Neurosecretory cells in the optic tentacles of certain...

Neurosecretory cells in the optic tentacles of certainpulmonates

By NANCY J. LANE

(From the Cytological Laboratory, Department of Zoology, University Museum, Oxford)

With two plates (figs, i and 4)

SummaryCells considered to be neurosecretory have been observed in the optic tentacles ofcertain stylommatophoran pulmonates. Such cells are divisible into three distincttypes, of which those called the 'collar' cells surround the central digitate ganglionand eye. The other two types, the 'lateral oval' and the 'lateral processed' cells, lielaterally in the tentacle, on the inner edge of the outer dermo-muscular sheath. Allthree cell-types have branching dendritic processes, containing granules. The den-drites of the collar and of the lateral cells apparently extend from the cell-body to thesurface of the epithelium. The axonal processes of all three types are thick and containgranules.

The ground cytoplasm of these cells is scarcely visible owing to the great numberof homogeneous, spheroidal granules that are present. The granules are sudanophil,and the ones in the collar cells contain phospholipid (probably cerebroside as well).All three types of cells contain a much smaller number of lipid droplets, with sudano-phil and osmiophil externum and sudanophobe and osmiophobe internum; these aredispersed through the cytoplasm. Special 'perinuclear bodies', also binary in structure,are present in the collar cells and lateral oval cells.

Cells of the types described in this paper have not been found in other sub-classesof the Gastropoda, nor in the Basommatophora, but only in the pulmonate order,Stylommatophora. They appear to form an area of active neurosecretion in the re-tractile tentacles of these animals.

Contentspage

I n t r o d u c t i o n . . . . . . . . . . . . . 2 1 1M a t e r i a l a n d m e t h o d s . . . . . . . . . . . 2 1 2R e s u l t s . . . . . . . . . . . . . . 2 1 3

S t r u c t u r e of t h e optic t e n t a c l e . . . . . . . . . . 2 1 3' C o l l a r ' cells . . . . . . . . . . . . . 2 1 5

S p h e r o i d a l g r a n u l e s . . . . . . . . . . . 2 1 7Dispersed lipid d r o p l e t s . . . . . . . . . . . 2 1 8P e r i - n u c l e a r bodies . . . . . . . . . . . 2 1 8

' L a t e r a l o v a l ' cells . . . . . . . . . . . . 2 1 8' L a t e r a l p r o c e s s e d ' cells . . . . . . . . . . . 2 1 9

D i s c u s s i o n . . . . . . . . . . . . . 2 2 0R e f e r e n c e s . . . . . . . . . . . . . 2 2 6

IntroductionI T has been observed that the optic tentacles of certain Stylommatophoracontain a number of rather large, distinctive cells, arranged in a characteristicfashion. The long axon-like processes of these cells, and their disposition[Quarterly Journal of Microscopical Science, Vol. 103, part 2 , pp. 211-26, June 1962.]

212 Lane—Neurosecretory cells in the

round the periphery of the central tentacular ganglion, suggest a nervousorigin. These cells appear to be secretory, for they contain such a very largenumber of granules that the ground cytoplasm is almost totally obscured.

These 'neurosecretory' cells, first observed by Flemming in 1870, arousedat that time a controversy as to whether they were glandular or nervous.Flemming (1872) concluded, from a study of material treated with gold, thatthe cells were neurones. In spite of this, many investigators remained uncon-vinced of the nervous origin of the cells, a fact readily explicable by the lackof any obvious connexion between the cells in question and the centralganglionic mass. They do appear to be merely glandular upon superficialexamination.

Physiological experiments (Lane and Pelluet, i960, unpublished), carriedout with the slugs Arion ater and A. subfuscus, and involving removal of theoptic tentacles, indicated that certain of the neurosecretory cells in the optictentacles secrete a hormonal substance that acts on the ovotestis in such away as to inhibit the differentiation of the oogonia and/or to stimulate thedevelopment of the spermatogonia from the undifferentiated germ cells of thegonad. Certain experimental results suggested that this hormone might alsoaffect the growth of the ovotestis as a whole.

It was considered desirable to examine these cells in greater detail. Theobject of the present investigation was to study fixed preparations of the optictentacles of a pulmonate in order to determine the micro-anatomical arrange-ment, to define the cytological types by position and cytoplasmic inclusions,and to examine and compare their inclusions histochemically.

Materials and methodsThe chief material employed in this study was the optic or superior tentacles

of the common snail, Helix aspersa. This animal was used for detailed con-sideration instead of the slugs with which the above-mentioned experimentalresults were obtained because of their availability in winter when this in-vestigation was carried out. The optic tentacles of H. aspersa are similar instructure to those of the slugs.

