the autonomic (enteric) nervous system of amphioxus ... · about the sympathetic nervous system of...

The Autonomic (Enteric) Nervous System ofAmphioxus Lanceolatus.

By

Prof. Dr. J. Boeke,Director of the Embryological and Hiatological Laboratory,

University of Utrecht.

With 16 Text-figures.

THE present paper contains in a more detailed and betterillustrated form the same facts as were described in a shortcommunication in the 'Proceedings of the Royal Academy ofScience of Amsterdam' of the year 1933 (vol. xxxvi, No. 10,December 1933).

About the sympathetic nervous system of A m p h i o x u svery little is known, common opinion among scientists beingthat A m p h i o x u s does not possess any sympathetic nervesat all in the sense of the sympathetic plexus of the highervertebrates. The dorsal roots send out rami dorsales and ramiventrales. From these rami ventrales branch off delicate ramiviscerales, running between the myotome and the pterygialmuscle; they divide into a ramus descendens and a ramusascendens. According to Legros (1902, 1910) the post-branchialrami ascendentes run inside the transverse septa, pass to themembranes covering the wall of the enteric canal and followthe course of the veins, running along the dorsal wall of theliver. Heymans and Van der Stricht already noted in 1893, inGolgi preparations, apparent nerve-fibres on both the parietaland visceral mucosa faces posterior to the abdominal pore, butthey were unable to find the origin of these nerves or to tracetheir course. They might come from the abdominal visceralnerves of Hatschek. They also noted in Golgi preparationsnerve-fibres supplying the intestine, and branches from thedorsal nerves of the anal region of the left side of the bodysupplying the anus. Dogiel (1902) described a delicate nerve-plexus in the wall of the rectum and the anus. Miss HarrietKutchin in 1913 described from, methylene blue preparations a

624 J. BOEKB

delicate nervous plexus on the walls of the gonadial pouches, inthe primary and secondary branchial bars, and on some of thelarger blood-vessels. Just as Dogiel she saw a few ganglioncells, and suggests too that further investigation may reveala visceral plexus with multipolar nerve-cells (I.e., p. 603).

Vascular nerves were also described by Howard Ayers (1921),who maintains that A m p h i o x u s possesses ventral roots,which are 'relatively as extensive as in the higher forms, i.e.run to the extreme limit of the muscle-organ to which they areassigned and are long branching nerves, not a brush of fibres'(I.e., p. 162). On a previous page (p. 156) Ayers gives the follow-ing description of his observations: ' The motor roots have beenthought to belong exclusively to the striated muscles of themyotomes, but there is a small bundle of fibres, which runs outwith the large bundle 3, but which does not enter the myotome,passing along its inner face, to join the nerve plexus of theaortic trunk. It is a splanchnic nerve.' On the following pageAyers describes this small bundle of fibres as running into thebody to join the nerve-plexus associated with the centralvascular mechanism (I.e., p. 157). Thus, as to the points inter-esting us here, the splanchnic nervous system described byAyers is the same as was described by Legros in 1910, andindicated already by Heymans and Van der Stricht. The viewsof Legros were accepted by Franz in his extensive monographof 1927, but although Franz accepts the existence of ramiviscerales even of the post-atrioporic nerves, running to thevisceral wall, he states that he never could see a nerve-fibre ora ganglion cell on the endodermal visceral wall; apparentlyFranz does not know the paper by Miss Kutchin. According tohim the ganglion cells described by Van Wijhe in 1913, whichare mentioned below (on p. 626), which were to be seen in amicro-photograph sent him by Van Wijhe, were nothing butmotor endings of the common spinal type on the atrial muscle-fibres. In this Franz is certainly in error, as will be discussedin the following pages.

As already mentioned, neither Dogiel (1903) nor Heymans andVan der Stricht (1898) had been able to find real ganglion cellsin the dorsal roots or peripheral sympathetic ganglion cells, and

NERVOUS SYSTEM OF AMPHIOXUS 625

because of these statements Franz entirely denies the existenceof a real' sympathetic' of the viscera. I may cite the followinglines of his monograph: 'paraspinale Ganglien reiner Einge-weidenerven oder aber ein Grenzstrang fehlen dem Akranier,und so sind seine Eingeweidenerven so wenig " sympathisch"im morphologischen Sinne wie die viscerale Verzweigung desVagus bei einem Kranioten' (I.e., p. 528).

It may be said in conclusion that the question of the presenceof an autonomic nervous system in A m p h i o x u s is stillundecided, and that most authors seem to believe that it doesnot exist. Thus it seems to me that my original statement istrue, that we know very little about the sympathetic system ofAmphioxus.

And this is the more interesting because, although about theancestry of A m p h i o x u s nothing definite is known, we maybe sure that this animal stands at the bottom of the chordates,between the vertebrates and the invertebrates, and all thevertebrates possess a distinct and well developed sympatheticsystem, whilst among the invertebrates in different classes awell developed and ganglionated sympathetic nervous systemis to be found (Heider, Hanstrom, Keim, Nillson, Sanchez,Patten, Carlson, Alexandrowicz). Thus it would be improbablethat in A m p h i o x u s , standing between those two groups,there is no sympathetic system at all.

With the exception of Dogiel and Miss Kutchin only Van Wijhedescribed real ganglion cells, and these in a cursory way, for ina footnote to his paper on the metamorphosis of A m p h i o x u s ,which he published in the ' Proceedings of the Eoyal Academyof Amsterdam' in 1913, he only mentions that he found under-neath the atrial epithelium covering the liver and the post-branchial prehepatic intestine (the' oesophagus') a great numberof beautiful multipolar ganglion cells, which sent their axiscylinder through the dorsal nerve-roots to the spinal cord.

Van Wijhe never published anything more about theseganglion cells, but at my request he has now sent me a micro-photograph of one of his preparations, which is reproduced herein Text-fig. 1, and in a letter accompanying this photographVan Wijhe wrote the following statement (November 16, 1933):

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626 J. BOEKE

'On the sympathetic in A m p h i o x u s I never published any-thing, but I possess preparations, in which neurones are seenunder the atrial epithelium covering the oesophagus and theliver. The ganglion cells show two or three short dendrites,

TEXT-FIG. 1.

