THE ANATOMY AND NEUROSECRETORY SYSTEM OF THE SUPRAOESOPHAGEAL
GANGLION OF HERMODICE CARUNCULATA (ANNELIDA:POLYCHAETA)
FITZSIMONS THE ANATOMY AND NEUROSECRETORY SYSTEM OF THE SUPRAOESOPHAGEAL
GANGLION OF HERMODICE CARUNCULATA (ANNELIDA:POLYCHAETA)
by
Patricia Gail Fitzsimons
A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfilment of the requirements for the degree of Master of Science in Zoology.
Zoology Department, McGill University
October 1964 ACKNOWLEDGEMENTS
The author wishes to express her thanks to Professor J. c. Marsden for her supervision of the problem. Part of this work was carried out in Barbados at the Bellairs Research Institute of McGill University.
I am grateful to the director of the Institute, Dr. John B. Lewis, for the hospitality and facilities he afforded me and for his advice and help in collecting and other matters. The work in Barbados was made possible by a Summer Demonstratorship from the Zoology Department of McGill University. My thanks are due to Mr. J. W. Pollock for the preparation of the photomicrographs. TABLE OF CONTENTS
STi\TEl1ENT OF PROBLEM •••••••••••••••••••••••••••••••••••••••••••• 1
SECTION I: BRAIN ANATOHY
INTRODUCTIOI~ •••••••••••••••••••••••••••••••••••••••••••••••••••• 4
General Introduction ...... 4 1~e Drain of Nephtys ••••••••••••••••••••••••••••••••••••••• 7
NATERIALS AND METHOUS ...... 11 Dissections ...... 11 Histological Work •••••••••••••••••••••••••••••••••••••••••• 12 OBSERVATIONS ...... 16
External Morphology of the Head •••••••••••••••••••••••••••• 16 Eye-Spots •••••••••••••••••••••••••••••••••••••••••••••••••• 18 Gross Morphology of the Brain •••••••••••••••••••••••••••••• 19 Cranial Nerves ...... 20 Microanatomy of the Brain ...... 24
Prostomial and Brain Cavities ••••••••••••••••••••••••• 24 Brain 11embranes ••••..•.•••••••••••••••••••••••••••.••• 27 Epidermal Sensory Cells ...... 28 (~neral Brain Architecture ...... 28 Neurons ...... 29 Neuroglia ...... 32 Internai Organization of the Brain •••••••••••••••••••• 34 General Brain Organization ••••••••••••••••••••••• 34 Circumoesophageal Connectives •••••••••••••••••••• 35 lionic Nue lei .•••••••..••..•••••••.•••..•.••• 35
DISCUSSION •••••••••••••••••••••••••••••••••••••••••••••••••••••• 47
SECTION II: NEUROSECRETION
INTRODUCTION ...... 54 General Introduction ...... 54 Morphological Characteristics of Neurosecretory Cells 54 Histochemical Aspects of Neurosecretion ...... 58 Functional Significance of Neurosecretion ...... 61 TABLE OF CONTENTS (contd.)
Neurosecretion and Diurnal Rhythms • • • • • • • • • • • • • • • • • • • • • • • • • 64
1~eurosecretory Cells in Polychaetes • • •• • • • • • •• • • •• ••• • • • • • • 66
MATERIALS AND METHODS ...... 69 OBSERVATIONS ...... 70
DISCUSSION • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 80
84
LITERATURE CITED ...... 86 REFERENCE LETTERS FOR ILLUSTRATIONS ...... 91 ILLUSTRATIONS, FIGS. 1 - 33 ...... 93 STATEMENT OF THE PROBLEM
Hermodice carunculata (Pallas) is an Amphinomid po1ychaete commonly found throughout the Caribbean area (Marsden, 1960). It also occurs in the Gulf of Mexico (Hartman, 1951) and off the coast of southern Florida
(Mullin, 1923).
The family Amphinomidae includes the first species of annelids to be described from the Western Hemisphere. ln the eighteenth century, some tvest Indian faunal collections were deposited in various museums of western Europe. At that time, a young physician, Peter Simon Pallas, became interested in these collections. Among the species which he studied and described from the collections, was one which he named
Aphrodita carunculata. According to Hartman (1951), the descriptions and illustrations made by Pallas in 1766 are still noteworthy for their accuracy and interest.
In 1857 the same species was found by Kinberg, who, considering it to be a new species, named it Hermodice carunculata, by which name it is known today.
Hermodice carunculata occurs on living coral reefs and also under stones inshore of reefs. Hartman (1951) states that it is also associated with drifting abjects in warm currents.
The literature on this common Caribbean species is surprisingly meager. It is restricted almost exc1usively to faunal records and des criptions of external morphology. As far as the author is aware, the only references to its internai anatomy and physiology are to be found in papers by Storch and Marsden. Storch (1912), in his publication on polychaete anatomy, includes a description of the gross anatomy of the nervous system of Hermodice carunculata. Marsden is interested in this species and has reported in detail on its feeding habits (1962a), and on the anatomy and histology of the digestive tract and the anatomy of the stomatogastric nervous system (1926b).
The literature apparently contains no descriptions of either the external or the interna! anatomy of the supraoesophageal ganglion, or brain, of Hermodice carunculata. Storch's (1912) illustrations of the nervous system are very diagrammatic, depicting the supraoesophageal ganglion simply as a circle inside the prostomium. The only cranial nerves indicated are the circumoesophageal connectives. Gustafson (1930), in his study on the anatomy of the Amphinomidae, includes a diagrammatic illustration of the Amphinomid supraoesophageal ganglion, showing the origin of the circumoesophageal connectives and stomatogastric nervous system. No other cranial nerves are shown, and the stomatogastric system is grossly overMsimplified.
The supraoesphageal ganglia of many polychaete species have been studied for neurosecretory activity. However, no such investigations have been carried out on Hermodice carunculata.
Hermodice carunculata is a predator of living coral (Marsden, 1962a) and shows a definite diurnal periodicity with respect to its feeding.
The worm is never found on the reef during the day. But, very regularly, late every afternoon, at about four p.m., it suddenly appears and is seen feeding on the living tissues of the coral. It may also be found on the reef in the early morning, but only until about eight a.m. at the latest. Between eight a.m. and four p.m. the worm cannot be found wandering on the reef. With very persistent searching, worms may be found resting in crevices in the coral and underneath rocks inshore of the reef. The same pattern of activity is shown by animals kept in the laboratory. They remain almost entirely motionless during the day, curled up in a shaded corner of the laboratory water table. In the late afternoon they become active and begin to crawl about over the bottom of the water table, feeding on any available bits of coral.
This activity and feeding persist throughout the night until early morning, and then the worms become quiescent again. Marsden* has suggested that this diurnal activity rhythm of Hermodice carunculata may be under the control of cerebral neurosecretory cells.
The literature on polychaetes appears to contain no references to experiments on the relationship between neurosecretion and diurnal biological rhythms. The concept of neurosecretory control of a diurnal activity rhythm is of interest in relation to the subject of biological clocks in general. Marsden's hypothesis has been treated by examining supraoesophageal ganglia for the presence of neurosecretory cells which show evidence of a diurnal secretory cycle which could be correlated with the diurnal activity rhythm displayed by the animal.
The purpose of this thesis is to present a description of the external and internai anatomy of the supraoesophageal ganglion and a general account of the neurosecretory sites within the supraoesophageal ganglion of Hermodice carunculata. Also, the hypothesis of a correlation between neurosecretion and the diurnal activity cycle has been investigated.
The thesis is divided into two sections. The first section is con cerned with the anatomy of the supraoesophageal ganglion, while the second section contains the work on neurosecretion.
* unpublished -4-
SECTION l: BRAIN ANATOMY
INTRODUCTION TO THE WORK ON BRAIN ANATOMY
The polychaete supraoesophageal ganglion is a complex organ showing great structural variation within the class. Racovitza (1896) has presented a very comprehensive account of head and external brain structure in polychaetous annelids. Of particular importance is his description of brain morphology in the Eunicidae. This farnily is generally considered to be closest to the ancestral polychaete stock
(Grassé, 1959) and hence Racovitza 1 s account serves as an introduction to the basic pattern of polycheate brain structure.
In the Eunicidae, the brain is contained entirely in the prostomiurn and i s si tuated in contact with the epiderrnis, from whicl~ i t i s separated only by the basement membrane of the epiderrnis.
Viewed externally, the brain is seen to be composed of three regions, situated behind each other: forebrain, midbrain and hindbrain. Racovitza has analysed the se portions <~ccording to the following table:
Brain Region Organs Innervated Nervous Centres Fonctions
Palpa! area or Palps and gustatory Palpai ganglia Touch and fore brain grooves tas te
Sincipital area Eyes and antennae Optic ganglia and Sight and or midbrain antennal ganglia ta ste
Nuchal area or Nuchal organs Nuchal ganglia Olfaction hindbrain
(Translated from Grassé, 1959)
Each part is composed of two syrnmetrical halves joined by a transverse commissure, except for the hindbrain. The two halves of the hindbrain are -5-
well separated by a posterior notch and are not joined by a commissure.
In other polychaete families, the brain does not appear in this
expanded Eunicid form which is considered to be anatomically primitive
(Grassé, 1959).
The structure and size of the brain depend on the development of
the eyes and sensory prostomial appendages. The loss of the eyes, the
reduction or disappearance of the palps or antennae, is acc~npanied by
simplification of the brain. This accounts for the structure of the brain
of the sedentary polychaetes which live burrowed in the mud or in a tube
and which have the prostomial appendages and eyes very reduced or com
pletely lost. The brain of the sedentary polychaetes shows a reduction
in size and a simplification in structure. The corpora pedunculata are
lacking, except in the Serpulidae, where they are present but very tiny
(Hanstrom, 1927). In the sedentary polychaete brain one cannat discern
subdivisions corresponding to those of the errant forms.
In studying the interna! structure of the polychaete brain, Holmgren
(1916) found that the neuron cell-bodies are located principally in the
periphery of the brain, while the fibers (axons and dendrites) occupy
the interior regions and constitute the fibrous structure known as the
neuropile. The majority of the neurons are unipolar, but bipolar neurons may be found in various parts of the brain.
The nerve cell-bodies may form a uniform mantle over the neuropile of they may be disposed in distinct clusters forming discrete brain centres known as ganglionic nuclei. These brain centres have been
studied in their entirety only by Holmgren (1916) in Nereis diversicolor
and by Clark (1958a,b,c) in Nephtxs. Many polychaetes possess specialized brain centres known as corpora
pedunculata or mushroom bodies, homologQS with those structures found in
certain arthropods. These have been studied more extensively than other
cerebral centres. The corpora pedunculata are situated in the most
anterior region of the brain, on each side of the sagittal plane. They
consist of one or more dense rounded masses (known as globuli) of nerve
cell-bodies forming a cap over a bundle of ascending or descending fibers
composed of the axons and dendrites of the cells of the globuli. The
neurons composing the globuli are morphologically distinct from ordinary
neurons (Hanstram, 1927) and are called globuli cells. They are very
small with little cytoplasm and chromatin-rich nuclei.
The corpora pedunculata are well•developed only in the errant
polychaetes. They are lacking in the sedentary polychaetes, except for
the Serpulidae, where they occur in a very rudimentary form. In fact, the
degree of development of the corpora pedunculata is a function of the
development of the visual organs and the chemoreceptive organs (palps
and antennae). Although we have no detailed knowledge of their function,
the corpora pedunculata are considered to be important associative centres,
correlating impulses received from the visual organs and the chemoreceptive
organs (Grassé, 1959). They are most highly developed in the Aphroditidae
and are also very well developed in the Neridae and the Hesionidae.
However, the corpora pedunculata are absent in the Eunicidae. This would
seem incongruous since these polychaetes possess a well developed forebrain, eye-spots, and a complete assortment of prostomial sensory appendages.