The tentacles and head regions of other Stylommatophora were examined,including Agriolimax, Limax, Arion, Cepaea, Oxychilus, and Milax. OtherGastropoda studied were the prosobranchs Patella, Crepidula, and Buccinum,the opisthobranchs Archidoris and Greilada, and the Basommatophora Plan-orbis and Limnaea.

The histochemical tests applied and the other techniques used are listedin the Appendix (p. 224). The silver impregnation method of Golgi (Guyer,1949) for nerve-cell ramifications was employed. For this, a dichromate/osmium mixture was found to be preferable to formaldehyde for fixation.Holmes's (1947) method for axons was also used, with certain modificationssuggested by Bone (1961). The differences from Holmes's original procedurewere these: a solution of methanol, formalin, and acetic acid (80:10:10) wasused for fixation; incubation was carried out in the 1 % AgNO3 solution for

optic tentacles of certain pulmonates 213

3 days; and reduction in hydroquinone was prolonged to 30 min. Variousother silver impregnation techniques were also tried, with little success(Peters, 1955; Baginsky, 1957; and Alexandrowicz, 1951).

finger-likeprocess ofCigitate gang/ion

dermo-muscula.layer

FIG. I. Diagram of a longitudinal section of the optic tentacle of a stylommatophoranpulmonate, showing the position of the collar cells, the lateral oval cells, and the lateral pro-

cessed cells in relation to the rest of the tentacular structure.

ResultsStructure of the optic tentacle

Both the optic and the inferior tentacles of Stylommatophora in general,including H. aspersa, are retractile and inversible. Their structure has beendescribed by many earlier authors, including Flemming (1870) and Yung(1911). The innervation of the optic tentacle has been much studied byRetzius (1892), Samassa (1894), Hanstrom (1925), and others.


The eye, along with a bulbous central ganglion, is situated at the distal endof the extended optic tentacle, inside a dermo-muscular sheath. Internally tothis outer sheath, between it and the eye and ganglion, lie various muscularbundles, constituting the tentacular retractor muscles (fig. i).

The dermo-muscular layer consists of an outer epithelial sheet and an innermuscular wall. The outer epithelial layer is covered with a cuticle. This skinis very smooth and unfurrowed at the tip, above the eye and ganglion, butlaterally, down the sides of the tentacle, it is raised into warty protuberances,which are not caused by contraction of skin muscles, but are always present.These become natter near the tip of the tentacle. The retractor muscles,which are bundles of smooth muscle-fibres, are attached to the inner distalend of the tentacle (fig. 2, A).

The central ganglion and the eye lie at the terminations of the large tentacu-lar nerve and the smaller optic nerve respectively. These two nerves areseparate throughout their length, originating in the lateral portion of thecerebral ganglion, as Hanstrom has described fully (1926).

The tentacular ganglion is digitate, that is, it sends out finger-like processescomposed of nerve-fibres towards the tip of the tentacle (figs. 1; 2, A). Thesefibres run to bipolar sensory neurones of two sorts (Demal, 1955), which liebeneath, and send dendrites into, the epithelial layer of the tentacle tip.Neurones lying at the periphery of the digitate ganglion send small axonalprocesses into the central fibrous mass.

Surrounding this ganglion and the eye there is a partial 'collar' of cells,forming an incomplete ring round these central structures. These cells arelarger than the ordinary ganglionic neurones, and their ends are drawn outinto long processes, which run together into fibrous tracts that are continuouswith part of the digitate processes or other portions of the central ganglion.This would seem to indicate that they are true nerve-cells. However, theydiffer from the normal neurones not only in size, being larger, but also in thattheir cytoplasm is crammed full with many large spheroidal granules, as wellas other inclusions. Thus they appear to be glandular nerve-cells, and hencemight be regarded as neurosecretory.

FIG. 2 (plate), A, longitudinal section of the optic tentacle of the snail, H. aspersa. Zenker/Heidenhain's iron haematoxylin. e, eye; dg, digitate ganglion; cc, collar cells; rm, retractormuscles; gn, ganglionic neurones. The scale inscribed on c applies also to A.

B, pigment cells (pc) lying alongside the bodies and processes of collar cells (cc) in the optictentacle of H, aspersa. Helly/PAS. Note dark pigment granules and elongated portions of thecells; note also PAS-positive spheroid granules in the collar cells. (The pigment cells werefocused; the collar cells are not all in focus.)

c, longitudinal section of the optic tentacle of H. aspersa. Helly/PAS. Note processes fromthe collar cells (cc) running towards the epithelial surface (sur) branching and dividing on theway. (The tentacle was retracted when fixed; hence the lateral portions (Ip) are pulled upabove the tip, instead of lying beside the collar cells as is the case when the tentacle isextended.)