Microphotograph by Van Wijhe, showing the stellate ganglion cellson the wall of the intestine of a full-grown A m p h i o x u s . Biel-schowsky preparation.

and a long axis cylinder (neurite) is seen ascending into thedorsal (septal) nerves. The ganglion cells are similar to thesympathetic cells described in P e t r o m y z o n , but theirposition directly underneath the atrial epithelium suggests thatthey are analogous to the ganglion cells which form the first twoseptal (dorsal) nerves under the dorsal covering of the rostrum.In no case can they be motor, because there are no muscle-fibres in the neighbourhood.' For a discussion of this last state-ment of Van Wijhe I can refer to the following pages.

NEKVOUS SYSTEM OF AMPHIOXUS 627

The cells described by Van Wijhe, which are to be seen in thephotograph of Text-fig. 1, with their long axis cylinders, I knowfrom my own Golgi preparations, in which a few were impreg-nated, but I must confess that I had not dared to identify themwith sympathetic elements. Yet, after having studied thephotographs and the description of his findings, I am sure thatthe cells mentioned by Van Wijhe are real sympathetic cells,and that Franz is entirely wrong when he thinks that theelements shown in the photograph are merely motor end-plateson the muscle-fibres of the atrial muscle (Musculus trapezius,Schneider, Legros). The motor end-plates (compare the drawingsof them in the ' Anatomischer Anzeiger', vol. 33, 1908) havequite another aspect than the elements impregnated in thephotograph of Van Wijhe (cf. p. 656).

For a series of splendidly impregnated Bielschowsky prepara-tions, longitudinal sections and cross-sections through adultanimals, revealed a quite astonishing abundance of stellatemultipolar nerve-cells on the wall of the liver and of the ad-joining parts of the intestine, the so-called 'oesophagus' andthe post-hepatic part of the intestine, often wrongly called the' stomach' but only comparable to the middle intestine of thevertebrates. These preparations were made years ago, and Ihave known these cells for more than twenty years, but I neverpublished anything about them, because I knew too little aboutthe comparative microscopical anatomy of the sympathetic inthe lower vertebrates to classify them rightly. It was only thestudy of the sympathetic system of the last three or four yearswhich enabled me, as I presume, to establish their true nature.

The complex nature of the enteric plexus itself is well shownin Text-fig. 2.

In it is drawn as accurately as possible from the preparationwhat a thin (15 ft) tangential section through the intestinal wallreveals of this plexus. Of the ganglion cells present in thesection only one was cut so as to enable the artist to draw itaccurately (Text-fig. 2, Gz.).

The form of the ganglion cells themselves, and the neuro-fibrillar structure of the cells and of their processes is well shownin Text-figs. 3-6. It is very clear and distinct. Coarser fibrillae

m.fG.z.-^

EXT-FIG. Z.

The enteric plexus of an adult A m p h i o x u s , as shown in a thinlongitudinal tangential section through the wall of the intestine.Bielschowsky preparation.


run through the cell-body from one process to another, butinside the cell-body they are everywhere connected by a number

rO

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TEXT-FIG. 3.

TEXT-FIGS. 3-6, Camera lucida drawings of the elements of thenervous plexus covering the wall of the liver and intestine of afull-grown A m p h i o x u s . Bielschowsky preparation, treated withchloride of gold, and stained weakly with haematoxylin.

of finer fibrillae forming a distinct network. The nucleus islarge, contains a large nucleolus and only a small amount ofchromatic substance.

630 J. BOBKE

The processes leave the cell-body in every direction, and theneurofibriUar network of the cell-body is continuous with that

-20

-30

-40

of the processes; in several places the processes themselves arebroader, flattened out, and in these places, as is shown in thefigures, the very delicate neurofibrillar network is as conspicuousas inside the cell-body. In short, the multipolar cells showexactly the same neurofibrillar structure as the nerve-cells ofthe spinal cord of A m p h i o x u s as it was described amongstothers by me in the 'Proceedings of the Royal Academy,'Amsterdam (April 1902, p. 695), the only difference being that,as a rule, the neurofibrillar structure of the multipolar cells


described here is finer and more delicate than that of the nerve-cells of the spinal cord. But it seems to me that there cannot

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TEXT-FIG. 5.

be the slightest doubt but that we have to do here with realmultipolar nerve-cells of the same character as the sympatheticnerve-cells of the vertebrates.

A difference between neurites and dendrites was not to befound, the processes of the cells being all of them of the samestructure and pattern, as is clearly shown in the figures accom-panying this paper. Even in this they resemble closely the

632 J. BOBKB

sympathetic cells of the higher vertebrates, in which accordingto Stoehr (1928, 1932) in most cases all the processes are so

TEXT-FIG. t>.

entirely of the same pattern, that there is no distinction betweena long axis cylinder, a neurite, and branched dendrites. In somecases, however, I found on the wall of the liver and of the post-hepatic small intestine bipolar cells with two processes running


in opposite directions (nearly in a longitudinal direction) alongthe wall of the liver, which cells showed exactly the same form

TEXT-FIG. 7.

Three bipolar cells from the wall of the intestine and of the liver of anadult Amphioxus . Bielschowsky preparation.

as the cells photographed by Van Wijhe, reproduced in Text-fig. 1. In Text-fig. 71 have drawn some of these cells from the wallof the intestine and of the liver as carefully as it was possiblefrom my preparations to show the details; but whether theyreally represent a different type which could be compared withthe second type of Dogiel (compare the papers by Lawrentjew

634 J. BOEKB

and Van Esveld for the details of this type of sympatheticcell) in the plexus of Auerbach of the intestinal Avail of thehigher vertebrates, is difficult to say. In the thin sections whichI had at my disposal, the form of these ganglion cells could onlybe studied where the wall of the intestine was cut tangentially.It is always possible that several of the longer processes mayhave been cut away, as was certainly the case with the thirdcell (to the right) drawn in Text-fig. 7. But I took care to drawonly those forms which had a shape apparently so deviatingfrom the common forms drawn in the foregoing figures, thatwe may suppose that they really represent a different type.That is what was meant by the drawings of Text-fig. 7. Atpresent we cannot go any further. That they resemble theforms shown in the photograph of Van Wijhe is apparent.

