velum to come free after a treatment of 70 minutes.Intermissions continued throughout the experiments.

(d) Conclusions.

In considering these experiments the complexity of the reaction which formstheir subject must be remembered. The reflex arc is complex and the failure of anypart of it or of the receptor or effector organs at its ends will produce the sameresult, namely, the inhibition of the reaction. It is therefore not surprising thatthe very various narcotics used in these experiments all produce this result.

Further, it is clear that the intermissions might be prevented either by a failureof the stimulus to reach the ciliated cell or by some change within the ciliated cellitself which rendered it insensitive to the stimulus. Too little is known of thechanges produced by the nerve-impulse within the effector cell to make this aprofitable subject for investigation, but it is clear that if it were shown that theintermissions could be prevented by treatment whose action could not be u p f lnerve-tissue, the effect might be due to action of such a kind.

In the following table the results of the experiments are stated:

On Nervous Control of Velar Cilia, of Nudibranch Veliger 23

Table V.

Drug

AlcoholEtherChloroformMorphine HC1Chloral hydratePhenyl-urethaneMentholNicotineStrychnine

Atropine

PilocarpineVeratrineCurare

Interval

Definite

Very markedApparently definite, but the inter-

missions reappear laterDoubtful and cloaked by the other

effects of the drugNone

Slight and doubtfulNone

With the exception of curare and of atropine all the typical narcotics producedthe effect in a marked degree. The experiments on atropine were inconclusive butindicated that the drug had an effect of the same kind as the typical narcotic sub-stances. Only in the case of curare were the experiments definitely negative. Itmay therefore be said that the experiments with narcotic substances support thethesis that the intermission is due to a nerve-impulse.

The inhibition of the intermissions produced by the other drugs, if it occurred,was always doubtful or untypical. The results of the experiments with veratrineand strychnine have been discussed above and reasons have been given for be-lieving that these results are not in opposition to the theory that the intermissionsare caused by a nerve-impulse.

Other experiments showed that changes in the reaction of the medium in thealkaline but not in the acid direction prevented the intermissions. This also hasbeen discussed and reasons have been given for the belief that this should be soon the theory that the stimulus is nervous.

It would seem, therefore, that it is not necessary to demand any action of thedrugs within the ciliated cell to bring these results into line with the theory of thenervous origin of the stimulus, but the possibility that action of this kind occurs isnot excluded. The fact that all the more typical narcotics inhibit the inter-missions very readily, and the fact that the inhibition was slight and doubtful, ifit occurred, when any other treatment was used, both support this view. It is alsosupported by the fact that the intermissions do not take place when the cells areisolated from the other tissues and by the fact that nerve-endings occur amongthese cells which resemble those of regulatory rather than of sensory nerves. Severallines of evidence, therefore, point to the conclusion that the control of these ciliais brought about by stimuli reaching them through the nervous system of theanimal.

24 G. S. CARTER

SUMMARY.

(1) The beat of the velar cilia of the Nudibranch veliger is intermittent andappears to be under the control of the organism.

(2) Fibres lying between the cells which bear the velar cilia are described. Theyend near the outer surface of these cells in fibrils which resemble those of themuscle-plate in structure. They stain with specific nerve-stains and were shownto be in all probability continuous with nerves lying deeper in the tissues. It isconcluded that these are nerve-fibrils.

(3) Within the ciliated cell at the level of the terminal fibrils of these fibresthere is a layer of granules which stain with methylene blue.

(4) The cilia of cells which have been separated from the tissues beat activelybut the beat is continuous and not intermittent.

(5) In the presence of various narcotics the intermissions are inhibited morequickly than the beat of the cilia is affected and at a lower concentration of the drug.

(6) Some drugs which are not known to act as narcotics were used and withcertain exceptions, which are discussed, did not have this effect.

(7) In an acid medium the beat is slower but the intermissions continue solong as the cilia beat.

(8) In an alkaline medium the intermissions are inhibited before the beat isaffected. This is discussed and it is suggested that it is in accordance with recentwork on the effect of changes in the reaction of the medium on nerve-tissue.

(9) It is concluded that the nerve-endings found among these cells are regula-tory and are concerned in the causation of the intermissions. The beat of the ciliais therefore under the control of the nervous system of the animal.