D, collar cells of H. aspera. Formaldehyde-calcium / Sudan black B. Note the processes (pr)of the various cells running into a common tract, and also the spheroid granules (gr) lyingwithin, along the length of these processes.


Rather similar cells are found lying laterally along the inner side of thedermo-muscular sheath, all the way from close below the tip of the tentacleto near its base (fig. 1). These cells are full of secretory granules. Their endsare drawn out into long processes. Because of their position far from thedigitate ganglion, no direct connexion with this ganglion is evident. However,connexions with nerve-fibres lying in the dermo-muscular area can be seen(fig. 4, A), for the drawn-out processes of these cells run into these fibrousnerve-tracts, and this suggests that they are neurones. Their dendritic pro-cesses run out towards the outer epithelial layer. These lateral cells can besubdivided into two separate groups, differing in shape and in the chemicalcomposition of their cytoplasmic inclusions.

The results of the histochemical tests are recorded in the Appendix (p. 224).It has been mentioned above that the secretory cells under consideration maybe divided into three categories according to position, size, shape, and inclu-sions, and this division is used in the Appendix.

Flemming (1870) described only two of these three types of cells in thetentacles of Helix. Besides the collar cells (Flemming's 'Z" cells, fig. i), he alsodescribed certain lateral cells 'Z', which here have been subdivided into twotypes, the lateral oval and the lateral processed cells.

Adjacent to all of these different cell types, around them, and along theirprocesses, lie pigment cells (fig. 2, B). These are long, thin, and branching.They contain black, dark brown, or yellow pigment granules. The proximityof these two cell-types—pigment and nervous—is reminiscent of the parsnervosa of the vertebrate pituitary. In an examination of a variety of the slug,Arion ater, that is colourless apart from the pigment in the retina, it wasobserved that, of the cells present, none resembled a pigment cell lackingpigment granules. Hence these cells were apparently absent, and, as theanimal was otherwise normal, they cannot be supposed to form an integralpart of the neurosecretory mechanism The facts suggest that pigment cellsshould not be considered as degenerating neurosecretory cells.

'Collar' cells

The collar cells are arranged singly or in groups round the central ganglion,as described earlier. They are oval or pyriform, and are sometimes compressedin such a way that they appear diamond-shaped in sections. Their ends aredrawn out into processes which often run alongside one another (fig. 2, D).They can, with difficulty, be seen to enter the nervous tract of the digitateganglion, or to merge imperceptibly into its outer layer of neurones.

These collar cells are bipolar, for their dendritic processes seem to extendinto the epithelial tip of the tentacle (fig. 2, c). These dendrites branch anddivide throughout their length and vary a good deal in diameter. In someareas they are threadlike and very thin; in others they are thicker and containspheroidal granules. So far as can be determined, they run to the surface ofthe tentacular epithelium, and here again the processes running parallelbetween the epithelial cells may be threadlike or may be wider and contain

2421.2 Q

2i6 Lane—Neurosecretory cells in the

granules. Possibly these different sorts of dendrites are in fact stages in thetransport of a secretory product.

The axonal processes of the collar cells are very thick for some distancefrom the axon hillock. The latter is often very elongated and the distinctionbetween hillock and actual axon is therefore difficult. These thick, granule-laden processes are often seen to lie side by side as they proceed from theircells of origin. After a certain distance they too show an inconstant diameter,

FIG. 3. Diagrammatic illustration of the nuclear and cytoplasmic inclusions of the collar cellsof H. aspersa,

becoming noticeably thinner, but in some cases still containing granules, andin others diminishing to threadlike proportions. Branching then occurs andthe thin processes can be seen to enter the ganglionic mass. Once here, theprocesses run into fibres present in the ganglion, or make contact with oneof the smaller ganglionic neurones lying within. At this point the granulesdisappear, and none are evident in the fibrous mass (Retzius's fibres mous-seuses).

The length of the body of these collar cells varies from about 30 to about70 /A. The nucleus is spherical or slightly elliptical, on the average about 12 /J.in diameter. It contains a great deal of chromatin in the form of threads,which stain heavily. It also includes two nucleoli, which may have smallerattached satellite nucleoli (fig. 3).

Mitochondria and Nissl substance cannot be demonstrated with certaintyin these cells by light microscopy on account of the sparsity of the cytoplasmand the consequent difficulty in distinguishing them amid the closely packed


granules. A study by electron microscopy is about to be undertaken and it ishoped that these points will be elucidated.

All parts of the cytoplasm are crowded with these spheroidal refractilegranules which appear to be secretion products. As mentioned earlier, thegranules lie not only within the cell-body itself, but also in the axonal pro-cesses (see figs. 2, D; 4, A).