An interesting feature of many of these cells is, as alreadymentioned, that in several places the thin cell processes arebroader, flattened out to an exceedingly thin membrane witha very delicate neuroflbrillar network, which condenses im-mediately afterwards again to the thin thread-like cell processes.The same feature was described by Lawrentjew (1929) andafterwards by Stoehr (1932) for the sympathetic ganglion cellsof the mammalian intestine ('Dendritlamellae', Lawrentjew),and it may be an argument for the classification of the elementsof A m p h i o x u s , described here, as sympathetic ganglioncells (cf. Text-figs. 9, 4, 6).

The processes themselves may end in ring-like or reticulatedsmall endings, which seem to lie freely in the tissue surroundingthe cells; at least any connexion with the surrounding elementscould not be distinguished in these cases; but it remains alwayspossible that there was a break in the impregnation, the ring-like or reticulated endings being in all the cases studied verysmall and only weakly stained.

The ganglion cells described above are very numerous and aredistributed very regularly in a thin layer on the wall of the liver,the oesophagus, and the post-hepatic middle intestine; they forma regular plexus, the cell processes extending in the thin sheathcovering the wall in every direction (viz. always in the samehorizontal plane), and intertwining to form plexiform bundles,

NEEVOUS SYSTEM OF AMPHIOXUS 635

as is shown in the figures. As far as I could gather from thestudy of the sections, with the exception of the few rarecases mentioned above, the processes of the multipolar cellsanastomose freely with each other; but, because I could studyonly thin sections, from 10 to 6/x, it was impossible to settlethis point definitely and without doubt, most of the anastomosesapparently being severed in the thin sections.

With regard to these anastomoses we have to distinguishbetween the connexions between the processes of the differentcells and the connexion of the cell processes with the coarsernerve-fibres running in bundles between the ganglion cells, thusbuilding up the enteric plexus itself (Text-fig. 2).

As to the second mode of synaptic contact, we find everywheresynaptic connexions between the cell processes and the coarsernerve-fibres running between the cells, of which more later on.In Text-fig. 8 are drawn as accurately as I could from the pre-parations several examples of these synaptic connexions. Wesee the dendrites of the cells (Text-fig. 8, d.) running towardsthe nerve-fibres of the plexus, bending alongside of them andforming synaptic junctions, .which present all the features ofa true syncytial connexion. They resemble strongly the picturesgiven by Bozler (1927, 1928), of the connexions and synapticjunctions between the nervous elements in R h i z o s t o m aand by Young (1933) of the synaptic connexions between thesympathetic cells and post-ganglionic nerve-fibres in Selachians.In the post-branchial plexus of S c y l l i u m c a n i c u l a Youngmade the following observation, which I may be allowed toquote from his paper: 'at the nodes of the post-branchial net-work there are often single cells which are not enclosed incapsules. Their dendrites make contact with neighbouringfibres without the latter terminating. In such cases it couldclearly be seen that there is no actual continuity of substanceat the synapse, since the dendrites were stained yellow-brownand the passing fibres an intense black' (I.e., p. 600).

Although in my opinion the cells of the enteric plexus ofA m p h i o x u s may be compared with the ' single cells' describedby Young, as to the question of the synaptic connexions Icannot entirely agree with him. I could study these synaptic

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TEXT-FIG. 8.

Post-branchial plexus with synapses between dendrites and nerve-fibres. Adult A m p h i o x u s . sy., synapses; d., dendrites;gc, ganglion cells; nf., nerve-fibres.

connexions in the enteric plexus of Amphioxus in thinsections with the highest power (cf. Text-fig. 8), and I am con-vinced that we have to do here with a true syncytialsynaptic connexion. In the first place, even when studiedwith the highest power and as accurately as possible, there


appeared to be not only a close contact between the two fibres,but a real continuity of substance and especially of the neuro-fibrillar substance, even when the staining of the two parts ofthe synapse is a little different. Different staining (yellow-brownand intense black in the common Cajal preparations, grey andblack in the preparations treated with chloride of gold, as inText-fig. 8) cannot be regarded as a conclusive argument fora different origin of the neurofibrillar strands stained. Howoften we find a ganglion cell with its dendrites stained yellow-brown, whilst the neurite is stained intensely black; and whenwe follow this neurite until it fuses with the cell-body, all atonce the black stain changes into a light yellow-brown.

We must be very careful in drawing conclusions about thesynaptic conditions from what we see in our silver preparations.It is possible that the impregnation with silver-salts dependslargely on the functional activity of the element in question,as does, for instance, the capacity for adsorbing rhodalit-white,used by Bozler in R h i z o s t o m a . Here it is still more difficultto reach a trustworthy conclusion, because the stain cannot befixed, and can only be studied for a few minutes! (Bozler). InK h i z o s t o m a , according to Bozler (I.e., p. 255), in some casesboth bipolar and multipolar ganglion cells were stained in thesame way, in other cases only the multipolar cells took the stain.When using silver-salt preparations we are not allowed to decreethat a different staining must depend on a different origin of thefibres. As I tried to demonstrate some years ago in a shortpaper in the ' Proceedings of the Eoyal Academy of Amsterdam'(1929), it is impossible to believe that the synapses betweenthe ganglion cells are built up of dead matter, of a 'stickysubstance', cement substance, as maintained by the neuronists,such as Cajal ('ciment unitif, une substance intermediaire,granuleuse ou vacuolisee', Cajal, 1908) and many others, andalso Windle and Clark (1928) for the end-bulbs of Auerbachon the surface of the cells, &c. How it would be possible to seein the synapse a connexion of high physiological importancewith varying physiological properties, as was demonstrated soconclusively by Foster and Sherrington when they founded theconception of the 'synapse', if it were considered to consist of