Hanstrom (1927) points out that the absence of corpora pedunculata in the
Eunicidae supports the hypothesis that the Eunicidae are those polycheates closest to the ancestral stock. He regards the absence of the corpora pedunculata in the Eunicidae as an archaic and fundamental characteristic and not as a feature related to a degeneration of the forebrain.
The most complete accounts of the microanatomy of the polychaete supraoesophageal ganglion are to be found in the papers of Holmgren (1916) and Clark (1958a,b,c; 1959). Holmgren describes the external and internai structure of the brain of Nereis diversicolor, while Clark deals with the brain structure of Nephtys, using twelve species of Neeht~s in his study. Clark (1958c) has drawn a careful comparison between the brains of Neehtys and Nereis and has found it possible to homologize many of the ganglionic nuclei in the brains of the two genera. In this thesis, a comparison will be made between the brain of Hermodice carunculata and the study by Clark on Nephtys.
The Brain of Neehtys
We owe our present knowledge of brain structure in Neehtys primarily to four papers by Clark (1958a,b,c; 1959), from whose accounts the following is taken.
The supraoesophageal ganglion of Neeht~s is clearly epidermal, being bounded laterally and ventrally by a connective tissue sheath which is continuous with the epidermal basement membrane. In most species of
Neehtys, part of the ganglion extends into the anterior body segements and in them the ganglion is suspended beneath the epidermis and is com pletely invested by its connective tissue sheath. Only that part of the ganglion which lies in the prostomium is in contact with the cuticle.
The sheath which invests the brain laterally and ventrally in the pro stomium is actually double, consisting of an inner laminated connective tissue layer which is continuous with the basement: membrane of the
epidermis and an outer extremely thin cellular layer.
The prostomial sensory apparatus of Neehtys is somewhat reduced.
The re are two pairs of antennae, two pairs of phot:oreceptors and a pair
of nuchal organs. The palps are absent. Also, there are numerous
cuticular sensory hairs, mainly on the dorsal surface of the prostomium.
The sensory nerves of all these structures enter the supraoesophageal
ganglion.
The supraoesophageal ganglion gives rise to the following nerves:
2 ... 8 tegumentary nerves and 2 pairs of antennary nerves from the antero
dorsal margin of the brain; 1 pair of optic nerves to the anterior eye
spots (the posterior eye-spots are embedded in the supraoesophageal
ganglion and there are no external nerves); 1 pair of nuchal nerves
arising from the lateral surface of the brain, about midway along its
length, and innervating the paired nuchal organs which lie at the postera
lateral margins of the prostomium; 1 pair of circumoesophageal connectives.
In Nephtys, the stomatogastric system arises from the circumoesophageal
connectives rather than directly from the brain.
The brain contains only truly ganglionic material (neurons and
neuroglia), with the exception of a series of thick fibers traversing
the brain in the mid-line and running dorso-ventrally from the cuticle to
the sheath investing the brain. These fibers correspond with the insertion
of the muscles on the other side of the sheath and presumably prevent too great distortion of the brain when the muscles are contracted. There are
no blood vessels or mucus cells within the brain tissue (mucus cells are
found in the "posterior lobes11 of the brain, structures which are not
truly part of the brain). The nerve cell·bodies are arranged to form a cortical layer over the top and sides of the central neuropile. Few nerve cell-bodies occur ventrally below the neuropile. The neuropile is composed of two main parts, anterior and posterior, and the posterior neuropile is sub divided into three parts.
Clark found that the nerve cell-bodies form an almost uniform mantle over the top and sides of the neuropile of most species, particularly the smaller ones, and the ganglionic nuclei are not very distinct.
Nevertheless, he was able to discern twenty-five paired nuclei. There is only minor inter-specifie variation in the size and disposition of the ganglionic nuclei. ln the brain of sorne speci.es of Nephtys, Clark finds globuli cells. However, they are not organized into true mushroom bodies. Clark reports that, in those species which possess them, the globuli cells have been evolved from three ganglionic nuclei, and different stages in the evolution of mushroom bodies can be found in various species.
Clark identifies three types of ganglion cell within the brain of
Nephtys: globuli cells; ordinary nerve cells of variable size; a third intermediate type, probably the cell-bodies of sensory neurons.
The brain of Nephtys contains one transverse commissure, the optic commissure situated in the midbrain. This commissure inter-connects with the optic nuclei on either side of the brain.
The circumoesophageal connectives emerge from the brain as single structures. However, internally they are composed of two main bundles of fibers, an inner bundle and an outer bundle. These two fiber tracts retain their separate identity throughout their course to the suboesophageal ganglion. The outer bundle of fibers arises from the second and third -10- posterior neuropiles, while the inner bundle arises from the anterior and the first posterior neuropile.
Clark has found a cerebro-vascular complex associated with the brain of Nepht~s. This structure is fundamentally the same as that which occurs in certain Nereids (Bobin and Burchon, 1952; Defretin, 1955), except that its structural organization is somewhat simpler than in the
Nereidae. Clark has produced evidence that the cerebro-vascular complex in ~htys, like that in Nereis, constitutes a route of elimination of hormones secreted by the supraoesophageal ganglion. -11-
MATERIALS AND :HETHODS FOR THE WORK ON BRAIN ANATOMY
The anatomy of the supraoesophageal ganglion of Hermodice carunculata was studied macroscopically by dissections and micro- scopically by serial sections.
(a) Dissections: Dissections were performed on both Bouin's• preserved material and on fresh material obtained during the author's stay at the Bellairs Research Institute.
With the preserved material, optimal results were obtained when the dissections were performed following 6-8 hours' maceration of the
0 heads in 5% aquaeous nitric acid at 37 C. Following incubation in nitric acid, the heads were washed with tap water and dissected in 70~ ethanol. The nitric acid caused selective digestion of muscular and connective tissue elements so that these could be teased away very readily without injury to the underlying nervous tissue.
In the dissections of fresh material, methylene blue was used as a selective vital stain for the nervous tissue (Henry, 1947). The procedure most suitable for Hermodice carunculata was worked out simply by trail and error and was found to be as follows. The living worms were placed in glass dishes containing sea water. By means of a hypodermic syringe, 2 cc. of .5% aquaeous methylene blue was injected into the coelomic cavity through the mid-dorsal surface of the body.
After two hours, the worms were slowly narcotized by the graduai addition of 90% ethanol to the dishes. This facilitated dissection by assuring that the worms assumed a completely relaxed position with the prostomium fully extended. After narcosis, the anterior ends of the worms were removed and dissected in 90% ethanol. -12-
(b) Histological Work: All material used in the histological work was treated in the same manner prier to staining. The specimens used were all medium-sized worms (i.e., about 10 cm. long). All specimens were fixed whole in Bouin's fluid (made up in sea-water to prevent shrinkage) for 12-24 hours, and subsequently stored in 70% ethanol. Prier to sectioning, the anterior ends of the worms were removed, dehydrated and double impregnated in celloidin and paraffin according to Peterfi's methyl benzoate method (Carlton and Drury, 1957).
Three staining techniques were employed for stuyding the micro structure of the brain. These were: Bodian's (1937) Protargol method;
Mallory's triple stain (Davenport, 1960); Gabé's (1953) paraldehyde fuchsin with Halmi's (1952) trichrome counterstain. This last tech nique, Gabé 1 s paraldehyde fuchsin followed by Halmi's trichrome counter stain, was introduced by R. B. Clark and was described in detail by him in 1955.
The specimens used in the Protargol and Mallory 1 s triple techniques were collected in Barbados by Marsden in the summer of 1960. The specimens used in the paraldehyde fuchsin-Halmi technique were procured by the author in Barbados during the summer of 1963.
The heads to be stained with Protargol and Mallory's triple stain were eut in 7 serial sections in transverse and frontal planes. The heads to be stained with paraldehyde fuchsin-Halmi's were eut in 7 seria! sections in the transverse plane only. A brief outline of these techniques follows.
(i) Bodian's Protargol technique is very suitable for study
of the polychaete nervous system. For Hermodice nervous tissue,
the best results were obtained when certain modifications and
precautions were observed. Bodian states that the sections may -13- be immersed in the Protargol bath for 12·48 hours. lnvariably it was found that optimal staining results were obtained when the sections were incubated in Protargol for the full 48 hours.
Following the silver impregnation, Bodian prescribed reduction in a solution composed as follows: hydroquinone .. . 1 gm •
sodium sulphite ••• 5 gm. distilled water .. . lOO cc • It was found that more favorable staining results were obtained when 10 gm. of sodium sulphite were used instead of the recommended
5 gm. Bodian recommends toning in 1% aquaeous gold chloride in order to impart a fine contrast between .the various tissue elements.
With Hermodice tissue, it was found that this concentration of gold chloride caused undesirable reddish tones in the sections. A more dilute gold chloride solution of .2% was far more satisfactory.
After immersion in gold chloride, Bodian prescribes transferring the sections to 2% oxalic acid for 2-5 minutes in order to intensify the stain (owing to the effect of oxalic acid in increasing the deposit of metallic gold on the already existing deposit that resulted from the gold which replaced the silver during the gold toning). However, in practice, it was found that submersion in oxalic acid for 2-5 minutes caused excessive darkening of the stain. As Bodian himself suggests, this difficulty is surmountable by giving the sections merely a brief rinse in oxalic acid and then passing them promptly to rinsing in several changes of distilled water. The Bodian technique calls for the presence of clean copper shot (washed in concentrated nitric acid and weil rinsed in distilled water) in the
Protargol solution during the incubation period. The role of the -14- copper is to inhibit the silver impregnation of collagenous fibers.
According to Davenport (1960), "•••••• The Protargol first impregnates both neural and connective tissues and the connective tissue is destained by the copper, since it is a common observation that a greater degree of differentiation between neural and con nective tissue elements is obtained when copper is usedu. However, the author found that the presence of the copper resulted in the deposition of greenish copper salts on the slides. To avoid this, it was necessary to caver the copper shot with cover-slips.
Carried out as outlined above, the Bodian technique was found to be a very specifie and informative stain for neural tissue. The nerve cell bodies and their processes appeared grey or lavender.
The neuroglial cells and their process stained in a similar manner.
All cell nuclei stained rather strongly, appearing deep lavender to black. The nucleoli were usually obscured. Six supraoesophageal ganglia were stained by the Protargol method of Bodian.
(ii) The second staining technique employed was introduced by R. B. Clark and described by him in detail in 1955. lt involves the use of paraldehyde fuchsin prepared according to the method of
Gabé (1953) followed by Halmi 1 s (1952) trichrome counterstain.
The component stains of this counterstain are light green (fast green), orange Gand chromotrope 2R. This method gives good differentiation of tissues in the brain, and furthermore, the paraldehyde fuchsin stains neurosecretory products. Therefore the technique has the advantage of demonstrating brain structure and neurosecretory products in the same preparations. The neuron -15-
cell-bodies and their processes stain very faint greyish-green
and neuroglial cells and their processes stain a darker green.
Nuclear chromatin appears purplish-brown to reddish-brown while
the nucleoli stain bright red with chromotrope 2R. Acidophilic
cytoplasmic granules and muscle tissue stain varying shades of
orange with orange G. Collagen and basement membrane are stained
green with the fast green. Neurosecretory products are stained
by the paraldehyde fuchsin and appear lavender to deep purple.
A total of twelve supraesophageal ganglia were stained by this
technique.
(iii) The Mallory's triple stain was employed according to
the method outlined by Davenport (1949). Satisfactory staining
results were obtained despite the fact that Bouin's fixative
was used instead of the recommended zenker's fixative. Three
supraoesophageal ganglia were stained with Mallory's triple.
In addition to the above material, the author bad at her disposai two supraoesophageal ganglia which bad been sectioned and stained by
Marsden. One ganglion bad been eut in seriai transverse sections of 5~ and stained with PA/S (following diastase digestion) and hematoxylin.
This ganglion was prepared from a specimen taken by Marsden in Barbados in the summer of 1960. The other ganglion bad been eut in seriai transverse sections of 5~ and stained with Mallory's triple stain.