Other inclusions are also present and visible after certain procedures,namely dispersed spheroids and peri-nuclear 'dictyosome' bodies. Thearrangement of all these cytoplasmic inclusions is shown in fig. 3.

Spheroidal granules. The spheroidal granules are made evident in certainpreparations by their very high refractive index. However, contrary toJobert's suggestion (1871), this is not due to the presence of calcareousmaterial. Their reactions to histochemical tests and various other proceduresare recorded in the Appendix (p. 224), as are those of all the other inclusionsto be described later.

Histochemical techniques indicate that these granules contain phospholipid,and imply that cerebroside may also be present. Although extraction with hotacetone does not lead to the negative PAS result, which would be expectedif cerebroside were at hand, this may merely signify that it is closely linkedwith a substance, probably protein, which is insoluble in hot acetone.

These granules are not readily dyed by acid or basic dyes; and when thecollar cells are stained, it often happens that only the cytoplasm takes up thedye, so that the appearance of a network is presented (as in fig. 4, D).

As far as can be seen by the present results, there is no positive evidenceof the presence of an)' other chemical constituent within these spheroidalgranules.

The granules vary in diameter from about 0-3 to 2-0 ju, which may suggestgrowth in addition to any qualitative change. The latter is indicated by theoccasional slightly mixed reaction of the droplets to any one test, a few drop-lets giving a different depth of positive coloration or a slightly more or lessintense reaction. This suggests cyclic secretory activity, as does the fact that

FIG. 4 (plate), A, lateral oval cell of H. aspersa. Brasil/Gomori's chrome-haematoxylin-phloxine. Note the processes (pr) of the lateral oval cells running towards the external surface(ext), and the presence of spheroid granules (gr) within these processes. Note also the lateralnerve-tract (tr) running at right angles past the cells and their processes. The actual junctionof the cells to the nerve-tract are not shown here. One axon (not shown) appears to originateat X and to run across to the tract.

B, lateral processed cells of Helix. Brasil/Gomori's chrome-haematoxylin-phloxinc. Notethe branching processes (pr) of the cells running to the exterior (ext), and note also the lateraloval cells (loc) lying just beneath the processed cells, their spheroid granules not staining.

c, collar cell of Helix. Mann-Kopsch. Note dispersed lipid droplets (did) scattered through-out the cytoplasm, showing strong osmiophilia; also note peri-nuclear bodies (pnb) in the vicinityof the nucleus.

D, collar cell of Helix. Kolatchev. Note network formed round osmiophobe spheroidgranules (gr).

E, collar cells of Helix. Mann-Kopsch. Note bodies (pnb) arranged around the nucleus, suchbodies being intensely osmiophil. The scale inscribed on A applies also to c, D, and E.

2i8 Lane—Neurosecretory cells in the

the cytoplasm at one end of the cell is sometimes vacuolated, and devoid ofgranules, these presumably having been somehow extruded.

Dispersed lipid droplets. Throughout the cytoplasm and scattered among thespheroidal refractile granules, are found various dispersed spheroids, rangingin diameter from 0-3 to 2-0 JJL. After certain procedures, such as osmium im-pregnation or coloration with Sudan black, they appear to be dictyosomes,in the typical form of rods, platelets, or spheres (see fig. 4, c, D). Thus theyare binary in structure, with a lipid externum and a non-lipid internum.Although these dispersed droplets are lipid, they do not contain phospholipidor cerebroside. Lillie's test (1957) indicates that protein is present.

When the spheroidal granules are dyed, for instance with acid fuchsine,these lipid droplets may be obscured and it is sometimes impossible to becertain whether they are present.

Gomori's neurosecretory stains are taken up by bodies with the same dis-tribution as the lipid droplets, and it is therefore possible that these droplets,or a part of them, form the visible and stainable neurosecretory product.

Some sort of cyclic activity may occur here, for gradations in intensity ofcolouring may be observed in these droplets, which is suggestive of alterationin, or accumulation of, products. There may also be a cyclic developmentbetween the spheroidal and these lipid droplets, the one differentiating intothe other. Also, aggregation may occur before extrusion of products, for thelipid droplets are occasionally seen to be clumped together (fig. 4, c).

Peri-nuclear bodies. In the area surrounding the nucleus, impregnation withosmium tetroxide, particularly by the Mann-Kopsch procedure, reveals large'dictyosomes', up to 3 [x, long (fig. 4, c to E). These I have called peri-nuclearbodies, to distinguish them from the smaller, dispersed lipid droplets. Thesebodies differ greatly from the dispersed lipid droplets in chemical composition,for only osmium impregnation produces a good demonstration of their struc-ture. Sudan black B colours them only very faintly, and Nile blue andSudan IV not at all.