638 J. BOBKB

dead matter, of a ' sticky substance', in which every action oralteration attributed to living matter alone is excluded, is reallybeyond my comprehension. When we study for instance thehighly magnified drawings given by Windle and Clark in 1928,the only conclusion to be drawn from them, is that either theend-bulbs are severed from the surface of the cell-body on whichthey lie by the inevitable shrinkage of the cells caused by theimpregnation methods used, or that there is a distinct con-tinuity of the (living) substance of the cell and of the end-bulbdrawn in the figure. What is ' ciment unitif',' sticky substance','Kittsubstanz', intermediate substance between the cells? Ican only repeat what was said in my paper of 1926: we havedragged this expression with us through the fields of histologynow for more than seventy years as a hereditary burden withoutknowing in the least what was meant by it. Isn't it time tobury it together with so many outgrown and obsolete scientificconceptions? The only way to u n d e r s t a n d r i gh t lythe phys io logica l independence of the neuronesand the i r being connected by a synapse , able toplay an i m p o r t a n t pa r t in the physiology of thenervous sys tem, is to i n t e r p r e t th is synap t i cconnexion as a connexion by l iving m a t t e r ,capable of va ry ing i ts conduc t ing and o therqua l i t i e s under va ry ing condi t ions ; only thenhave we a r ight to speak of a syncy t i a l connexion .The most important question of future neurological researchwill be, as Lawrentjew expressed it (1934), the question ofsynaptology. 'Die Synapse, ihre Morphologie und Physiologiefesseln unsere Aufmerksamkeit', says Lawrentjew, 'weil sicheine Reihe von bedeutsamen Vorgangen eben an diesem Orteabspielt. Mit der Struktur der Synapse hangen allem Anscheinnach die Erscheinungen von Veranderung im Charakter dernervosen Erregung, die Irreprozitat der Leitung und hoehst-wahrscheinlich die Hemmungserscheinungen zusammen' (I.e.,p. 71), and farther on: ' der Kern des Synapsenproblems ist nichtim Ubertritt oder Nichtubertritt der Neurofibrillenim Bereich derSynapsen, sondern in der Frage zu suchen: ist die Synapse einbesonderer spezifischer Innervationsmechanismus oder nicht?'


As so clearly expressed in these words, this is the real synapticproblem, as I described it in my paper of 1929. It is onlypossible to solve it, yes, to see it even as a problem, when weconsider the synapse as consisting of living matter with varyingproperties and not as a dead intermediate substance of a stickynature! Perhaps I may be allowed to quote a few lines from mypaper of 1929 (I.e., p. 689): 'in the synapse the stimulus is notstopped but simply altered, polarized. . . . The living matterwhich must connect the neuronic elements, must be able toconduct this stimulus, and therefore must connect in a certainway the neurofibrillar structures of the two elements connected.If we could regard these interneuronal junctions as of the samenature as the periterminal network of the different peripheralnerve-endings, the physiological independence of the neuronesin relation to drug-action and function, and the peculiar wayin which the synapse differs from both the nerve-cell and theterminal branches of the nerve-fibres, would be satisfactorilyaccounted for, together with the anatomical continuity ofstructure (and connexion by living substance) between themwhich we have to acknowledge in the light of modern histology.Even the hypothetical synaptic membrane of Sherrington mustbe an arrangement of units of the l i v i n g substance, and thisarrangement may be present in the periterminal network, notas a real visible membrane, but as a biphasic condition of theliving substance itself.'

But ' revenons a nos moutons'. I fear that we have made anexcursion too far from the broad and even path of our discussionof the sympathetic plexus of A m p h i o x u s , but this wasnecessary in order to pave the way for further arguments.

Thus, in the first place, the ganglion cells are in continuousconnexion with the nerve-fibres of the enteric plexus, as isshown in many of the drawings accompanying this paper. Inthe second place the processes of the multipolar ganglion cellsthemselves may anastomose with each other, as is also shownin the figures. As was said already, it was difficult to settle thispoint definitely, because in the thin sections I could study mostof these anastomoses were cut through. At all events the cell-bodies are often lying at the knot-points of the plexus (Text-

640 J. BOBKB

figs. 3-6, 11), at the nodes of the network formed by the post-branchial nerve-fibres, and in this they remind us strongly ofthe' single ganglion cells', described by Young in the sympatheticpost-branchial plexus of Selachians. I never found them lying

TEXT-FIG. 9.

Stellate ganglion cell with thin lamellar expansions of the dendrites('Dendritlamellae'). From the wall of the post-hepatic intestine,adult A m p h i o x u s .

in groups as the encapsuled sympathetic cells of the verte-brates, the well-known ganglion cells of the plexus of Meissnerand of Auerbach. We have to refer to this later on.

Before discussing the nature of these ganglion cells, I willdraw the attention to the ganglion cells drawn in Text-figs. 10and 11, in which the peculiar shape, which they often have, and


their synaptic connexion with the nerve-fibres of the plexusis well shown. They do not need a long description.

As already mentioned, the ganglion cells described above are

TEXT-FIG. 10.

Stellate ganglion cell with part of the cell-body flattened to a thinmembrane, the processes being connected with a nerve-fibre.

very numerous and are distributed very regularly in a thinlayer on the wall of the liver, the oesophagus, and the post-hepatic intestine. They form a regular plexus, the processesextending in the thin sheath covering the wall in every direction(viz. always in the same horizontal, tangential plane), andintertwining to form plexiform bundles, as is shown in thefigures (cf. Text-fig. 2). No capsule cells could be distinguished,the ganglion cells lying freely between the connective-tissueelements. The long processes of the cells, the post-ganglionicnerve-fibres, often run in small bundles together with the pre-ganglionic fibres from the visceral nerves, and in this way a very

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642 J. BOEKE

regular plexus is formed. On the wall of the liver thickerbundles are seen running mostly in an oblique direction towards

TEXT-EM. 11.