This specimen was also taken in Barbados by Marsden in the summer of
1960. -16-
OBSERVATIONS FOR THE WORK ON BRAIN ANATOMY
The External Mor~Èology of the Head
A thorough description of the external morphology of the head is desirable before proceeding with an account of brain anatomy. The external morphology of Hermodice carunculata has been described fully by Mullin (1923).
The head of Hermodice ~U..!l~!l-lata is a typical polychaete head
(Fig. 1, 2). It is composed of two divisions, the prostomium and the peristomium. The peristomium is continuous with the segmented trunk and appears as part of it except for the fact that it is considerably smaller than the following trunk segments. Like the trunk segments, the peristomium bears notopodia, neuropodia, gills, setae, dorsal and ventral cirri. The peristomium is the first setiger. The prostomium is squarish and flattened dorso-ventrally and sits on the dorsal surface of the peristomium. Two large, parallel, fleshy lips begin at the anterior face of the prostomium and extend over the ventral surface of the peristomium and backwards to the anterior edge of the mouth. The lips are hollow. The mouth is ventrally situated and oval. The anterior circumference of the mouth opening is in the middle of the third setiger and the posterior circumference is on the posterior edge of the fourth setiger. During feeding the pharynx is everted through the mouth, thereby forming a proboscis through which the animal ingests its food.
At their anterior ends, where they arise from the prostomium, the two lips are confluent, while at their posterior ends they remain separate and turn into the anterior end of the mouth cavity.
The following structures are found on the prostomium: Two pairs -17-
of eye~spots; three antennae; two palps and a caruncle (Fig. 1). The eye-spots are on the dorsal surface of the prostomium. One pair is located on the anterior dorsal surface and the other on the posterior dorsal surface. The posterior pair is smaller than the anterior pair and also slightly further apart than the anterior pair. The eye-spots appear as small brick-red protuberances of the epidermis. In the centre of each eye-spot is a round black spot. The three antennae are found on the dorsal surface of the prostomium and the two palps on the anterior surface. Two of the antennae are paired and the third is single. The paired antennae project from the antero-dorsal edge of the prostomium~ just behind the origin of the lips. The single prostomial antenna is situated more posteriorly, arising from the mid-dorsal surface of the prostomium, midway between the anterior and posterior pairs of eyes.
The single antenna is larger than the paired antennae.
The paired palps are directed anteriorly and arise from the anterior face of the prostomium, flanking the lateral edge of each lip elevation.
(Fig. 2).
The caruncle, located dorsally, is oval and cushion-like (Fig. 1).
It extends posteriorly from the posterior edge of the prostomium to half way~ong the sixth setiger. The caruncle is transversely folded to give two rows of eight converging laminae. According to Racovitza (1896), the caruncle is actually the paired nuchal organs which have become very enlarged and elaborated and joined via a medium mass. The caruncle is considered to be an olfactory organ (Grassé, 1959), and owes its sensitivity to the presence of ciliated cells on its lateral borders.
The median mass insensitive. When referring to the caruncle, Hartman
(1951) calls it the "prostomial carunclen, thereby inferring that the -18- caruncle is attached solely to the prostomium. However, the author invariably found the caruncle to be attached not only to the prostomium, but also to the peristomium and the second and third setigers. In other polycheates, the nuchal organs are confined to the prostomium.
The Eye-Spots
No description of the histology of the eye of this species could be found in the literature. The author's histological preparations revealed that ali four eyes are structurally identical. The eyes of Hermodice carunculata are highly developed and are structurally nearly identical to those of Nereis. The eyes of Hermodice carunculata are cup•shaped structures. The eye-cup is nearly closed. It is lined by a light- sensitive surface, the retina, and encloses a lens-like body which is separated from the retina by a vitreous layer. The retina is a single celled layer. It is comprised of bipolar primary neurons separated from each other by pigmented supporting cells. The supporting cells are pyriform. the lower (proximal) end of each supporting cel! is drawn out into a long, tenuous, slightly coiled filament. The nucleus is large and elongated and is situated toward the proximal end of the cell. The entire cytoplasm is densely packed with tiny dark brown spherical pigment granules. Each pigmented cell is separated from its neighbour by one bipolar neuron.
The neuron cell•bodies are situated on a lower level than the cell-bodies of the adjacent pyriform pigmented cells. They are on a level with the bases of the filamentous extensions of the pigmented ce lls. The axons are proximal and converge to form a fiber tract which runs downwards to enter the brain. The distal dendrites of the bipolar neurons run upwards between the pigmented cells. According to Grassé, in Nereis the distal processes can be followed along their entire course between the pigmented -19- cells and can be seen to terminate as tiny fibrils projecting into the hyaline vitreous layer. In the histological preparations of Hermodice carunculata it was not possible to follow the course of the distal processes. They could only be seen to disappear between the pigmented cells. In Nereis, the supporting cells are pigmented only in the distal portion of the cell body, while in Hermodice carunculata the entire cell-body, including the proximal fibrillar extension, is packed with pigment granules.
The eyes of Hermodice carunculata are of an entirely epidermal nature, being bounded laterally and ventrally by sub•epidermal basement membrane
(Fig. 4).
The Gross Morehology of the Brain
The gross morphology of the brain is shown in Fig. 3. The brain is entirely prostomial in Hermodice carunculata. lt commences in the anterior end of the prostomium, slightly anterior to the first pair of eyes, and extends back usually to the leve! of the posterior eyes. In large specimens
(length about 20 cm. or over), the brain extends back to a level slightly posterior to the second pair of eyes.
Like the brain of Eunice (Grass~, 1959), the brain of Hermodice carunculata can be divided into three parts, on the basis of external morphology. One can readily discern a forebrain, a midbrain and a hindbrain situated behind one another. Each part is composed of two symmetrical halves. The brain expands laterally and dorso-ventrally at a level approximately a third of the distance between its anterior and posterior extremities. This point of expansion marks the division between the fore brain and the midbrain. The hindbrain is smaller than the forebrain and midbrain. Its two halves are well separated by a posterior notch. The hindbrain, which gives the impression of being an appendage of the midbrain, ·20- superficially resembles the "posterior lobes" of Nephtys (Clark, 1958b), although the two structures are not homologous. In his diagrammatic illustration of the Amphinomid brain, Gustafson (1930) does not depict this bifurcation at the posterior end of the brain. In Eunice (Grass~,
1959), the two halves of the forebrain and also the midbrain are joined by a transverse commissure. In Hermodice carunculata there is only one transverse commissure, that which joins the two halve s of the midbrain.
There is no comtnissure joining the two halves of the forebrain. In both Eunice and Hermodice, the two halves of the hindbrain, separated by the posterior notch, are not joined by a commissure.
The epidermal origin of the brain is clearly manifest in Hermodice carunculata (Fig. 4). The brain is bounded laterally, ventrally, anteriorly and posteriorly by a connective tissue sheath which is con tinuous with the epidermal basement membrane. Only the dorsal surface of the ganglion is not delimited by such a membrane. The dorsal surface of the brain is sunk below the dorsal prostomial epidermis from which it is separated by a layer of transverse muscle tissue.
The cranial nerves emerging from the brain are also intimately connected with the epidermis, being bounded by extensions of the sub epidermal basement membrane.
The Cranial Nerves
Cranial Nerves Arising from the Dorsal Surface of the Brain (Fig. 3)
The cranial nerves arising from the dorsal surface of the brain are listed as follows, in the arder of emergence, passing from the anterior to the posterior end of the brain:
(a) The Tegumentary Nerves (Fig. 19A)
These are a pair of nerves emerging from either side of
the dorsal surface of the forebrain and running directly into -21-
the overlying prostomial epidermis. Upon entering the epidermis
they ramify extensive ly to form innumerable free nerve endings
(sensory no doubt) throughout the dorsal prostomial epidermis.
(b) The Nerves to the Paired Antennae
These are two cranial nerves, one emerging from the dorsa
lateral surface of each half of the midbrain and entering the
antenna on the same side.
(c) The Optic Nerves to the Anterior Eyes (Fig. 19E)
Each anterior eye is innervated by a nerve tract leaving
the dorsa-lateral surface of the midbrain, just behind the
emergence of the nerves of the paired antennae.
(d) The Nerves to the Single Dorsal Antenna (~ig. 5, 17A)
The median unpaired antenna is innervated by two slender
nerve tracts which leave the dorsal surface of the midbrain on
either side of the median line. Before entering the antenna,
these nerves cross each other to form a chiasma•like configura
tion at the base of the antenna (Fig. 5). In the cross roads
of this chiasma, a portion of the fibers of the right nerve tract
cross to the left tract, and vice versa.
(e) The Nerves to the Caruncle
The brain sends eight nerves to the caruncle. These nerves
all arise from the hindbrain. The first four nerves arise from
the dorsal surface of the hindbrain. These four caruncular
nerves occur in two pairs, one pair emerging behind the other.
The first pair of nerves is considerably larger than the second
pair.
The second set of four caruncular nerves actually consists -22-
of extensions into the caruncle of the posterior extremity of
the brain. As previously mentioned, there is a slight bifurcation
at the posterior end of the brain due to the separation of the
two halves of the hindbrain by a notch. Within the prostomium,
the se two extensions are directed upwards towards the overlying
caruncle. At its posterior extremity, each extension tapers and
divides into two nerve tracts which enter the caruncle.
Cranial Nerves Arising from the Lateral Surface of the Brain (Fig. 3)
(a) The Optic Nerves to the Posterior Eye s
Each posterior eye is innervated by a nerve which arises
from the mid-lateral surface of the midbrain, at a level just
posterior to the point of emergence of the nerves to the single
antenna.
(b) The Lateral Epidermal Nerves (Fig. 6)
These are a pair of slender nerve tracts, one arising from
either side of the mid-lateral surface of the midbrain, slightly
anterior to the emergence of the circumoesophageal connectives.
These nerves enter the lateral prostomial epidermis, where they
ramify extensive ly to form innumerable free nerve endings in the
epidermis.
(c) The Circumoesophageal Connectives (Fig. 71 17A, 19F)
The largest, most conspicuous nerve tracts leaving the
brain are the circumoesophageal connectives. These arise from
the ventro•lateral surface of the midbrain at approximately the
same level as the antennal nerves to the paired antennae. After
leaving the brain, the connectives travel posteriorly and ventro- -23-
medially to converge on the suboesophageal ganglion in the
seventh setiger. Each connective appears to emerge from
the brain as a single structure. However, histological
sections reveal that each connective has a double root.
There is a dorsal and a ventral root, each having a different
origin within the brain.
Cranial Nerves Arisil'!.&..-~.rom the Anterior Surface of the Brain (Fig. 1)
( n) The Pal~al Nerve s (Fig. 17B)
The palps are innervated by a pair of nerves arising
from the antero•ventral surface of the forebrain.
(b) The Stomatogastr_i_c;___ Nerves (Fis• 8 1 9A 2 17B) From the antero•ventral edge of the forebrain, just below
the emergence of the palpai nerves, there arises a mass of
nerve fibers directed ventrally and posteriorly. Almost
immediate ly after leaving the brain, this mass of nerve fi bers
divides into two thick tracts. These are the stomatogastric
nerves. The stomatogastric nerves innervate the digestive
tract. Through complicated ramifications, they supply the
lips, the buccal mass, the pharynx and the entire alimentary
canal. The distribution of the stomatogastric nerves has been
described in detail by Marsden (1962b).
"Tetraneurie" in Hermodice carunculata
The following is a translation, by the author, of a passage from
Grassé.
nin the Amphinomid brain, apart from the perioesophageal connectives, there extends a pair of lateral nerves, which, in certain species (e.g. Hermodice), extend to the posterior extremity of the body. In each segment these nerves bear a ganglion, the pedal ganglion, which is the nervous centre of the parapodium and which is in communication with the medioventral chain by means of a transverse commissure. Storch (1912) classifies as 11 Tetraneurestt the Amphinomids which possess such a structure (Tetraneurie) and as uoineures" the other polychaete annelids. n
In all the dissections and histological preparations studied, the author has found a discrepancy between her observations and the descrip- tions by Storch and Grassé. The same discrepancy has been noted by
Marsden. * lt sppears that, in actual fact, the longitudinal pedal tracts, bearing the pedal ganglia, do not arise directly from the brain, as contended by Storch and Grassé. The author is convinced that they arise from the circumoesophageal connectives about midway along their length.