BIBLIOGRAPHY.(1) ADRIAN, E. D. (1920). Journ. Physiol. 54, 1-31.(2) APATHY, S. (1897). Mitt. Zool. Stat. Neapel, 12, 495.(3) BORGER, O. (1890). Zeit. wiss. Zool. 50, 1.(4) CARTER, G. S. (1924). Proc. Roy. Soc. London, B, 96, 115.(5) CLARK, A. J. (1921). Journ. Pharm. Exp. Ther. 18, 423.(6) DIXON, W. E. (1906). A Manual of Pharmacology. Arnold, London.(7) EISIG, H. (1887). F. u. Fl. d. Golfes v. Neapel, 16.(8) ENGELMANN, T. W. (1880). Arch. ges. Physiol. 23, 505.(9) GRAY, J. (1920). Q. Journ. Microsc. Set. 64, 345.

(10) (1922). Proc. Roy. Soc, London, B. 93, 104.(11) (1922). Proc. Roy. Soc, London, B, 93, 122.(12) (1923)- Proc. Roy. Soc, London, B, 95, 6.(13) (1924). Proc Roy. Soc, London, B, 96, 95.(14) HATSCHEK, B. (1886). Arb. Zool. Inst. Univ. Wien, 6, 121.(15) HEIDENHAIN, M. (1876). Centralbl. med. Wiss., 1876, No. 24 (quoted by Schmidt).(16) KLEINENBERG, N. (1886). Zeit. wiss. Zool. 44, 1.(17) KOLACEV, A. (igio). Arch. mikr. Anat. 76, 349.(18) KONDO, S. (1923). Act. Soc. Med., Kyoto, 3, 19 (abstract in Physiol. Abstrs. 4, 281).(19) KUPELWIESER, H. (1907). Zoologica, Stuttgart, 19, Heft 47.(20) LAQUEUR, E. (1909). Schr. Physik. Ges., Konigsberg, 1.(21) LEE, A. BOLLES (1921). The Microtomist's Vade-mecum, 8th ed., edited by J. B. Gatenby.

J. and A. Churchill, London.

JOURNAL OF EXPERIMENTAL BIOLOGY VOL. IV, PLATE I.

/

CARTER—ON NERVOUS CONTROL OF VELAR CILIA OF NUDIBRANCH VELIGER (pp. 1-26).

JOURNAL OF EXPERIMENTAL BIOLOGY VOL. IV, PLATE II.

Fig 9O 5 10 20 39a

0 10

ep-

-epFig 10 O i 10 SOIL

20 4O/1

Fig. 7

mbg.

vU5O 10 20 W/U w 5 O 3 10/1

CARTER—ON NERVOUS CONTROL OF VELAR CILIA OF NUDIBRANCH VELIGER (pp. 1-26).

On Nervous Control of Velar Cilia of Nudibranch Veliger 25(22) MANSFELD, G^(i92i). Arch. ges. Physiol. 188, 247.

•MERTON, H. (1923). Arch. ges. Physiol. 198, 1.METALNIKOFF, S. (1900). Zeit. miss. Zool. 68, 262.

(25) MEYER, E. (1882). Arch. mikr. Anat. 21, 769.(26) OVERTON, E. (1901). Studien iiber die Narkose. Gustav Fischer, Jena.(27) (1904). Arch. ges. Physiol. 105, 176.(28) PARKER, G. H. (1896). Bull. Mus. Comp. Zool., Cambridge, Mass. 29, 107.(29) (1919). The Elementary Nervous System. Lippincott, Philadelphia.(30) PATON, D. NOEL, and PEMBREY, M. S. (1924). Physiological Histology. Arnold, London.(31) PRENANT, A. (1912-14). Journ. anat. et physiol. 47-9.(32) PROUHO, H. (1890). Arch, zool., Paris, 2e serie, 8, 409.(33) (J892). Arch, zool., Paris, 2e sgrie, 10, 557.(34) RABL ROCKHARD (1868). Arch. Anat. Physiol. 72.(35) SAGUCHI, S. (1917). Journ. Morph. 29, 217.(36) SCHMIDT, C. (1882). Arch. mikr. Anat. 20, 123.(37) SHARP, R. G. (1914). Univ. Cal. Publ. Zool. 13, 4.(38) TAYLOR, C. V. (1920). Univ. Cal. Publ. Zool. 19, 403.(39) VIGNON, P. (1900). Arch. Zool., Paris, 3e sdrie, 8, iii.(40) —•—-(1901). Arch. Zool., Paris, 3

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280).(42) YOCUM, H. B. (1918). Univ. Cal. Publ. Zool. 18, 337.(43) ZOTTERMANN, I. (1923). Journ. Physiol. 57, xxi.