Stellate ganglion cell with a long process, and a synaptic connexionwith a nerve-fibre. Adult A m p h i o x u s . Wall of the post-hepatic intestine.

the beginning of the small intestine where the liver is connectedwith the intestine. From my sections I got the impression thatthere where the liver communicates with the intestine, the nerve-plexus covering the wall of the liver is connected with the visceralnerves. This would be the natural connexion, because the liver


is formed as an outgrowth from the intestinal wall. Althoughaccording to Van Wijhe the elongation of the liver duringgrowth is in a cranio-caudal direction, the foremost point ofthe liver remaining fixed in relation to the surrounding elements(gill-slits, &c), the liver as a whole is directed craniad, and whenstudying the course of the nerve-bundles of the plexus coveringthe wall, it gives the impression that the nervous plexus coveringthe wall of the liver has grown out from the point of origin of theliver outgrowth together with this formation. Thus we findthe thicker bundles of the nervous plexus on the liver runningin an oblique ventro-dorsal direction towards this point ofconnexion with the intestine; on the wall of the post-branchialintestine we find the same disposition of the nervous plexusand of the coarser connecting bundles. Here, too, they are foundrunning in an oblique dorso-ventral direction, and in some casesI could follow them farther on, until they were passing into thevisceral nerve-bundle going to the dorsal segmental nerves.When we compare the ganglionic plexus described here with thesympathetic system of the higher vertebrates, perhaps we areentitled to compare the visceral nerves with at least a part ofthe rami communicantes and of the splanchnic nerves. Butof a formation which could be compared with the sympathetictrunks running along the ventral side of the vertebral column,no trace was to be found. In this connexion I may mentionthe fact, that according to the investigations of Young (1933)even in Selachians there are no long pre- or post-ganglionicpathways in the sympathetic and therefore no true sympatheticchains, though the ganglia of adjoining segments are sometimesconnected (cf. Chevrel, 1887; Botazzi, 1902). In A m p h i o x u sa true sympathetic chain (sympathetic trunk) is entirely absent,as far as I can judge from the study of my preparations.

But when we compare the enteric plexus of A m p h i o x u swith the sympathetic visceral system, viz. with the plexus ofAuerbach and Meissner, what are the elements with which theganglion cells and their processes are connected ? Are the nerve-fibres constituting the plexus of an efferent or an afferent nature ?

According to the statement of Van Wijhe, mentioned above(letter of November 16, 1933), the stellate ganglion cells, which

644 J. BOBKB

he saw on the wall of the liver and intestine, could not be motor,because there are no muscle-fibres in the neighbourhood.

On this point I cannot agree with him. On the wall of theliver and of the oesophagus and post-hepatic intestine there isto be found a very conspicuous and regular layer of long spindle-shaped cells, running parallel to each other, and chiefly in acircular direction around the gut. In Text-figs. 4, 6, 12, forinstance, they are very conspicuous; often the bundles of thesecells run in peculiar vortices, which are difficult to describewithout a large number of accurate drawings, but for thesedetails I may refer the reader to the general literature aboutA m p h i o x u s . These spindle-shaped cells are well known, andhave been described either as smooth muscle-cells or as con-nective-tissue cells. The most recent author in this field (Franz,1927) describes them as fusiform connective tissue cells, althoughhe admits that the van Gieson stain they took in his prepara-tions was not very strong ('nicht sehr ausgesprochen'). In thewall of the blood-vessels (bulbilli, endostyle-artery) they arealso to be found (Joseph, a.o.), and here they are commonlyadmitted to be real smooth muscle-cells. In my opinion t h e s ee l o n g a t e d fusiform cells or f ibres are t y p i c a ls m o o t h musc le - f ib res ; they have exactly the same formand structure as the smooth muscle-fibres of the higher verte-brates, they possess the same oval or rod-shaped nucleus, inthe preparations they take the same stain as the smooth muscle-fibres in the higher vertebrates, only they are a little thinnerand do not lie so closely aggregated. But in the wall of the liverand of the post-hepatic intestine they form a very regular andcontinuous, though thin, layer just outside the intestinal epithe-lium, with only a small number of connective tissue cells betweenthem, and they are covered by the nervous plexus mentionedabove and by the splanchnopleure. In the figures these spindle-shaped cells or fibres are very conspicuous, just as they appearin the preparations, where they can be studied with the utmostclearness. In my opinion we are justified when we comparethe entire regular layer of the cells or fibres with the muscularcoat of the intestinal wall. And in this connexion it is extremelyinteresting that the nervous plexus described above seems to


be connected everywhere with these elements. When studiedwith the highest power and strong light we see very delicateneurofibrillae branching off from the nervous strands and endingin fine nets on the spindle-shaped fibres (Text-figs. 4, 6). Insome cases I could distinguish extremely small but very sharplydefined end-rings, lying closely to the spindle-shaped fibres, inother cases no end-rings could be distinguished but very delicatereticular formations were to be seen; in all cases there seems toexist a distinct terminal connexion of the nervous strands of theintestinal plexus with the elongated spindle-shaped cells, viz.the smooth muscle-fibres.

But there is still more. When we study these formations inlongitudinal sections, there appears, as I mentioned before, asmall amount of connective tissue covering the layer of spindle-shaped fibres and the intestinal epithelium. In this tissue wemay distinguish a still more delicate network of neurofibrillae,given off by the nervous strands of the denser plexus, and lyingbetween the connective tissue cells. In this second layer ofnervous strands we also find ganglion cells (Text-fig. 12,Gl.Z.M.) of the same stellate shape as those described above,but with a still more delicate neurofibrillar structure. In Text-fig. 12 I have reproduced a drawing (camera lucida drawing byour artist) of a tangential section through the wall of the post-hepatic intestine, which shows the layer of spindle-shapedsmooth muscle-fibres (mf.) with a ganglion cell lying on them(to the right of the figure), and the neurofibrillar network lyingsomewhat deeper between the connective tissue elements (tothe left). Here too a ganglion cell (Gl.Z.M.) is to be seen, but theneurofibrillar structure of this cell was so very delicate that ithad to be a little exaggerated to become visible in the repro-duction of the drawing. Nevertheless it was very conspicuous,and the drawing gives exactly what was to be seen in the section.

This second plexus lying in the thin layer of connective tissuecovering the layer of smooth muscle-cells and the intestinalepithelium, is more delicate in structure and somewhat lessregular in formation and extension than the denser plexuscovering the smooth muscle elements. I have not been able todetect a distinct connexion of the neurofibrillar strands of the

GI.L

TEXT-FIG. 12.

Tangential longitudinal section through the wall of the post-hepaticintestine of an adult Amphioxus , showing the nervous plexuscovering the smooth muscle-cells (TO/.), and the more delicateplexus lying underneath it between the connective tissue-cells.Camera lucida drawing from a section 15 /j. thick. Ol.Z.M.,ganglion cell of connective-tissue plexus.


plexus and the epithelium of the intestine. Sometimes I got theimpression that small ring-like or club-shaped nerve-endingswere lying against the base of the epithelium cells, but it wasimpossible to come to a definite conclusion on this point, theformol-fixation not giving sufficiently clear pictures of theepithelial structures. Text-fig. 12 gives an example of what couldbe distinguished.