The Microanatomy of the Supraoesophageal Ganglion
Prostomial and Brain Cavities
Neither the brain nor the prostomium are solid structures. The brain is partially surrounded by a prostomial cavity, and there is a cavity within the brain itself. Also, there is a cavity in the caruncle which invades the bra.in tissue to a considerable extent.
Transverse sections of the posterior regions of the brain show that, ventrally and laterally, the brain is not in contact with pros- tomial tissue. Instead it is suspended above a cavity in the prostomium
(Fig. 4, 9B). This cavity is continuous with the coelomic cavity of the body. The cavity tapers off anteriorly~ and ends at the level of the anterior part of the midbrain. Anterior to this point, the ventral surface of the brain is in contact with the prostomial sub-epidermal
* Unpublished -25- basement membrane (Fig. 9A). Just before it terminates, this coelomic cavity beneath the brain sends off two finger-like extensions which turn upwards and run anteriorly along each side of the brain, and extend as far forward as the anterior extremity of the prostomium (Fig. 9A, 10).
The coelomic cavity beneath the posterior regions of the brain is lined by a double membrane (Fig. 9 , 11, 31 ). This is comprised of an outer connective tissue sheath which is continuous with the basement membrane of the lateral prostomial epidermis, and an inner syncytial layer of protoplasm which borders the lumen of the cavity (Fig. 11).
The syncytial layer is a 3-5~ thick. In this inner layer the nuclei are large and chromophobic and nearly s~rical (slightly irregular) and are evenly spaced in a single layer. In the region of the posterior extremity of the brain, the thickness of this syncytial layer increases to 35-40}Â and there are found many small blood vessels running through it (Fig. 31). These vessels are derived through repeated branching of the dorsal blood vessel. The anterior extensions of the infra-cerebral cavity are not lined by a double membrane. There is only a single connective tissue lining which is continuous with the prostomial sub• epidermal basement membrane (Fig. 9A, 10).
In addition to the prostomial vacuities described above, there is also a cavity enclosed within the brain tissue itself. This cavity takes the form of a long narrow tubular canal running longitudinally through the ventral portion of the neuropile, in the median line. It commences in the forebrain and extends posteriorly as far as the middle of the mid brain (Fig. 10, 12). This cavity in the brain is reminiscent of a
"ventriclen of the vertebrate brain. It is lined by a thick connective tissue membrane exhibiting staining properties identical to those of the -26- basement membrane. The lumen of this canal is chromophobic. This suggests that the original contents, possibly sorne sort of fluid, were dissolved out in the histological procedures employed. The canal contains neuroglial cells and fibers and occasional clusters of small, spherical, strongly fuchsinophilic granules.
The caruncle is a hollow structure and the cavity in the stalk of the caruncle extends downwards into the brain tissue below, where it expands laterally. The result of this downward extension of the caruncular cavity is a shallow wide depression or concavity in the dorsal surface of the brain (Fig. 9B). The depression extends from the level of the middle of the midbrain to the posterior extremity of the brain. The depression becomes wider posteriorly. The caruncular cavity and the dorsal depression in the brain are lined by a single uninterrupted layer of basement membrane. The caruncular cavity and the dorsal brain cavity are separated by a barrier of several transverse strands of connective tissue. Each strand projects from the basement membrane lining on one side of the cavity and extends across the cavity to become continuous with the lining on the opposite side
(Fig. 9B).
As previously noted, the dorsal surface of the brain is separated from the overlying epidermis by a transverse barrier of muscle tissue.
This barrier is interrupted in the area of the caruncular cavity and the dorsal brain cavity. Inside both the caruncular cavity and the dorsal brain cavity are found droplets and granules of fuchsinophilic material and fibroblasts. The fibroblasts are large fusiform cells with an acido• philic cytoplasm. They have a large spherical nucleus with a single nucleolus. The dorsal brain depression contains sorne neuroglial cells.
These are most abundant in the ventral portions of the depression which are in close proximity with the brain tissue. -27-
Brain Membranes
The supraoesophageal ganglion is epidermal. It is bounded by the sub-epidermal basement membrane of the prostomium or by extensions of this basement membrane. In the anterior regions of the brain, the basement membrane of the ventral prostomial epidermis serves as the ventral brain sheath (Fig. 9A). Posteriorly, where the ventral surface of the brain is suspended above a coelomic cavity (Fig. 4, 9B), the ventral brain sheath is formed by an inward extension of the lateral prostomial basement membrane. Here the ventral brain sheath is actually double and is identical to that which lines the coelomic cavity below the brain in the same region. It consists of an inner connective tissue sheath which is continuous with the basement membrane of the lateral prostomial epidermis, plus an outer pericapsular membrane (Fig.
4, 11). This outer membrane consists of a very thin syncytial layer of protoplasm adhering to the connective tissue sheath and containing, at regular intervals, a single row of large, nearly spherical chromophobic nuclei (Fig. 11). The pericapsular sheath extends along the sides of the brain, but here it is rouch less prominent, detectable only as occasional nuclei and strands of cytoplasm adhering to the connective tissue sheath.
The syncytial layer which forms the inner lining of the coelomic cavity beneath the brain and the syncytial layer which forms the pericapsular sheath of the brain both appear to represent modified coelomic epithelium.
At the sides of the brain, where the two syncytial layers come into contact, they ~re confluent with each other. Also, these two layers are connected by numerous cytoplasmic strands which extend across the inter vening coelomic space (Fig. 11, 31). -28 ...
Epidermal Sens~!Y Cells
There are numerous tufts of cilia on the lateral borders of the ca rune le and on the dorsal surface of the prostomium (Fig. 13 ). The se tufts arise from sensory cells embedded in the epidermis of the caruncle and prostomium respectively. Morphologically these cells are typical invertebrate sensory cells. They occur in groups of 3 or 4 cells in a row. They are slender goblet-shaped cells bearing a fringe of cilia on their distal borders. The cilia protrude through tiny pores in the cuticle. The large, oval, chromophobic nucleus is situated towards the proximal end of the cell and contains a single round nucleolus. Proximal to the nucleus, the cell narrows abruptly into a long tenuous filament.
These filaments travel downwards through the epidermis and disappear into the dorsal surface of the brain (either fore-, mid-, or hindbrain, depending upon the location of the sensory cells).
General Brain Architecture
The brain of Hennodice carunculata contains only truly ganglionic material (i.e., nerve cells and neuroglia). There are no connective tissue or muscular elements within the brain itself. There are no blood vessels penetrating the brain tissue. Mucus-cells, which are abundant in the prostomial epidermis, do not occur in the brain.
The neuron cell-bodies are located peripherally, forming a cortex
or mantle over the top and sides of a central fibrous neuropile (Fig. 10).
Neurons are also found in the ventral part of the brain beneath the neuropile, but these are relatively few. There are no neuron cell .. bodies within the mass of the neuropile.
Neuroglial tissue is plentiful. Typical neuroglial cells and fibers occur randomly throughout the cortical layer (Fig. 25). They are also scattered throughout the neuropile (Fig. 7, 22). Neuroglial tissue is most abundant in the ventral regions of the neuropile.
Neurons
Within the brain, five different types of neurons may be distinguished:
(1) Ordinary neurons. These are of two sorts:
(a) Ordinary neurons with small pyriform cell•bodies
(b) Ordinary neurons with large subspherical ce ll•bodie s
(2) Globuli cells
(3) Bipolar neurons
(4) Giant neurons
These cell types are illustrated in Fig. 14.
The Ordinary Neurons
These are typical neurons, having a large amount of cytoplasm, and a clear chromatin•poor nucleus. There appear to be two types of ordinary neurons: those with small pyriform cell-bodies (Fig. 14A, B;
25 ) and those with larger, subspherical cell•bodies (Fig. 14C, 29).
The small pyriform neurons are about 7JJ. long and 4)-A. wide at the broadest portion. The subspherical neurons are about 8~ long and 8~ wide in the proximal end of the cell-body.
These two types of neurons will be referred to as "ordinary pyriform" and "ordinary subspherical" neurons respectively. The ordinary pyriform neurons are by far the most abundant nerve cells in the brain. Both cell types possess a single axon and severa! dendrites.
The pyriform neurons have a single ovoid or bean-shaped nucleus (length -30-
about 4~ ; width about 3~) which is usually located in the proximal portion of the cell-body (Fig. 14A, B).
The sub-spherical neurons (Fig. 14C) have a single, spherical nucleus (diameter about 4~) in the centre of the cell-body. Both types of ordinary neurons may exhibit fuchsinophilia. Sorne of the fuchsinophilic pyriform neurons were characterized by the absence of dendrites and/or the presence of the nucleus in the axon hillock rather than in the proximal portion of the cell-body (Fig. 25). In most cases, the nuclei of both types of cell contain a single small nucleolus. The nucleolus was characteristically located off .. centre, near the nuclear membrane. Sorne neurons of each type had two or three such nucleoli. There was no correlation between the number of nucleoli and the staining reactions of the neurons. One or more collateral branches extended from the axons of many of the ordinary neurons.
The Globuli Cells (Fig. 8t l4D)
The globuli cells are specialized neurons which are restricted to the corpora pedunculata of the brain. Clark (1958c) describes globuli cells as follows:
"• ••• The se bodies (i.e., the corpora pedunculata) are found in the anterior part of the supra-oesophageal ganglion of many polychaetes, including the Aphroditidae, Hesionidae, Nereidae, Eunicidae and Serpulidae (Hanstrom, 1927), and of course, in the higher crustaceans and insects. The corpora take the form of large numbers of small globuli cells with little cytoplasm and chromatin•rich nuclei arranged in a mushroom body with a stalk composed of the axons of these cells. There are many dendritic branches of the axons in the stalk (Holmgren, 1916; Hanstrêim, 1927). 11
The globuli cells of Hermodice carunculata are quite typical. The spherical cell-body is small (diameter about 2.5)1) and poor in cytoplasm, with a chromatin-rich nucleus (diameter about 1.8)J. ). The nucleoli were obscured by the dense, darkly•staining chromatin. These characteristics readily distinguish the globuli cells from the ordinary neurons with their plasma-rich cell-bodies and chromatin-poor nuclei. The globuli cells and the stalks of the corpora pedunculata have a greater staining affinity than ordinary neurons and are therefore very conspicuous.
Both the neuroplasm and the axoplasm are strongly argentophilic and acidophilic. No fuchsinophilic globuli cells were found.
The axons of the globuli cells converge to form the stalks of the corpora pedunculata (Fig. 8). The axons have numerous collateral branches, while the cell~bodies have conspicuous dentritic extensions.
The Bieolar Ne2rons (Fig. 14E, 15)
These occur in small numbers in the brain, where they are restricted to one specifie site (nucleus VII) in the outermost edge of the lateral cortex in the midbrain. These neurons have a number of distinctive cytological characteristics. They possess one axon and one long main dendrite projecting from opposite ends of the fusiform cell~body. The cell•body is plasma-rich and slender and fusiform in outline (length about 9_p.; width about 3.5p ). Strictly speaking, the se neurons are not truly bipolar, since, in addition to the single main dendrite, there are other short minute dendrites extending from the cell-body. The large oval nucleus is centrally located. It is about 4f(long and 2.5~ wide.
The staining reactions of the bipolar neurons differ from those of the ordinary neurons. The cytoplasm is intensely acidophilic, staining darkly with fast green. There are numerous very fine granules distributed throughout the cytoplasm. The se are also acidophilic, but have an affinity for orange G rather than fast green. Sorne of the bipolar neurons -32-
were fuchsinophilic. Unlike the clear, chromatin-poor nuclei of the ordinary neurons, the nuclei of the bipolar neurons contain a large amount of basichromatin. The chromophil material of the nucleus obscured the nucleoli.