Studies of living cells, to be carried out in the near future, will probablyindicate the true nature of these bodies in the living state.

The inclusions of these collar cells have not yet been studied at differentstages of development of the animal. Examination of animals at various levelsof body-growth, as well as at different stages of ovotestis differentiation, willbe carried out in order to determine if any definite cyclical secretory activityis taking place.

'Lateral oval' cellsThe lateral oval cells (fig. 4, A) are very similar to the collar cells in shape

and sorts of inclusions, but differ in their distribution, histochemistry, andsize-range. As described earlier, they are situated laterally down the sides ofthe dermo-muscular sheath of the tentacle, and are usually oval or pyriform.These cells are somewhat smaller than the collar cells (generally 20 to 30 JJ.long).


They are apparently neurones, for, as mentioned earlier, their axonal pro-cesses can be seen to run into the fibrous nerve-tracts which are found in thetentacular wall, having branched off from the tentacular ganglion. Granulescan be seen in these axons up to the point of their actually entering the nerve-tract. The dendritic processes of these cells lead out in the direction of theexterior, extending to the epithelium of the dermo-muscular layer. They arevery thick where they leave the neurone, and gradually taper down in diameter.However, they do not appear to attain threadlike proportions, as far as canbe seen, but maintain a considerable width, and contain granules throughout.

The cytoplasmic inclusions of the lateral oval cells are similar to those ofthe collar cells, except that the spheroidal granules are markedly different inchemical composition. These do not contain phospholipid or cerebroside, buta faint sudanophilia indicates the presence of some form of lipid. They canbe coloured by Rawitz's 'inversion' technique. These spheroidal granules alsodiffer from those in the collar cells in that they take up iron haematoxylin andcertain other basic dyes, notably safranine and toluidine blue.

The dispersed lipid droplets in the lateral oval cells are the same as thosein the collar cells except that the acid haematein test gives a positive reaction.Again, as in the collar cells, it is often difficult to distinguish these lipiddroplets when the spheroidal granules are stained heavily, as with ironhaematoxylin.

The peri-nuclear bodies are similar in distribution and reactions to those inthe collar cells, and the ground cytoplasm gives very much the same reactionsas that of the collar cells, except that it takes up some dyes more strongly.

I have called these cells lateral oval cells, because their shape serves usuallyto distinguish them from the lateral processed cells. Both kinds of cells haveprocesses, but these are usually much more striking in the 'processed' thanin the 'oval' cells. This is perhaps mainly due to the different staining pro-perties of the granules contained in the processes, which cause those in thelateral processed cells to colour more intensely, thereby vividly tracing thepath of their processes to the exterior.

' Lateral processed' cells

These cells are so called in order to differentiate them from the lateral ovalcells, the distribution of which is very similar. They differ from the lateraloval cells histochemically, and also in shape and size. These lateral processedcells are somewhat irregular in shape, more elongated, and smaller than thelateral oval cells. Their cell-body measures 10 JJ. or more in width, and theyhave thick processes which are as full of inclusions as are the cells themselves.The shape of these granules in fixed preparations varies from spheroidal tonearly rectangular or cubical.

The lateral processed cells seem to have the same axonal connexion withthe fibrous nerve-tracts as the lateral oval cells, and their dendrites can beseen running to the edge of the dermo-muscular sheath (fig. 4, B). Theseprocesses branch and divide, but maintain their granular component and are


fairly wide throughout their course. They run between and parallel to theepithelial cells to the outer side of the sheath, where they terminate justbeneath the cuticle.

Histochemically, these granules are similar to those of the collar cells, withcertain exceptions. They are PAS-positive and also strongly chromotropic;this suggests the presence of acid muco-polysaccharides. They react positivelywith Rawitz's 'inversion' staining method. Phospholipid may perhaps bepresent, for they give a positive reaction to the acid haematein test bothnormally and with the pyridine extraction control, and they are also colouredblue with the oxazine of Nile blue.

Heidenhain's iron haematoxylin is strongly taken up by these granules, incontrast to those of the collar cells, and they also take up certain basic dyesintensely. Another definite difference from those in the collar cells is thatwhen stained with the commonly accepted 'neurosecretory' stains of Gomori,these inclusions are far more intensely dyed.

These lateral processed cells contain only dispersed lipid droplets, in con-trast with the other two cell-types, for no peri-nuclear bodies can be seen.It is impossible to distinguish these lipid droplets after certain stains, suchas Gomori's, PAS, and acid haematein; but it may be that they are hidden bythe intense coloration of the spheroidal granules.

Studies of the reactions of all these cells with vital dyes have not yet beencompleted. These are now being carried out, and, when finished, shouldprovide a more accurate description of the true state of the various inclusionsin these cells, particularly the dispersed droplets and peri-nuclear bodies.