This second plexus lying in the connective-tissue layer iseverywhere connected with the neurofibrillar strands of thedenser plexus described above. If we compare this denserplexus covering the smooth muscle-cells with the plexus ofAuerbach of the higher vertebrates, I should feel inclined tosuggest that the delicate plexus lying in the connective tissuemight be compared perhaps with the plexus of Meissner, whichin the higher vertebrates, too, is so much finer than the plexusof Auerbach.

Further study will be "required here to settle these points,and it will also be necessary to study more closely the innerva-tion of the blood-vessels. All I can mention here is that alsoon the wall of the larger blood-vessels, endostyle artery, bulbilli,aortae, venous blood-vessels running alongside the dorsal andventral wall of the liver-sack, here and there a network of verydelicate nervous fibres could be distinguished, connected withthe plexus mentioned above, but this network was so extremelydelicate that it was impossible to come to a definite conclusionabout the details of it, and I must confine myself to these generalstatements.

It seems to me, however, that these observations are suffici-ently reliable to justify the conclusion that we have to alterthe statements mentioned in the beginning of this paper, andthat A m p h i o x u s possesses, in a very primitive form, asympathetic system, which may be compared with the entericplexus of the higher vertebrates.

There is still one point which has to be discussed, viz. whetherwe are entitled to compare the ganglion cells described abovewith the ganglion cells of the enteric plexus of the highervertebrates or not. What is the nature of the stellate ganglioncells composing the enteric plexus of A m p h i o x u s ?

In order to discuss this question I must in the first placeemphasize the fact that the ganglion cells of the enteric plexusof A m p h i o x u s are everywhere lying in a syncy tial arrange-ment. Not only, as I have tried to demonstrate in the previous

TEXT-FIG. 13.

Two stellate ganglion cells of the plexus entericus of an adultAmphioxus , with synapses on a post-ganglionie nerve-fibre(nf.) and anastomosing processes.

pages, are they in syncytial synaptic connexion with the pre-or post-ganglionic nerve-fibres, but in many places their shorterprocesses are undoubtedly anastomosing with each other. InText-figs. 13 and 141 have given examples of these anastomoses,


drawn as exactly as it was possible from the preparations. ToText-fig. 14 I shall refer later.

In the second place, we have to bear in mind that they arelying at the nodes of the plexus exactly like the ' single ganglion

TEXT-FIG. 14.

Two anastomosing ganglion cells in the wall of the post-hepaticintestine of an adult Amph ioxus . Highly magnified.

cells' described by Young in the enteric plexus of Selachians.They are not enveloped by capsule cells as the larger ganglioncells of the selachian sympathetic plexus and of the plexus ofAuerbach of the higher vertebrates.

In these two points they strikingly resemble the neuroblastsin a tissue culture. In Text-fig. 15 1 have reproduced a drawingof Grigorieff, from his recent paper on tissue cultures of nervous

650 J. BOEKE

elements (Grigorieff, 1932, Taf. 6, fig. 1). In it are drawn threeanastomosing ganglion cells (neuroblasts) in a tissue culturein v i t r o of a small piece of the brain of a chick embryo,impregnated after Bielschowsky.

The picture is very clear. We see the neurofibrillar structure

TEXT-FIG. 15.

Three anastomosing ganglion cells in a culture in v i t r o of a pieceof the brain of a 7-days chick embryo. Culture of 72 hours. AfterGrigorieff, 1932.

of the neuroblasts and the anastomosing of the shorter processes,as it Avas demonstrated by LaAvrentjew at the Anatomical Con-gress in 1930. The long processes were groAving out into theculture fluid, Avhere they too form exquisite plexiform nets(cf. Mossa, Shiro, 1929). Such pictures are veij frequent; innearly every culture in v i t r o of neuroblasts we can find them.I could have reproduced just as well a drawing from our OAvntissue-cultures of neuroblasts (Bijleveld), but I preferred to takeit from the publication of another author. The similarity

NERVOUS SYSTEM OP AMPHIOXUS 651

between Text-figs. 14 and 15 is very striking, and in my opinionit has a deeper meaning.

To make my meaning clear, I may be allowed to reproduce

TEXT-PIG. 16.

Two Sohwann cells (*.) of the plexus of Meissner from the humanstomach, with terminal reticulum (t.). After Stoehr, 1934.

in Text-fig. 16 a drawing from the latest paper by Stoehr(1934, Abb. 7), in which are shown two Schwann cells from thenervous plexus of the wall of a human stomach (neonatus) withthe neurofibrillar structure and the very delicate network, which

652 J. BOEKB

Stoehr calls the 'terminal reticulum'. Here, too, we encounteran entirely syncytial arrangement of the elements in questionand of the neurofibrils, and when we compare Text-figs. 14,15, and 16, it is interesting to note the conformity of the generalcharacter of the structures in Text-fig. 16 with those of Text-figs.14 and 15.

In my opinion the cells drawn by Stoehr in Text-fig. 16represent the syncytial elements called 'interstitial cells' byLawrentjew, the old interstitial neurones, 'neurones sym-pathiques interstitiels' of Cajal (1893,1911).