The Giant Neurons (Fig 14F, 30)
There are a very few giant-sized neurons scattered randomly throughout the brain. Except for the ir large size ( length about 15}1 ; width about 6~), they are morphologically very similar to the ordinary pyriform neurons. The cell-body is pyriform, with a prominent axon hillock. The cell-body of the giant neuron is proportionally more slender and elongated than that of the ordinary pyriform neuron. There are tiny dendrites extending from the cell-body. The cytoplasm may show any of a wide variety of staining reactions, ranging from chromo phobia to varying degrees of acidophilia, or various intensities of fuchsinophilia. The aval nucleus is chromatin-poor and there is usually more than one nucleolus.
Neuroglia
Neuroglial cells are scattered among the neuron cell-bodies of the cortex (Fig. 25). They also occur throughout the neuropile (Fig. 7, 22) and even inside the canal which runs through the centre of the brain
(Fig. 12). The neuroglia is more abundant in the neuropile than in the cortex.
On the basis of morphology, all the glial cells of the brain are of the same type (Fig. 14G). They are readily detected by their nuclei which are irregular in outline (as opposed to the regular outline of the nuclei of the neurons) and slightly elongated. In all the techniques used, the nuclei of the neurolial cells stained more deeply than those -33-
of any type of neuron. The glial nuclei are about 4~ long and 3JUL wide.
The PA/S-hematoxylin preparations revealed that the nuclear membrane is notably thicker in the glial cells than in the neurons. There is little cytoplasm around the nucleus and it is invariably acidophilic.
The cell-body is highly irregular in outline (much more so than the nucleus) and bears numerous, highly branched cytoplasmic extensions.
These extensions are the neuroglial fibers and are at least as long as the cell itself.
Neuroglial cells were found in 3 different types of relationship with the nerve cells:
1. They occur in close proximity to nerve cell-bodies and fibers, without coming into actual contact with the neurons them selve s.
2. The glial fibers terminate upon the surface of nerve cell-bodies and axons.
3. The glial cells and their fibers surround fiber tracts within the brain, thereby forming a narrow reticular sheet about the tract. Sorne of the cells may send fibrous extensions directly into the tract.
With the staining techniquesused, it was not possible to determine whether any of the neuroglial ce lls se nd cytoplasmic extensions right into neuron cell-bodies, thereby forming a trophosponge (Defretin, 1955).
There are three sites where the neuroglial cells are specially oriented. When they surround fiber tracts within the brain, the neuroglial cells are usually longitudinally oriented. There is a row of neuroglial cells bordering the ventral edge of the brain, just inside the connective tissue membrane and another row of neuroglial cells along the dorsal edge of the brain. ln both these neuroglial borders, the cells -34-
are horizontally oriented (Fig .. 32). These neuroglial cells form a very thin reticular layer along the dorsal and ventral edges of the brain.
Except for the three areas mentioned above, the orientation of the neuroglial cells appears to be entirely random.
The lEEernal Organization of the Brain
General Brain Organization
In Hermodice carunculata the neuropile is a single continuous structure. It is not divided into an anterior and posterior neuropile
As in Nephtys (Clark, 1958c). The neuropile begins just behind the anterior end of the brain and runs uninterrupted to the posterior extremity of the brain. The relative amount of neuropile tissue and cortical tissue varies along the length of the brain. Anteriorly the neuropile mass is small, with a relatively wide mantle of neuron cell bodies (Fig. 10). As it runs posteriorly, the neuropile expands, due to the reception of more and more axons from the cortical layer. Con current with the expansion of the neuropile is a progressive reduction of the cortex to a narrower and narrower zone. Thus, proceeding from the anterior to the posterior end of the brain, there is a progressive displ~cement of cortex by neuropile. This displacement reaches a climax in the posterior portion of the hindbrain, where the neuropile cornes to comprise nearly thA entire mass of the brain tissue (Fig. 4). At the end of the hindbrain t!tc neuropile termina tes by becoming subdivided into 4 nerve tracts which run dors:1lly into the caruncle (Fig. 3).
The two si des of the midbrain are joined by a transverse commissure
(Fig. 16 t 19C). Th~_Circumoesopha&eal Connectives
Viewed externally, the circumoesophageal connectives emerge as a single root on either side of the midbrain. However, histological sections show that, in actual fact, each connective commences as two roots, a dorsal root and a ventral root (Fig. 7, 17A). The two roots are separated by a small, connective tissue-lined space and have different origins within the brain. Almost immediate ly after leaving the brain, the two fiber tracts unite to become a single tract. The two roots are so short and so close together that, unless one takes histological sections, one would conclude that the connectives emerge as single roots. The ventral fiber bundle is derived entirely from the neuropile.
The dorsal tract has a heterogeneous origin, deriving its fibers from three different sources: (1) From the neuropile; (2) From a fiber tract which is formed by the axons of a cluster of cortical neurons in the lateral side of the midbrain (nucleus VIII); (3) From the axons of a small cluster of neurons situated just lateral to the root of the dorsal fiber bundle of the connective (nucleus VI). The circumoesophageal connectives are bounded by extensions of the sub-epidermal basement membrane.
The Ganglionic Nuclei (Fig. 18. 19A-H)
As in Nereis (Holmgren, 1916) and Nephtys (Clark, 1958c), so in
H. carunculata, the neurons of the cerebral cortex are arranged in paired ganglionic nuclei. The neuroglial cells are evenly dispersed throughout the cortex and do not serve to de lirnit the various ganglionic nuclei. Furthermore, the nerve cells tend to be uniformly distributed.
Consequently, few of the ganglionic nuclei are clearly separated from -36-
their neighbours. In sorne nuclei the axons enter the neuropile in
discrete bundles, so that the positions and extent of the nuclei can
be readily discerned. However, in other nuclei, the axons enter the
neuropile singly rather than converging to form discrete fiber tracts.
In such cases it is diificult to determine accurately the boundaries of
the nuclei in question. The situation is somewhat simplified by the
fact that the nuclei usually occupy the entire width of the cortex, so
that few nuclei are situated in overlying positions. However, many
nuclei overlap each other slightly.
The following is an account of the ganglionic nuclei of the brain.
An arbitrary system of numbering with Roman numerals has been devised
for designating the pairs of nuclei. lt should be emphasized here that
the numbers used for Hermodice carunculata do not correspond to those
used by Holmgren (1916) for Nereis diversicolor. Furthermore, the
numerical sequence of nuclei in H. carunculata is not intended to
correspond to the alphabetical sequence of nuclei described by Clark
(1958c) for Nephtys.
(A) Nuclei in the Forebrain (Fig. 18)
I, II. (Fig. 8, 178, 19A). These two pair of nuclei
constitute the corpora pedunculata and are the anteriormost nuclei of
the brain. I is situated median and slightly dorsal to II. The axons
from each nucleus are directed downwards and form short discrete fiber
tracts. The fiber tracts from nuclei I and II unite to form a single wide, ventrally-directed tract running through each half of the forebrain.
Most of the fibers in these tracts run straight through the brain and emerge from the antero-ventral surface of the brain. Just as they leave the brain, they unite to form a single mass of fibers, and just after leaving the brain they separate again into two separate fiber tracts.
The single mass of fibers is the root of the stomatogastric nervous system (Fig. 3) and the two separate tracts into which it divides are the stomatogastric nerves (Fig. 3). However, not all of the axons from nuclei I and II contribute to the stomatogastric system. Sorne of them are diverted along other paths (Fig. 17B). A small proportion of them are directed posteriorly, thereby constituting the anteriormost fibers of the neuropile, i.e., the neuropile is initiated by nuclei I and II.
Also, just before uniting to form the stomatogastric root, each fiber tract sends off a slender fiber tract from its anterior edge. These tracts are the palpai nerves. They run anteriorly and enter the paired palps.
Nuclei I and 11 are composed of small, plasma-poor, chromatin-rich globuli cells (Fig. 8, 14D). Globuli cells are found nowhere else in the brain. Nuclei I and II, together with their axon tracts, constitute the corpora pedunculata of the brain. Thus, in Hermodice carunculata, the corpora pedunculata are bilobed, consisting of two globuli (nuclei I and II) on each side of the brain. The peduncles of the globuli are the axon tracts of nuclei I and II. On each side of the brain the peduncles of the two globuli merge with each other to form a single ventrally directed fiber tract, the course of which has been described above.
The palpai nerves, the bulk of the stomatogastric system and the anteriormost fibers of the neuropile are all derived from the corpora pedunculata. -38-
III. (Fig. 8, 19A). These are a pair of very diffuse nuclei.
The cell-bodies are sparsely scattered over the tops and sides of nuclei
I and II and also between nuclei I and II. The axons do not form any discrete fiber tract and do not run into the neuropile. They run dorsally towards the dorsal edge of the brain. As they reach the dorsal edge of the brain, the êXons on each side converge to form the main tegumentary nerve innervating the prostomial epidermis on the same side of the body. In other words, the two main tegumentary nerves arise from nucleus III, the right nucleus giving rise to the right tegumentary nerve and the left nucleus giving rise to the left tegumentary nerve.
III is a very heterogeneous nucleus, being composed of ordinary pyriform and subspherical neurons and also a few giant pyriform neurons.
IV. (Fig. 19A). These are a pair of very small, laterally compressed nuclei. They are situated side by side between the bases of the two stomatogastric nerves. The axons run laterally and enter the stomatogastric nerves. Nucleus IV is composed entirely of ordinary pyriform neurons.
(B) Nuclei in the Midbrain (Fig. IR)
v. (Fig. 19c, 20). These are large paired nuclei which commence in the antero-dorsal end of the midbrain and extend posteriorly to overlap the roots of the circumoesophageal connectives. They are very voluminous, mushroom-shaped nuclei which produce prominent bulges in the sides of the midbrain. The cell-bodies are packed together very closely and are of the ordinary pyriform type. The axons run medially to form a short stout fiber tract which enters the side of the neuropile. -39-
VI. (Fig. 7_,_ 19F', 3_Q_2_. The se are small, paired, laterally
compressed nuc.lei situated lateral to tbe h."lse of the dorsal root of
the circumoesophageal connectives. The axons are directed medially
and run into the dorsal root of the connective s. The axons do not
unite to form a discrete fiber tract. Each axon enters the connective
root independently. This nucleus is composed of ordinary pyriform and
subspherical neurons and also includes a few giant pyriform neurons.
VII. (Fig. 15, 19C). These are a pair of very small nuclei
comprised of a single row of bipolar nE~urons arrayed along the outer
edge of nucleus v. The fusiform cell-bodies occur in an almost vertical
position, but are slightly inclined from the vertical. The cells are
all inclined in the same direction and at approximately the same angle.
One main process, the upper one, is directed obliquely outwards tmvards
the brain membrane, while the other main process, the lower one, slopes mediall y towards nue le us V. Bath the main proce sse s of the bi polar
neurons appeared identical with the staining techniques used and it was
not possible to determine which WGS the axon and which \vas the dendrite.
The main processes directed towards nucleus V do not run into the
neuropi le. They termina te in the formation of synaptic connections with nerve cell-bodies in nucleus V. The main processes directed towards
the lateral brain membrane are highly branched. Some of the branches
are in contact with the brain membrane, while others terminate on the cell-bodies of neighbouring bipolar neurons. Bipolar neurons are found
nowhere else in the brain.
VIII. These are a pair of small, laterally- compressed nuclei situated median and ventral to nucleus v. The axons -40- converge to form a ventrally-directed fiber tract which runs into the dorsal root of the circumoesop~ageal connective. The fiber tract from nucleus VIII and the fiber tract from the neuropile enter the dorsal root of the connective side by side, just above the entrance to the axons of nucleus VI into the dorsal root. Nucleus VIII is comprised of ordinary pyriform neurons.