DiscussionThese cells, as briefly mentioned earlier, were first observed by Flemming

in 1870, who commented on their strange combination of glandular andnervous features. This is perhaps one of the earliest references ever made tothe subject of neurosecretion. Jobert, in 1871, also observed these cells anddecided that they were specifically glandular. One year later Flemming pub-lished a second paper in which he claimed that by using a gold method, hehad shown that the collar cells were nervous in origin. The lateral cells healso concluded to be ganglion cells and he believed them to be peripheralnervous centres of the nerves and ganglion concerned—by which presumablyFlemming meant the tentacular ganglion and nerves. Simroth (1876) assertedthat the cells were purely glandular, but Retzius (1892) described and drewthe collar cells as ganglionic neurones. Yung (1911) described them as large'cellules conjonctives', and suggested a lubricating action as their function.Demal (1955) briefly noted their presence in certain pulmonates, referring tothem as secretory cells.

Thus from the moment of their discovery these cells have aroused interest,and they have provoked discussion from time to time. The main questiontoday is whether they can properly be regarded as neurosecretory.


Neurosecretion is generally understood to occur when a nerve-cell showscytological features indicative of a glandular function; that is, when a neurone,early in its differentiation, acquires the characteristics of a gland cell. In mostcases the nervous nature of the cell is still evident, as the axonal process isseen to be in connexion with the central nervous system. Specialization, how-ever, may lead to the loss of various nervous characteristics. Certain cells,definitely nerve-cells in origin, have, in the course of development, changedfrom typical neurones into glandular cells, lacking any nervous propertieswhatsoever (Scharrer and Scharrer, 1937).

In the cells under consideration here, certain preparations show processesof collar cells running into a fibrous tract which enters the central tentacularganglionic mass. Silver methods for axons show fibres running from the collarcells to this digitate ganglion. These fibres can also sometimes be seen inmaterial fixed in formaldehyde and coloured with Sudan black or various dyes.Hence it seems unquestionable that these collar cells are nerve-cells. It maybe that, after a nervous origin, a subsequent differentiation occurred, causingtheir alteration to a glandular type. The term 'neurosecretory', as defined bymost investigators, seems to be applicable to the collar cells of Helix.

The lateral oval and processed cells, which also have dendritic processesrunning from the external surface of the dermo-muscular layer, are obviouslyfarther removed from the central digitate ganglion, and no direct innervatingconnexion with the ganglion has yet been demonstrated. However, smalltracts of nerve-fibres have been stated to exist in these areas and connexionsare present between these nerve-fibres and the lateral secretory cells. Agersborg(1925) has described, in the oral tentacles of certain nudibranchs, cells show-ing a general similarity to the lateral processed cells. They are innervated bysmall nerve-fibres at the base of the dermo-muscular layer. A situation thatis in some respects comparable may be present here.

It is likely that the cells described in the present paper are not merelyinnervated cells, but actual nerve-cells; for Veratti (1900) described in Helixlateral sub-epithelial 'ganglion cells' which gave rise to intra-epithelial nerve-endings. These nerve-endings he found in association with the sense cells(Sinneszellen) of the epithelial sheath, but he was unable to determine whetherthe sub-epithelial processes arose from the sense cells or were merely attachedto them as free nerve-endings would be. However, the manner in which hedescribes the processes which run to sub-epithelial ganglion cells, leads oneto believe that these cells may be the same as the lateral processed cellsdescribed here. Retzius (1892) also mentions the presence of lateral nerve-cells in Helix, similar to those in question here, and describes the sparse con-nective tissue of the tentacles as being functionally involved in bindingthese nervous swellings to the muscular sheath.

Thus it may be that during differentiation, alterations have been initiated,and development has progressed towards a glandular appearance, withconcurrent loss of certain salient nervous features, but maintenance of con-nexions with the fibres within the ganglion.


Neurosecretory cells are often arranged as 'neurohaemal' organs, with asingle or many secretory neurones or terminations of neurosecretory fibrescollected near a blood-vessel or sinus. A definite terminal organ may some-times be formed, to which a neurosecretory pathway runs from a neuro-secretory centre, as in the intercerebralis / corpus cardiacum system of insects,and the X-organ / sinus gland arrangement in Crustacea. In the case underconsideration here, no terminal organ resembling the corpus cardiacum orsinus gland has been observed, but blood-vessels do extend into the optictentacles, dividing and branching throughout. Also, part of the haemocoelicsinus is here present, ostensibly aiding in extension of the tentacles, butperhaps also serving another function.