As is well known, Lawrentjew in 1926 has taken up the oldproblem of the interstitial cells of Cajal, proclaims them to bean important element of the end formation of the sympatheticplexus, and describes them as the constant syncytial terminalelements of it, from which grow out the endings on the smoothmuscle-fibres, &c. According to Lawrentjew the finest branchesof the sympathetic plexuses show a syncytial structure, theneurofibrillar strands being enclosed in a protoplasmic sheathwith dispersed nuclei of Schwann, but without definite cellularsheaths. 'Verfolgt man,' says Lawrentjew, 'ein solches Biindellangs seinem Verlauf inmitten der glatten Muskelelemente, sofindet man immer an bestimmten Punkten eine Anhaufung vonProtoplasma und darin einen Kern; von diesem Punkte ausstrahlen die Neurofibrillenbundel unter grossen Winkeln nachverschiedenen Eichtungen auseinander, indem sie dabei nochdauernd innerhalb ausserst dtinner Protoplasmastrange bleiben.Das sind die interstitiellen Zellen, von ihnen aus zieht dasGrundbundel von Neurofibrillen weiter zu einem anderensolchen Punkte (einer "interstitiellen Zelle"), wahrend eineganze Eeihe anderer feiner Biindel nach verschiedenen Richtun-gen ausstrahlt, zu den glatten Muskelzellen hinzieht und inihnen die motorischen Endigungen bildet' (I.e., p. 474). In thisconception he was followed by Van Esveld (1929), who showedby his remarkable pharmacological experiments the highphysiological importance of these elements, by Leontowitsch, bySchabadasch a. o., and in a series of contributions to the histo-logy of the end-formation of the sympathetic system (' Innerva-tionsstudien' I-IV, in the 'Zeitschr. f. mikr.-anat. Porschung',


vols. 33, 34, 35, 1933, 1934), I tried to show, that these inter-stitial syncytial elements are a constant structure, that theyare to be regarded as true interstitial parts of the sympatheticend-formation, and that from them grows out the intra-protoplasmic so-called periterminal network, by which thenervous impulse is transmitted to the various organs of response.The more I worked out this conception, the more I becameconvinced that there is no fundamental difference between thesesyncytial elements of the end-formation of the sympatheticplexuses, which conduct the nervous impulses, and the elementscarrying the impulse to or from the different end-organs (tactilecells, elements of the core of the sensory corpuscles, endingson the smooth muscle-fibres, the gland-cells, their myo-epithelialcells, &c.); it has to be regarded as a syncytial arrangement,building up the terminal formation1 in a protoplasmic connexionwith the neurofibrillar strands and with the organ of response.

But we must bear in mind that the neurofibrillar bundles andthe more delicate neurofibrillae of this end-formation, lyinginbedded in a syncytial protoplasmic sheath ('Protoplasma-strange' of Lawrentjew and Stoehr) are nothing but the nervousprocesses growing out from the sympathetic ganglion cells lyingeither inside the medulla or in the peripheral ganglia or the wallof the gut. Therefore we have to draw the conclusion, that theend-formations of the nervous sympathetic pathway, the so-called 'interstitial cells', and the ganglion cells, the beginningsof this pathway, are in protoplasmic continuity. I could in thisconnexion refer to a drawing by Stoehr in his publication onthe nervous elements in the human stomach-wall (1931, Abb. 56,p. 149, 'Zeitschr. f. Zellf.', Bd. 12), in which the protoplasmiccontinuity of the cell-body of a ganglion-cell and the cell processt o g e t h e r wi th its sheath of Schwann is shown very clearly,and to different drawings by Leontowitch and Schabadasch intheir recent publications.

1 For more detailed information on the sympathetic end-plexuses andtheir connexion with the end-organs, and the various interpretations ofthe sympathetic problem I may refer the reader to a paper 'Die periphereEndausbreitung des sympathischen Systems', in the 'Nova Acta of theAcademia Leopoldina', 1934/1935, pp. 209-57.

654 J. BOEKE

Thus it seems to me that there is room for the supposition,that in order to arrive at a true interpretation of the ganglionicsympathetic plexus of A m p h i o x u s (and of the higher verte-brates) we have to give up the classic and traditional categoricaldistinction between nervous elements and merely conductingelements, and that we have to acknowledge that there may existintermediate elements between them.

Yes, it would perhaps be pardonable to go yet one stepfarther, and to compare the interstitial elements of the sym-pathetic end-formation with primitive ganglion cells, as alreadysuggested by Cajal, when he called them' neurones sympathiquesinterstitiels' and attributed to them the faculty to give offimpulses to the end-organs (smooth muscle-cells, &c), 'perhapsunder the influence of the sympathetic plexus' (Cajal, 1911).

In this light it would not be too bold to compare the inter-stitial neurones of Cajal and Lawrentjew with primitive ganglioncells, such as we find in the nervous plexus of invertebrates (forinstance, E h i z o s t o m a according to Bozler, A s t a c u s ,Alexandra wicz).

The pharmacological experiments of Magnus and VanEsveld (1929) have taught us the importance of the interstitialelements for the physiological impulses (rhythmic contractions),and the relative independence of this end-formation of thesympathetic plexus in relation to the ganglion cells of Auerbachand Meissner; strips of smooth musculature of the gut, whichon histological examination did not contain a single ganglioncell but only the so-called interstitial elements, were seenretaining their regular rhythmical contractility for hours ata stretch and were even influenced by different drugs (VanEsveld, 1929).

In the sympathetic nervous plexus (' Grundplexus') we maysee a very old and very conservative nervous system, which haspreserved many—speaking phylogenetically—old and primitivecharacters. From it the encapsulated cellular nervous elementsof the sympathetic plexuses of the higher vertebrates havedifferentiated. Is it very improbable that the cellular elementsof the enteric plexus of A m p h i o x u s are primitive elements,comparable to the cellular elements of the nerve-net of the lower


invertebrates; primitive elements, which we see preserved inthe sympathetic plexus of the higher vertebrates as 'inter-stitial cells' and as non-incapsulated cells in the plexus of theSelachians, and which we see regaining their true character inthe neuroblasts as they are developing in the cultures inv i t r o of Lawrentjew and Grigorieff ?

Seen in this light the similarity between Text-figs. 14,15, and16 would have more and deeper meaning than a simple similarityof form. In it would come to light a fact of phylogenetic im-portance. We would be able to understand better the beginningsand the evolution of the sympathetic in the various classes ofthe invertebrates; and the development of the sympatheticplexus in A m p h i o x u s would not remain standing apart andisolated, but would become comprehensible as an intermediatestage between invertebrates and vertebrates.

With regard to the question of the sympathetic innervationof the cross-striated muscle-fibres the following observation maybe of interest:

Just behind the branchial arches, ventrally to the chordadorsalis there is found in my sections (longitudinal sagittalsections of adult Amphioxus ) a very thin triangular muscle,which is composed of cross-striated muscle-fibres, and isdirected in a ventro-caudal direction, the two muscles (rightand left) enclosing the post-branchial gut. This muscle can benothing else than the m u s c u l u s t r a p e z i u s of Legros (1902),which was discovered by A. Schneider in 1879,- and has beenstudied since by Legros (1902), who called it the trapezianmuscle, by Zarnik (1904) and Franz (1925).