EXPLANATION OF PLATES I, II, AND III.

b.g. basal granules; c. velar cilium; e.g. cerebral ganglion; e.f. external fibres of the cilium; ep. epithe-lium of the body and velum;/, foot; i.f. internal fibres; m. mouth; m.b.g. granules which stain withmethylene blue; m.c. mantle cavity; TO. d.n. microdissection needles; m.f. muscle fibres of the velum;n. nucleus; n.e. nerve-endings; n.f. nerve-fibrils between the ciliated cells; n.f.' terminal branchesof these fibrils; n.f." nerve-fibrils passing into the tissues of the body; n.t. end-organs on the fibrilsbetween the ciliated cells; oes. oesophagus; ot. otocyst;^. point of attachment to shell; r. rectum;s. shell; st. stomach; t.p. triangular plates of cilium in side-view; v. velum; v.c. velar cilia; v.h. visceralhump; v.c.c. cells of the velar cilia; x. area surrounding the basal granules which stains with haema-toxylin.

PLATE I.

FIG. I . Aeolis drummondi. Transverse section of the velum. The section passes through a singlefrayed cilium on each side. Iron haematoxylin, 6 f».

FIG. 2. Archidoris tuberculata. Part of a similar section. Somewhat overstained; the area surroundingthe basal granules has stained. Iron haematoxylin, 5 /x.

FIG. 3. Archidoris tuberculata. Longitudinal section of the cells of the velar cilia perpendicular tothe plane of the beat. The area around the basal granules is stained in part of the section. Ironhaematoxylin, 5 y..

FIG. 4. Archidoris tuberculata. Living. Cells of the velar cilia held apart by the microdissectionneedles. The broad and narrow fibres at their base are shown.

FIG. 5. Archidoris tuberculata. Living. A velar cilium and its cell in side-view with one of thenarrow fibres at its base.

PLATE II.

FIG.6 . Archidoris tuberculata. Living. Cellsofthevelarciliastainedt'ntra wfamwithmethyleneblue.

'IG. 7. Aeolis drummondi. Longitudinal section of the cells of the velar cilia showing the fibreseen the cells. Golgi's rapid bichromate of potash-silver nitrate method. 12 /*.

FIG. 8. Archidoris tuberculata. Similar section. The granules which stain with methylene blue arestained. Golgi's rapid bichromate of potash-silver nitrate method. 5/t.

26 G. S. CARTER

FIG. 9. Aeolis drummondi. Section parallel to the disc of the velum. The fibres between the ciliatedcells are shown. Gold chloride (Lowit's method). 12 ft.

FIG. 10. Aeolis drummondi. Longitudinal section of the cells of the velar cilia showing the fibresiBHend-organs between the cells. Gold chloride (Lowitt's method). 12/1.

FIG. 11. Aeolis drummondi. Similar section. Semi-diagrammatic. The fibres pass some distancebelow the cells. Gold chloride (Lowitt's method). 12ft.

PLATE III.

FIG. 12. Doto coronata. Transverse section of the velum. Nerve-supply of the velum from thecerebral ganglion is shown. Gold chloride (Lowitt's method). 5 /t.

FIG. 13. Aeolis drummondi. Longitudinal section of the cells of the velar cilia. Semi-diagrammatic.Structure of the end-organs of the fibrils shown in more detail. Gold chloride (Lowitt's method). 12 ft.

FIG. 14. A Nudibranch veliger at the time of its escape from the egg-capsule.

FIG. 15. Diagrams of the structure of the velar cilia and their cells, a, side view of cilium; b, endview.

FIG. 16. A reconstruction of a veliger to show the nerve supply of the velar cilia.

JOURNAL OF EXPERIMENTAL BIOLOGY VOL. IV, PLATE III.

~ -* Recoverybear

a. Fig. 15 t.

CARTER—ON NERVOUS CONTROL OF VELAR CILIA OF NUDIBRANCH VELIGER (pp. 1-26).

JOURNAL OF EXPERIMENTAL BIOLOGY VOL. IV, PLATE IV.

Fig. 3. Fig. 8.PATRICK—EXPERIMENTAL STUDY OF CELLS OF HEPATO-PANCREAS OF LIGIA

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