The neurosecretory product, then, in this case, might be extruded into theblood-stream or body-fluid from these cells, which must behave like merocrineglands, since no remnant of any destroyed cell has been observed. Dropletshave been seen in the dendritic and also in the axonal processes, presumablyin the course of movement. However, it does not appear that the productmoves back as far as the cerebral ganglion, for severing the tentacular andoptic nerves leads to no accumulation of secretory products at the woundsurface, nor do the physiological experiments carried out earlier (Lane andPelluet, i960) support this view. Hence the secretory product must movedirectly into the blood-stream or body-fluid from the tentacle; either fromthe cells themselves, or from the tentacular ganglionic mass, to which it maymigrate.

It has been mentioned in various reviews on neurosecretion (Gabe, 1954;Scharrer and Scharrer, 1954) that the secretory product very often can onlybe observed as far as the end of the axon, and often vacuolation occurs in thecytoplasm, after intra-cytoplasmic 'redissolution'. This implies extrusionof the neurosecretion in dissolved form. This may perhaps be the processinvolved in the cells described in the present paper.

A preliminary survey of the distribution of these neurosecretory cells withinthe class Gastropoda has shown that they are not present in the head ortentacular regions in representative members of the Prosobranchiata or theOpisthobranchiata examined. The order Basommatophora of the Pulmonataalso lack these cells. Further studies will be made of more representatives tocheck this. However, all the Stylommatophora so far investigated, comprising7 genera, contain these cells, not only in the optic tentacles, but also in thesmaller, inferior tentacles. The kinds of presumably neurosecretory cells inthe inferior tentacles appear, upon preliminary investigation, to be similarto those in the superior or optic tentacles, except that the collar cell of theinferior tentacle is smaller and the histochemical reactions of its granules aresomewhat different.

A consideration of the literature, and an examination of the whole bodyhistologically, appear to indicate that although there are some cells showinga superficial resemblance to the three cell-types described here, there are noexactly similar types.


The Leydig cells {Plasmazellen of Brock, 1883) and Mast cells found in theconnective tissue of gastropods, which have been described by various authors(Leydig, 1865; Semper, 1857; Cuenot, 1892; Brock, 1883; Bergonzini, 1891;Ballowitz, 1891; and Kisker, 1924), are somewhat like the lateral oval cells,but the former two types do not have nervous processes and the chemicalcomposition of the inclusions is dissimilar.

The cells of the pedal mucous gland of Stylommatophora are similarmorphologically to the lateral processed cells, as are the 'ganglion cells' ofVeratti (1900) seen on the lateral tentacular walls of Helix. The oral tentacularcells of certain nudibranchs, described by Agersborg (1924), are also rathersimilar to the lateral processed cells discussed here.

The collar cells are the most likely of the three types to produce a hormoneconcerned with the cytodifferentiation of the ovotestis (Lane and Pelluet,i960). I have not found any cells comparable to these in any other part of thebody, nor have other workers described such cells elsewhere. It would there-fore appear that these neurosecretory cells are characteristic of and restrictedto the tentacles of Stylommatophora.

I am greatly indebted to Dr. J. R. Baker, F.R.S., for his supervision andconstant encouragement during the course of this work. I wish to thankProfessor A. C. Hardy, F.R.S., for accommodation in his Department, inwhich this investigation was done.

This research was carried out during the tenure of an Overseas Scholarshipfrom the Canadian branch of the Imperial Order of the Daughters of theEmpire.

Appendix

A summary of the histochemistry of the inclusions of the three cell-types in the optic tentacles of H. aspersa

Test

Xylidine red (0-5% aq.)Eosin(01% aq.)Orange G (0-5% aq.)Methyl blue (0-5% aq.)Acid fuchsine (20% in sat.

aniline solution)Basic fuchsine (0-5% aq.)Crystal violet (0-5% aq.)Safranine(0-5% aq.)Dahlia (0-5% aq.)Methyl green (1% aq.)MetznerNissl stain (toluidine blue,

1 % aq.)

Toluidine blue after sulpha-tion

Metachromasia (toluidineblue, l%aq.)

PMGPMG after trichloroacetic

acidFeulgen

RawitzKolatchevMann-Kopsch (Weigl)Silver impregnationPASPAS after salivary amylase

Tests appliec

Ze3 hCaZe3 hZe 3 hHe

Ze 3 hZe 3 hCaCaCaAlt; He + PCCa

a

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Reference

———.—.