IX. (Fig. 19D). This is a pair of small, laterally-compressed nuclei situated median to the posterior portion of v. The axons are directed medially as they approach the neuropile, and they turn sharply upwards to form a fiber tract which innervates one side of the single dorsal antenna. The two fiber tracts, arising from the paired nuclei IX, cross each other to form a chiasma configuration before entering the single dorsal antenna (Fig. 5). Nucleus IX is composed of ordinary pyriform neurons.
x. (Fig. 19B .. G). This is an unpaired nucleus consisting of a thin layer of neurons scattered below the ventral surface of the neuropile. The nucleus is very diffuse. It commences just behind nucleus IV and runs posteriorly as far as the end of the midbrain.
The axons do not form any discrete fiber tracts. They are directed dorsally, and each axon enters the neuropile independently. The neurons are of the ordinary pyriform type.
XI. (Fig. 19G, 21). This is a pair of long wide nuclei comprising almost the entire dorsa-lateral cortex of the midbrain.
They begin in the anterior region of the midbrain and extend posteriorly as far as the end of the midbrain. Nue le us XI over-arche s th at dorsal depression in the brain which is formed by the downward extension of -41- the caruncular cavity. The axons from each nucleus XI form a stout fiber tract which enters the neuropile dorso~laterally, just beside the lateral connective tissue lining of the dorsal brain depression.
These nuclei are composed mainly of ordinary pyriform and subspherical neurons, but include a few giant pyriform cells. An unusually high proportion of these ordinary pyriform neurons have bean-shaped nuclei.
XII. (Fig. 16, 19C). This is a pair of small spherical nuclei in the antero-dorsal region of the midbrain. Nuclei XII are situated above the transverse comraissure of the midbrain. The axons of these nuclei do not form discrete fiber tracts. They run ventrally and then turn medially to enter the transverse commissure. The neurons are a mixture of pyriform and subspherical types.
XIII. (Fig. 19C). These are a pair of smal1 nuclei situated between the neuropile and the transverse commissure of the midbrain.
Each nucleus consists of a band of neurons strung out along the top of each side of the neuropile. The axons are directed dorsally and enter the transverse commissure above. The commissure itself is intimately connected with the neuropile. On each side of the midbrain there is a stout fiber tract connecting the neuropile and the commissure (Fig. 19C).
The neurons of nucleus XIII are of the ordinary pyriform type.
XIV. Q'_i~· 19G). This is a single, unpaired nucleus situated in the mid·line in the posterior region of the midbrain. It is a diffuse nucleus, comprised of subspherical neurons scattered over the dorsal surface of the neuropile immedinte ly below the dorsal brain depression.
The axons do not form a discrete fiber tract. They are directed dorsally and impinge upon th~ connective tissue lining of the dorsal brain -42- depression above. Sorne of the axons may be seen to penetrate the lining and extend their endings into the cavity of the dorsal brain depression
(Fig. 28) •
This is the only ganglionic nucleus to be found beneath the dorsal brain depression. Anterior and posterior to nucleus XIV, the neuropile runs directly below the dorsal brain depression.
xv. (Fig. 19C, 22). This is a pair of compact nuclei situated below the anterior portion of nucleus v. These nuclei lie directly anterior to the roots of the circumoesophageal connectives. The axons from each nucleus form a short slender fi ber tract which runs medially to enter the lateral side of the neuropile. This nucleus is composed entirely of ordinary pyriform neurons.
XVI. (Fig. tyo, E; 22). This is a pair of dorso-ventrally elongated nuclei situated ventral to and slightly median to nuclei XV.
Nuclei XV and XVI are separated by a fairly distinct oblique barrier of neuroglial fibers. This is the only instance where a zone of neuroglia intervenes between adjacent nuclei. The axons are directed medially towards the neuropile. The cortical area is very narrow in the region of nucleus XVI, with the result that the cell-bodies are not sufficiently distant from the neuropile for any fiber tract to be formed. In fact the axons are difficult to detect, because they penetrate the neuropile almost as soon as they leave the cell~bodies. A few of the innermost cell-bodies of this nucleus are actually situated in the periphery of the neuropile mass. In other words, nucleus XVI is partially embedded in the neuropile. The neurons are all of the ordinary pyriform type. -43-
XVII. This is a pair of small, compact, spherical nuclei situated just ventral to nuclei XVI. The axons are directed medially, and converge to form a very short fiber tract which enters the ventre-lateral side of the neuropile. The cell-bodies are all of the ordinary pyriform type.
XVIII. (Fig. 19G, 22). The se are small, paired, roughly spherical nuclei flanking either side of the mid-ventral nucleus X. The axons are directed dorso-medially and run into the ventro•lateral side of the neuropile. The neurons are of the ordinary pyriform type.
XIX. (Fig. 22). These are very small paired nuclei hemmed in be tween XVII and XVIII. The axons are directed dorso-medially and form a slender fiber tract which enters the ventro-lateral side of the neuropile. This fiber tract remains intact as it runs dorso-medially through the neuropile. It is flanked by neuroglial cells and fibers and its path through the neuropile can be traced until it approaches the median line. As it approaches the median line, it branches extensively and becomes lost in the general neuropile mass. The cell bodies of nuclei XIX are of the ordinary pyriform type.
xx. (Fig. 19E). These are two pair of small nuclei associated with the optic nerves to the anterior pair of eyes. The optic nerve of each anterior eye is formed mainly by the axons from the primary bipolar retinal nerve cells. These axons converge to form a fiber tract which emerges from the base of the eye-cup and runs downwards to enter the brain and disappear into the dorsal edge of the neuropile. There are
2 small nuclei, one lying on either side of this optic nerve, at the point where it enters the neuropile. The nucleus flanking the lateral -44-
side of the optic nerve has been designated as nucleus XX, while the nucleus flanking the median side of the optic nerve has been designated as nucleus XXI. The axons of each nucleus form a short fiber tract which runs into the optic nerve. These axons appear to travel upwards in the optic nerve, towards the eye, but could not be traced to their terminals. Hence the optic nerves of the anterior eyes contain fibers running in two directions. The bulk of the fibers run towards the neuropile and are the axons from the primary bipolar neurons in the retina. However, a small proportion of the fibers of the optic nerves runstowards the eye itself and these fibers are the axons from nuclei
XX and XXI. Nuclei XX and XXI are both composed entirely of ordinary pyriform neurons.
XXII, XXIII. (Fig. l9G). These are two pair of small nuclei associated with the optic nerves to the posterior pair of eyes. The optic nerves to the posterior eyes arise from the lateral edge of the neuropile, posterior to the origin of the anterior optic nerves. The relationship of nuclei XXII and XXIII to the posterior optic nerves is identical to the relationship of nuclei XX and XXI to the anterior optic nerves. That is, one nucleus flanks each side of the base of the optic nerve and sends its axons into the optic nerve. The axons travel upwards in the optic nerve, towards the eye, but they cannot be traced to their terminais. Nucleus XXII flanks the outer side of the base of the posterior optic nerve, while nucleus XXIII borders the median side. As in the anterior optic nerves, the bulk of the fibers in the posterior optic nerves consists of axons running towards the brain from the primary bipolar neurons of the retina. Whereas the neurons of nuclei XX and XXI are of the ordinary pyriform type, those of nuclei XXII and XXIII are of the ordinary subspherical type. -45-
XXIV. (Fig. 19G). This is a pair of very small nuclei flanking the outer edge of the posterior optic nerve~, just before these nerves leave the cerebral cortex. The axons are directed dorso medially and enter the optic nerves. The axons appear to run towards the eyes, but they can be traced for only a short distance after entering the optic nerves. Each nucleus is composed of only about
4 or 5 ordinary subspherical neurons.
(C) Nuclei in the Hindbrain (F~.."_,_l2J..
XXV. (Fig. 23). The transition from midbrain to hindbrain is marked by a vast and abrupt reduction of the cortical area. In the hindbrain there are no nerve cell-bodies beneath the neuropile. At the commencement of the hindbrain, the dorsal cortex disappears and the lateral cortex is reduced to a single cluster of cell-bodies on either side of the neuropile. This pair of neuron clusters is nucleus XXV.
Their axons do not run into the neuropile. Instead, the axons from each nucleus form a fiber tract which is directed dorsally and some what laterally and which emerges from the dorsal surface of the hind brain and runs into the caruncle above. These fiber tracts are the first of four pairs of nerve tracts running from the hindbrain into the caruncle. Nucleus XXV is composed entirely of ordinary pyriform neurons.
XXVI. (Fig. 19H, 24) • There is a pair of neuron clusters in the median side of each half of the forebrain, bordering the notch which separates the two halves of the hindbrain. These clusters constitute nuclei XXVI. The axons of each nucleus are directed laterally ~46- and run into the fiber tracts constituting the second pair of nerves to the caruncle from the hindbrain. However, most of the fibers of these nerve tracts are derived from the neuropile rather than nucleus
XXVI. Nuclei XXVI are composed entirely of ordinary pyriform neurons.
Nuclei XXVI are the posterior-most nue lei. Posterior to nue lei XXVI, the brain is composed entirely of fibrous neuropile. The last two pairs of nerve tracts to the caruncle are derived entirely from the neuropile. As described earlier, these two pair of nerve tracts are actually extensions of the posterior extremity of the neuropile (Fig. 3). -47-
DISCUSSION OF THE WORK ON BRAIN ANATOMY
In its external morphology, the brain of Hermodice carunculata is very similar to that of Eunice (Grassé, 1959). In both worms, the brain is wholly prostomial and separated from the epidermis only by the pros tomial sub•epidermal basement membrane. As in the brain of Eunice, three brain regions, forebrain, midbrain and hindbrain, can be readily distinguished in the brain of Hermodice carunculata. Each part is composed of two symmetrical halvest the two halves of the hindbrain being separated by a posterior notch. In the Eunicid brain, there are two transverse commissures, one joining the two halves of the forebrain and one joining the two halves of the midbrain. However, in Hermodice carunculata, there is only one transverse commissure in the brain, that which joins the two halves of the midbrain.
The heads of Eunice and Hermodice carunculata both bear a full complement of eyes and sensory appendages and the general arrangement of the cranial nerves innervating these structures follows the same pattern in both forms, i.e., the cranial nerves to the palps arise from the forebrain, the cranial nerves to the eyes and antennae arise from the midbrain, while the cranial nerves to the nuchal organs (represented by the caruncle in Hermodice carunculata) arise from the hindbrain.
The expanded Eunicid-type brain form in Hermodice carunculata is in accordance with the primative taxonomie position of the Amphinomidae.
Internally, the brain of Hermodice carunculata shows the typical polychaete brain architecture, with the neuron cell-bodies located peripherally and their axons and dendrites occupying the interior regions and constituting the neuropile. -48-
Corpora pedunculata are present in Hennodice carunculata. They are bilobed and situated in the antero-dorsal region of the forebrain, the typical position for polychaete corpora pedunculata. In the bi partite nature of its corpora pedunculata, Hermodice carunculata resembles Nereis pelagica, Nereis virens and Lepidonotus (Grassé, 1959).
The presence of corpora pedunculata suggests that the brain of
Hermodice carunculata is anatomically less primative than that of
Eunic~, the neurons in the forebrain having undergone the specialization and grouping necessary to produce corpora pedunculata. However, the forebrain itself is relatively small and the corpora pedunculata do not constitute the voluminous cerebral centres they do in the Aphroditidae, the Nereidae and the He sionidae.
Comparison with the Brain of Nephtys
The brains of Nephtys and Hermodice carunculata differ markedly in external appearance. The brain of Nephtys (Clark, 1958a,b,c; 1959) does not appear in the expanded Eunicid forrn as does that of Hermodice carunculata. There is no external lobation to demark separate brain regions in the former. The brain of Hermodice carunculata is confined to the prostomium, whereas in most species of Nephtys the brain extends into the anterior body segments.