It is composed of strong bundles of myofibrillae (not muscle-plates!), which show a very distinct cross-striation and arelying in a thin layer against the endothejium of the post-branchial gut. Its function seems to be to elevate and constrictthe gut; according to Zarnik the contractions of this muscle tendto show a certain rhythm (I.e., p. 621), at least Zarnik saw in theliving animal rhythmical contractions of the post-branchial gut,which could be attributed to contractions of this muscle.

656 J. BOBKE

On the surface of this muscle, covering the layer of muscle-fibres, there was found the same neurofibrillar plexus withstellate ganglion cells as that which covers the wall of the gut,and so far as I could gather (the impregnation of this plexusand especially of the final branches of the neurofibrillar strandswas not very strong) the processes of these ganglion cells werein connexion with the muscle-fibres, either by means of smallend-rings or of very delicate reticula, end-nets, which seemed topass into the substance of the cross-striated muscle-fibres butthen became invisible and could not be followed any farther.At all events I came to the conclusion, t h a t th is musc le ,as far as I could make out , is i n n e r v a t e d by thesame p lexus and in the same way as the smoothm u s c u l a t u r e of the gu t .

The th in spa tu l a - l i ke motor e n d - p l a t e s , soc h a r a c t e r i s t i c of the segmenta l c ro s s - s t r i a t edmusc le - leaves of Amphioxus (see Penfield, 'Cytology ofthe Nervous System', vol. i, p. 256, 1932) were en t i r e lyabsen t in th i s muscle , a l though they could beseen very well in the somat ic musc le -p la tes inthe same p r e p a r a t i o n s .

SUMMARY.

In this, paper is described a nervous plexus on the wall of theliver and the adjoining parts of the intestine of Amphioxusl a n c e o l a t u s with numerous stellate ganglion cells, whichmay be compared with the plexus of Auerbach of the highervertebrates. A layer of smooth muscle-cells is present, withwhich the processes of the ganglion cells and the nerve-fibresof the plexus are in synaptic connexion. Covering this layerof smooth muscle-cells a thin layer of connective tissue is present,in which is found a more delicate nervous plexus, connectedwith the first plexus, analogous to the plexus of Meissner.The nature of the synaptic connexions of the ganglion cellswith the pre-ganglionic and post-ganglionic nerve-fibres of theplexus is discussed. The ganglion cells may be compared withthe interstitial elements of the sympathetic plexus of the highervertebrates.


The cross-striated trapezius muscle (Legros) is innervated bythe same plexus as the muscular coat of the intestine and notby the somatic nerves.

The enteric plexus is connected by means of the visceralnerves and dorsal roots with the central nervous system.

BIBLIOGRAPHY.

Only the papers cited in the text are inserted.Ayers, Howard (1921).—' Journ. of Compar. Neurol.', vol. 33.Boeke, J. (1902).—'Proceedings Roy. Acad. of Science', Amsterdam.

(1908).—'Anat. Anz.', Bd. 32, 33.(1929).—'Proceedings Roy. Acad. of Science', Amsterdam, vol. xxxii,

no. 6.(1933, 1934).—"Innervationsstudien, I-VI", 'Zeitschr. f. mikr.-

anat. Forschung', Bd. 33, 34, 35.(1933).—'Proceedings Roy. Acad. of Science', Amsterdam, vol. xxxvi,

no. 10.'Nova Acta der Deutsch. Leopoldin. Academie der Naturf.' Halle,

1935.Botazzi, E. (1902).—'Zeitschr. f. Biol.', vol. 43.Bozler, E. (1927).—'Zeitschr. f. Zellf.', Bd. 5.

(1927).—'Zeitschr. f. vergl. Physiol.', Bd. 6.Dogiel, A. S. (1903).—'Anat. Hefte', Heft 66.Chevrel, H. (1887).—'Arch, de Zool. exp. et gen.', vol. 5 (bis).Van Esveld, L. W. (1928-9).—'Zeitschr. f. mikr.-anat. Forschung', Bd. 15.Franz, V. (1925).—' Jenaische Zeitschr.', Bd. 61.

(1927).—'Ergebn. der Anat. u. Entwgesch.', Bd. 27.Grigoriefl, L. M. (1931-2).—'Arch. f. Exper. Zellforschung', Bd. 11 and 12.Heymans and Van der Stricht (1898).—'M6m. cour. de l'Acad. R. Belg.',

vol. 56.Kutchin, H. L. (1913).—'Proc. Am. Acad.', vol. 49.Lawrentjew, B. J. (1926).—'Zeitschr. f. mikr.-anat. Forschung', Bd. 6.

(1929).—Ibid., Bd. 18.(1931).—Ibid., Bd. 23.(1934).—Ibid., Bd. 35.

Legros, R. (1902).—'Mitt. Zool. Stat. Neapel', Bd. 15.(1910).—'Anat. Anz.', Bd. 35.

Schabadasch, A. (1934).—'Zeitschr. f. Zellf.', Bd. 21.Schneider, A. (1879).—'Beitrage vergl. Anat. u. Entw. der Wirbeltiere.'

Berlin.Shiro, E. (1929).—'Zeitschr. f. wiss. Mikrosk.', Bd. 46.Stoehr, Ph. jr. (1028).—'Zeitschr. f. Anat.', Bd. 78.

(1931).—In 'Handbook of Mollendorff', Bd. 4.

NO. 308 X X

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Stoehr (193(M).—'Zeitschr. f. Zellforsch.', Bd. 12, 16, 21.Windle and Clark.—'Journ. of Compar. Neurology', 1928.Van Wijhe, J. W. (1913).—'Proceeding Roy. Acad. of Science', Amsterdam,

vol. 16.Meeting of April 1913.

Young, J. Z. (1933).—'Quart. Journ. Micr. Science', vol. 75, part iv.Zarnik, B. (1904).—'Anat. Anz.', Bd. 24.

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658 J. BOEKE




the autonomic (enteric) nervous system of amphioxus ... · about the sympathetic nervous system of...

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