—————

Metener and Krause (1928)Young (1932)

Bignardi(1940)

Baker (unpublished)

Jordan and Baker (1955)Pearse (1954)

Feulgen and Rossenbeck

(1924)Przei?cka(1959)Kolatchev (1916)Weigl (1910)Aoyama(1929)McManus (1948)Pearse (1954)

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226 Lane—Neurosecretory cells of certain pulmonates

References

AGERSBORG, H. P. K., 1925. Acta. Zool. Stockholm, 6, 167.ALEXANDROWICZ, J. S., 1951. Quart. J. micr. Sci., 92, 163.AOYAMA, F., 1929. Z. wiss. Mikr., 46, 489.BAGINSKY, S., 1957. Bull. Micr. appl., 7, 19.BAKER, J. R., 1944. Quart. J. micr. Sci., 85, 2.

1947. Ibid., 88, 115.1949. Ibid., 90, 293.1956a. Ibid., 97, 161.19566. Ibid., 97, 621.

BALLOWITZ, E., 1891. Anat. Anz., 6, 135.BERGONZINI, C , 1891. Ibid., 6, 595.BIGNARDI, C, 1940. Boll. Soc. ital. Biol. sperim., 15, 594.BONE, Q., 1961. Personal communication.BROCK, J., 1883. Z. wiss. Zool., 39, 1.CASSELMAN, B. W. G-, and BAKER, J. R., 1955. Quart. J. micr. Sci., 96, 49.CAIN, A. J., 1947. Ibid., 88, 383.CLARK, R. B., 1955. Ibid., 96, 545.CUENOT, L., 1892. Arch, de Biol., 12, 683.DANIELLI, J. F., 1946. J. exp. Biol., 22, no .DEMAL, J., 1955. Acad. Roy. de Belgique, 29, 1.FEULGEN, R., and ROSSENBECK, H., 1924. Z. physiol. Chem., 135, 203.FLEMMING, W., 1870. Arch. mikr. Anat., 6, 439.

1872. Z. wiss. Zool., 22, 365.GABE, M., 1954. Anne'e biol., 30, 6.GATENBY, J. B., and BEAMS, H. W., 1950. The microtomist's vade-mecum, n th edition.

London (Churchill).GOMORI, G., 1941. Amer. J. Path., 17, 395.

1950. Amer. J. Clin. Path., 20, 665.1952. Microscopic histochemistry. Chicago (University Press).

GUYER, M. F., 1953. Animal micrology, 5th edition. Chicago (University Press).HANSTROM, B., 1925. Acta. Zool., 6, 183.

1926. Zool. Anz., 66, 197.HERXHEIMER, G. W., 1901. Dtsch. med. Wschr., 36, 607.HOLMES, W., 1947. Recent advances in clinical pathology. London (Churchill).JOBERT, 1871. J. de l'Anat. et de la Phys., 2, 611.JORDAN, B. M., and BAKER, J. R., 1955. Quart. J. micr. Sci., 96, 177.KEILIG, I., 1944. Virchows Arch. Path. Anat., 312, 405.KISKER, L. G., 1924. Z. wiss. Zool., 121, 64.KOLATCHEV, A., 1916. Arch, russes d'Anat. d'Hist. d'Embriol., 1, 383.LANE, N. J., and PEIXUET, D., 1961. Results not yet published.LEYDIG, F., 1876. Arch. Naturg., 42, 209.LILLIE, R. D., 1957. J. Histochem. Cytochem., 5, 528.LISON, L., 1953. Histochimie et cytochimie animates, Paris (Gauthier-Villars).METZNER, R., and KRAUSE, R., 1928. In Abderhalden's Handbuch der biologischen Arbeits-

methoden, Abt. 5, Teil 2, 1 Halfte, 325. Berlin (Urban und Schwarzenberg).MCMANUS, J. F. A., 1948. Stain. Tech., 23, 99.PEARSE, A. G. E., 1954. Histochemistry, theoretical and applied. London (Churchill).PETERS, A., 1955. Quart. J. micr. Sci., 96, 323.PRZEEECKA, A., 1959. Ibid., 100, 231.RETZIUS, G., 1892. Biol. Unters. Neue Folge, 4, n .SAMASSA, P., 1894. Zool Jagrb., Abt. Anat., 7, 593.SCHARRER, E., and SCHARRER, B., 1937. Biol. Rev., 12, 185.

1954- 'Hormones produced by neurosecretory cells', a chapter in Recent progress inhormone research, vol. 10, p. 183, edited by G. Pincus. New York (Academic Press).

SEMPER, C, 1857. Z. wiss. Zool., 8, 340.SIMROTH, H., 1876. Ibid., 26, 227.VERATTI, 1900. Memorie del Reale Istituto lombardo di Scienze e Lettere. 18, Fasc. 9.

Quoted from SMIOT, H., 1902. Anat. Anz., 20, 495.YOUNG, J. Z., 1932. Quart. J. micr. Sci., 75, 1.YUNG, E., 1911. Rev. Suisse Zool., 19, 338.

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