The brain membranes and general internai brain organization are very similar in Hermodice carunculata and Nephtys. The epidermal nature of the brain is mani fest to a grea ter extent in Nephtys than in Hermodice carunculata. In Nephtys~ the prostomial portion of the brain is dorsally in direct contact with the cuticle.
Although globuli cells are present in sorne species of Nephtys, they are never organized into corpora pedunculata as they are in Hermodice carunculata. -49-
As in Nephtys, so in Hermodice carunculata, the supraoesophageal
ganglion is organized on a longitudinal basis with the ganglionic nuclei
succeeding one another in an anterior-posterior series, rather than
having the nuclei piled up on top of each other as is the case in Nereis
(Holmgren, 1916).
The structure of the neuropi le is much simpler in Hermodic~ ~
cu lata than in Nephtys. In the former worm, it occurs as a single
fi brous structure, whi le in the latter it is composed of three separate
parts which are interrelated in a complicated manner.
The brain of Hermodice carunculata differs from that of Neehtys in
the possession of bipolar neurons. These are very few and are confined
to nucleus VII. These bipolar neurons do not appear to be comparable
with the bipolar neurons in the brain of Nereis, since, in Hermodice
carunculata, these neurons are associated with nucleus v, while in
Nere they are associated with the nuchal organs.
The cranial nerves are similar in Nephtys and Hermodice carunculata.
Palpai nerves and lateral epidermal nerves are lacking in Nephtys but
present in Hermodice carunculata.
The stomatogastric system of Hermodice carunculata arises from the
forebrain whereas that of Neehtys arises from the circumoesophageal
connectives.
In Neehtys the nuchal organs are small, spherical structures
situated on the postero-lateral margins of the prostomium. They are
between 10 and 50JL in diameter, depending upon the size of the species
in question. In Hermodice carunculata the nuchal organs have become
fused and highly elaborated to form a large complex structure known as
the caruncle. Concomitant with the extensive development of the nuchal -50-
organs in Hermodice carunculata, there has been an elaboration of the nervous supply from the brain to the nuchal organs. In Nephtys the nuchal organs are innervated by a single pair of small nerve tracts, whereas in Hermodice carunculata there are 8 large nuchal nerves arranged in 4 pairs.
ln both Nephtys and Hermodice carunculata, the circumoesophageal connectives have a double origin in the brain, arising from t'Y'O tracts of fibers. In Nephtis the two connectives emerge as single structures, with the two fiber tracts retaining their identity within the connectives, whereas in Hermodice carunculata the connectives arise as double structures, short dorsal and ventral fiber tracts which fuse to form a single tract almost immediately after leaving the brain. In Nephtys, the circumoesophageal connectives arise wholly from the neuropile, while in Hermodice carunculata only the ventral fiber tract arises wholly from the neuropile. The dorsal fiber tract is derived from two ganglionic nuclei (VI and VIII) as well as from the neuropile.
Lateral epidermal nerve s, arising from the side of the brain and innervating the adjacent prostomial epidermis, are found in Hermodice carunculata, but not in Nephtys. It is doubtful t.:rhether the paired epidermal nerves of Hermodice carunculata are homologous to the nerves of the same name found in the brain of Nereis. Although the epidermal nerves in both worms have a similar distribution, they have different origins within the brain. The epidermal nerves of Nereis arise from a ganglionic nucleus, nucleus XIX (Holmgren, 1916) while the epidermal nerves of Hermodice carunculata emerge from the neuropile.
In Hennodice carunculata the relationships between the cephalic -51-
nerves and the ganglionic nuclei are more complicated than in Nephtys.
In Nephtys, the cranial nerves tend to emerge from the ganglionic nuclei, whereas in Hermodice carunculata most of them emerge from the neuropile, wi th the result that it is usually impossible to trace the cephalic nerves to the specifie ganglionic nuclei from which they originate.
It is difficult to attempt to homologize nuclei in the brain of
Nephtys and Hermodice carunculata since the origins and relationships of the cephalic nerves and the interconnections between the ganglionic nuclei are not sufficiently known in either worm. Hat-lever, a few homologies between nuclei do appear to be evident.
The nuchal nuclei, nuclei V in Nephtys, nuclei XXV and XXVI in
Hermodice carunculata, are recognized by their posterior position in the brain and by their association with nuchal nerves. In Nephtys, there is only one pair of nuchal nerves, one nuchal nerve arising from each nuchal nucleus. In Hermodice carunculata, there are four pairs of nuchal nerves, th~ first pair arising from nucleus XXV, the second pair arising partly from nucleus XXVI and partly from the neuropile and the third and fourth pairs arising entirely from the neuropile.
Judging from their positions in the brains of the two worms 1 it would appear that nucleus XXV of Hermodice carunculata is homologous with nucleus V of Nephtys. A transverse commissure, the nuchal commissure, joins the two nuchal nuclei in ~ephtys, but this is not present in
Hermo~}~ carunculata. Nucleus XXVI of Hermodice carunculata does not have a counterpart in the brain of NeJ?~!Y! and may be considered to be a new development vssociated with the evolutionary elaboration of the nuchal organs in this worm. -52-
It is likely that nuclei A of Nephtys are homologous with nuclei
III of Herm_!>dice carunculata, since these nuclei occupy the same position in the brain and are the source of the tegwnentary nerves in both worms.
Nuclei A in Nephtys also give rise to the antennal nerves, bot this is not the case in Hermodice carunculata. In the l.<.tter, the antennal nerves have an entirely different source. The nerves to the paired antennae arise from the neuropile, while the nerves to the single a:ltenna arise from nuclei IX. These antennal nuclei are not represented in the br:1 in of ~htys.
The large nuclei S in Nepht~~ are structurally very similar to the nuclei V in Hermodice carunculata and are composed of similar typ0.s of neurons. However, as these nuclei are not associated with sense organs, the homology is uncertain.
Nephtys and Herrnod~~ carunculata differ markedly in the arrangement of the ganglionic nuclei associated with the optic nerves. Both worms have four· eye-spots, an anterior pair and a posterior pair. In Nephtys, all the eye-spots are associated with the optic nuclei, U, while in
~ carunculata the anterior and posterior pairs of eye-spots are associated with different sets of ganglionic nuclei. In Nephtys, there i s a transverse commissure joining the two op tic nue lei, whereas in
Hermodice carunculata none of the five pairs of nuclei associated with the eye-spots are connected by a transverse commissure. Such anatomical differences be tween the op tic centres of the t'vo worms are to be expected in view of the differences in their behaviour with respect to light. Nephtys is strictly photonegative (Clark, 1958c), while Hermodice carunculata exhibits a more complex behaviour pattern in relation to light stimuli (described on PP• 2-3). .. 53-
The single transverse commissure found in the brain of Hermodice carunculata arises from nuclei XII and has no counterpart in the brain of Nephtys.
The findings indicate that the brains of Hermodice ~.!lculata
and Nephtx._s are constructed on similar patterns. In its external appearance,
the brain of Herrnodice carunculata is much closer to the ancestral
Eunicid forrn th an th at of Nephtys. Internally, the brain of Herrnodice
carunculata shows a higher degree of development than that of Nepht_ys.
In Herrnodice caruncu lilta the globuli ce 11 s are organized into copora pedunculata, the nuchal centres are greatly elaborated, and the optic centres are much more complex than in Nephtys. It is suggested that the higher leve! of organization of the brain of Herrnodice carunculata
is reflected in the behaviour of this worrn compared with that of Nephtys.
HermEdice carunculata leads an active predatory life. It is inevitably exposed to a greater variety of environmental stimuli than Nephtys which lives a quiescent life buried in the sand. Also, it is probable that the greater complexity of the optic centres in Herrnodice carunculata is associated with the precise diurnal activity rhythm displayed by this worm, in contrast to the constantly photonegative behaviour of Nephtys. -54-
SECTION II: NEUROSECRETION
INTRODUCTION TO THE HORK ON NEUROSECRETION
General Introduction
The phenomenon of neurosecretion is a relatively new field of study which presents many challenging problems to be solved. The existence of neurosecretory cells was discovered by E. Scharrer as recently as 1928, when he described secretory nerve cells in the nucleus preopticus of the hypothalamus of the teleost Phoxinus laevis. Since their discovery by E. Scharrer, neurosecretory cells have been found in a wide variety of animais. The discovery of neurosecretory cells in invertebrates is attributed to B. Hanstrom (1931), who found secretory nerve cells, the so-called "X organ", in the central nervous system of several Crustacea. Since then, neurosecretory cells have been found in the Insecta, Cephalopoda, Polychaeta (bath sedentary and errant),
Oligochaeta, Hirudinea, various Selachians and many mammals including man.
It is now apparent that neurosecretion is an integral function of nervous systems and the study of neurosecretion has opened a new era in the understanding of the nervous system.
Morphological Characteristics of Neurosecretory Cells
By definition, a neurosecretory cell is a nerve cell which produces and releases a hormone (E. & B. Scharrer, 1937). Like the classic verte brate endocrine gland hormones, these neurohormones are released into the circulation, are quite stable and affect structures and functions at many different points throughout the body ('1-laterman, 1961).
In the initial stages of an investigation, the criteria for neuro secretory cells are necessarily morphological (Clark, 1959). A true -55-
neurosecretory cell has the characteristics of a neuron plus the characteristics of an endocrine cell. ln other words, it is a '~land- nerve celln. Like an ordinary neuron, the neurosecretory cell possesses an axon, neurofibrils and Nissl bodies. The dendrites may be lacking
(Clark, 1956). Neurosecretory cell axons are capable of conducting e lee trical impulse s. The endocrine characteristic s of neurosecre tory cells have been described by E. & B. Scharrer (1937), from whose account the following surnmary has been taken:
(i) All gland-nerve cells, both of invertebrates and vertebrates, produce granules and/or droplets of colloid. In sorne cases these secretions appear in the cytoplasm itself (Aplysia, Pleurobranchaea, Bufo), in other cases they are included in Vâëüoles (Raia, Cristiceps).
(ii) A marked nuclear polymorphism is characteristic of many nerve-gland cells. Thus the nuclei of such cells in the spinal cord of Raïa, in the nucleus lateralis tuberis of Esox and Tetrodon, in the midbrain gland of Phoxinus, etc., are lobed and branched, so that the aspect of poly morphonuclear leucocytes or multinucleate giant cells is given in sections. Doubtless the active metabolism of secretory nerve cells requires a large nuclear surface, supplied here, as is often the case in gland cells, by the lobed and branched form of the nucleus.
(iii) Gland-nerve cells are often closely related to blood vessels, and this relation is likewise con nected with their active metabolism. Thus 4 or 5 capillaries sometimes surround a large gland nerve cell in the spinal cord of Raia, and a capillary may be enclosed by the cell body. Such pericellular and endocellular capillaries have also been observed in the secretory diencephalon nuclei of vertebrates.
Neurosecretory cells have been studied in a wide variety of animals, and it has become increasingly clear that colloid-containing vacuoles and colloidal inclusions occur too commonly in neurons for their presence to -56-
to be taken uncritically as evidence of neurosecretory activity (Shafiq,
1954; Malhotra, 1956; Chou, 1957). Moreover, while it is true that numerous vacuo1ations in the cytop1asm is a common feature of neuro secretory cells (Clark, 1959), it is well known that vacuolation also characterizes the trophospongial phenomena which result from the invasion of neuroglial cell processes into neurons. Such close contacts appear to be nutritional (Grassé, 1959), and are particularly numerous in polychaetes in which the central nervous system has no vascular supply
(Herlant-Meewis & Van Damme, 1962). lt is now realized that, in arder to justify its classification as such, a suspected neurosecretory cell, lilce an endocrine gland cell, must exhibit a definite intracellular secretory cycle, involving sorne cyclical pattern of accumulation and discharge of the neurosecretory material. Histological studies on many animais have shawn that the neurosecretory material leaves the cell by one or bath of two different routes: (1) by movement dawn the axon to its terminais and/or (2) by direct diffusion through the membrane of the perikaryon. The first route (axonal transmission) is indicated by the presence of droplets or granules of stainable neurosecretory material along the course of the axoplasm, while the second pathway is marked by the presence of droplets or granules of neurosecretory material on the outside of the cell membrane and in the surrounding neuroglia.
Clark (1956) points out that the neurosecretory material must have ready access to storage-release organs or to the blood stream or coelomic fluid, or directly to sorne effector organ. Typical examples of such storage-release organs are the vertebrate neurohypophysis (Palay, 1953), the sinus glands of Astracuran and Brachyuran Decapod Crustacea (Bliss, -57-
Durand and Welsh, 1954), and the corpus allatum of the insecta
(Thomsen, 1948). In vertebrates, the neurohypophyseal hormones are actually formed in the neurost>cretory cells of the supra-optic and para ventricular nuclei of the hypothalamn~. The axons of these secretory neurons constitute the hypothalamo-hypophysec. 1 tract and the hormones which they ela borate are transported along the fi bers oi. the hypothalamo hypophyseal tract to the neurohypophysis, ~1ere they are storerl prior to re le a se into the b lood stream. In the case of the Astracuran and
Bracyuran Decapod crustaceans, the sinus glands are storage-release organs for the products of the neurosecretory cells of the X-organs in the ventral region of the medulla terminalis of the brain. The neuro secretory substance is tr~nsported from the cells to the sinus gland along the axons which constitute a large nerve tract. It is stored in the sinus gland until released into the blood stream. An analogous situation exists in the lnsecta. Here the products of neurosecretory cells in the pars intercerebralis of the protocerebrum are transported via the axons to the corpus allatum where they are stored prior to release into the blood stream.
Neurosecretory products are not always transported to storage• release centres. They may be released directly from the cell into the blood stream or coe lamie fluid. Absorption of the secretory product i s facilitated by a close association between the axon terminais and the blood vascular system or coelomic fluid. ln many cases it can be shown th at the axon terminal s are modified for storage and re le a se of the neurosecretory material into the circulation. The modified terminal processes are of 2 major types (Waterman, 1961): -5R-
(1) Bulbous endings as found in the crustacean sinus glands.
(2) Highly br;mched terminais. The multibranched endings would appear to provide more surface for release of a neurohormone.
In some instances the neurosecretory product is released directly to the effector organ, without the intervention of a storage-release centre or circulatory system. Thus in crustacean hearts, the terminais of the neurosecretory cells producing a heart-regulating substance are in direct contact with the heart wall (Haterman, 1961).
Hi stochemical Aspects of Neurosecretion
It is difficult to make general statements about the histochemical properties of neurosecretory cells. The situation is complicated by the following factors: neurosecretory cells in different animais may exhibit different staining affinities; within the same animal there may be different types of neurosecretory cells showing different staining reactions; the same cell in a given animal may exhibit different stain- ing affinities at different stages of its secretory cycle. In at least one instance it has been shown that the staining properties of the neurosecretory substance varies with age (Dawson, 1953).
Neurosecretory material may be demonstrated by a number of histologi- cal stains. It is usually readily stained by the hematoxylin in Gomori's
(1941) chrome hematoxylin phloxine method. It has been observed to stain metachromatically with azan (Clark, 1959). Sometimes, but not always, neurosecretory products are stained by phloxine, osmium tetroxide, Sudan black B, and periodic acid-Schiff coloration (Clark, 1959). However, none of the foregoing methods are specifie for neurosecretory products. -59-
There are two stains which have been found to exhibit a very
highly selective affinity for neurosecretory products. These are alcian
blue and Gabé 1 s paraldehyde fuchsin. Adams and Sloper (1956) showed
that in vertebrates, alcian blue, after performic acid oxidation, stains
neurosecretory material specifically. So far, this observation bas not
been confirmed in invertebrates. The most widely used stain for neuro
secretory material is Gabé's (1953) paraldehyde fuchsin. It bas been
shawn empirically to have a strong affinity for neurosecretory products
in vertebrates, arthropods and annelids. It stains the neurosecretory
substance deep purple and may be used with a variety of different
counterstains. Until a recent study by Clark (1963), Gab~'s paraldehyde
fuchsin was considered to be a specifie stain for neurosecretory products
(Gabé, 1953; Clark, 1959). Clark (1963) now maintains that it is doubtful
that all neurons containing fuchsinophilic material are truly neuro
secretory. If this is indeed the case, the task of interpreting
histological material is more complicated than was previously supposed.
However, the fact still remains that, in the initial stages of investi
gation, the criteria for neurosecretory cells are necessarily morpho
logical and histological, even if this results in numbers of nerve cells which do not secrete biologically active substances being included,
perhaps unjustifiably, within the category of neurosecretory cells.
ln 1937, E. and B. Scharrer wrote, "•••• It is as yet unknown what
substances are secreted by glandular regions of the central nervous
system •••• ". Today the chemistry of neurosecretory material is better
understood, but rouch remains to be learned.
Neurosecretory material is not one specifie substance whose chemical -60-
nature is identical in all secreting neurons. In actual fact it com prises a class of different substances having certain chemical similari ties. Conventional histological stains have provided sorne data on the general chernical nature of neurosecretory rnaterial. The staining affini ties of neurosecretory material suggest an acid mucopolysaccharide corn panent with a protein carried substance (Clark, 1955).
Clark has made a detailed study of the neurosecretory system of the supraoesophageal ganglion of Nephthyid polychaetes. He identified three types of neurosecretory cells which he denoted as types A, B and
C. In both the B and C cells he found evidence of two percursor subN stances for the neurosecretory product. These substances were a PA/S positive polysaccharide and an osrniophilic lipid. In the same two cell types, Clark found a sudanophilic lipid cornponent of the neurosecretory substance, demonstrable only towards the final stages of the secretory cycle, when the cells are fully charged with the secretory product.
Sorne workers report that the neurosecretory material is basophilie
(e.g., Bliss, Durand and \.Jelsh, 1954), while others, working with different animals, find that it is acidophilic (e.g., Knowles, 1953). In fact, in the Crustacea, there is evidence that the secretory product transforrns from a basophilie to an acidophilic condition as it passes from the point of origin to the point of release (Carlisle and Knowles, 1959). This suggests that the acidophil condition is the storage condition.
The concept of a progressive change in the chernical nature of the neurosecretory material during the course of the secretory cycle is further supported by the findings of Arvy (1954). Studying the supraoesophageal ganglion of Apomatus similis (Serpulidae, Polychaeta Sedentaria), she found -61-
that, after staining with azan, certain neurons appeared clear blue, others bright red, while others showed a whole range of intermediate violets and purples. This spectrum of metachromatic colors reflects a corresponding range of chemically different secretory products. Arvy interpreta this range as an indication of the progressive change in the chemical nature of a single secretory product during the course of its elaboration and accumulation within the nerve cells.
Little is known about the chemistry of the carrier protein of the acid mucopolysaccharide neurohormone. In the Decapod Crustacea, sorne information has been obtained by means of electrophoretic separation and chromatographie and countercurrent methods (Waterman, 1961). Four types of protein carrier substances have been detected in Decapod Crustaceans.
There are four known cases where the neurosecretory substance is not an acid mucopolysaccharide at all. Two of these are oxytocin and vasopressin, the hormones produced by the neurons of the supra-optic and paraventricular nuclei of the vertebrate hypothalamus. Oxytocin and vasopressin have been identified as octapeptides and their exact chemical structure is known. These are the only neurohormones whose precise chemical formula has been e lucidated. They can now be synthe sized as well as isolated in pure form (Haterman, 1961). Also, it has been shawn that the red pigment-concentrating hormone and the Uca-darkening hormone are different but related polypeptides.
The Functional Significance of Neurosecretion
Even the criterion of an intracellular secretory cycle is not entirely valid for the designation of neurosecretory cells. Clark,
(1963) gives the example of the four types of neurosecretory cells -62-
originally described in the brain of Nereis (Schaeffer, 1939). Although
all these cell types can be shown to undergo a secretory cycle, only
one cell type now appears unequivocally neurosecretory. The final,
decisive criterion for designating a neuron as neurosecretory is a
physiological one. It must be demonstrated that the stainable substance
which it produces and secretes possesses true hormonal activity. Thus
there must be a correlation between the secretory activity of the post
ulated neurosecretory cells and sorne physiological activity in the body
of the animal. Research in both vertebrate and invertebrate neuro
secretory systems has succeeded in revealing the precise physiological
roles of at least sorne of the neurosecretory products.
In the vertebrates, Bargmann and Scharrer (1951) and Palay (1953)
have shown that the posterior pituitary hormones, vasopressin and oxy•
tocin, are formed by neurosecretory cells in the supraoptic and para
ventricular nuclei of the hypothalamus. The hormonal effects of both
these substances are well known, vasopressin acting as a blood pressure
raising and antidiuretic agent, and oxytocin causing contraction of
uterine smooth muscle.
In the Crustacea, neurosecretory systems are highly developed, and,
along wi th endocrine glands, they serve to control such fondamental
physiological proce sse s as moul ting, growth, maturation, and regeneration
(Watennan, 1961).
Neurohormones have similar vital roles in insects, where they have
been studied extensively with regard to their control of moulting,
differentiation and reproduction. For example, Higgle sworth (Thompsen,
1948) has shown that in Rhodnius, the moulting hormone is produced by neurosecretory cells whose products control ovarian development and egg maturation, by virtue of their activating effect upon the corpus allatum.
In the insect brain, she has also located other neurosecretory cells whose secretions exert an activating effect directly upon the avaries.
In the anne lids, neurosecretory centres, located in the brain or supraoesophageal ganglion, exert a controlling influence over important physiological processes. The experimental work of Durchon (1956), Clark and Bonney (1960), Hauenschild (l96û) and Herlant-Heewis and Van Damme
(1962) has clearly shown that, in Nereid polychaetes, the supraoesophageal ganglion secretes hormones that are essential for wound-healing and re generation. There is also evidence that the same is true of the
Nephtyidae (Clark, 1959). The endocrinplogy of regeneration in Nereis diversicolor has been studied in detail by Clark and Rustan (1963a).
These authors have shown that a hormone necessary for the regeneration of segments begins to accumula te in the supraoe sophageal ganglion within a few hours after amputation of the posterior end of the worm. The hormone content of the ganglion rises to a maximum on the third d;·y and is gradually released into circulation on the fourth day. By the fifth day the ganglion again contains very little hormone. Intact worms also contain i1 small quantity of the hormone which may therefore control normal as well as regenerative segment proliferation. Berrill (1952) reports that similar hormones are secreted by the brains of Lumbricid oligochaetes.
Experimental >-mrk on the Nereidae and Nephtyidae has shown that the interrelated processes of growth, genital maturation and epitoky are control led by cerebral hormone s. The supraoe sophageal ganglion of immature Nereids and Nephtyids secretes a hormone that inhibits sexua] -64-
maturation of the worms (Durchon, 1952; Cl.n:k, 1956). This hormone is knovm as the juvenile hormone. Removal of the supn10e sophageal ganglion causes the precocious appearance of su ch soma tic changes as nonnally occur when the worms are sexually mature (epitoky, heteronereidian trnnsformation) (Clark, 1961), and the gametes undergo a premature and accelerated development. These changes can be prevented by the implanta tion of the ganglion of an inw1ature worm into the decerebrate worm
(Durchon, 1952). Furthennore, the onset of nonnal metamorphosis can be delayed in an intact animal by the implantation of inw1ature ganglia
(Clark and Rustan, 1936b). In Nereis divery!~olor and Platynereis dumerilii, it has been shawn that, at the onset of sexual maturity, after the inhibitory influence of a high level of juvenile hormone has been lifted, a continued secretion of small amounts of juvenile honnone is necessary for vitellogenesis (Clark and Rustan, 1963b).
Clark and Rustan (1936a) have shown that, during the life of
Nereis diversicolor, the supraoesophageal ganglion ceases to secrete the bonnone ne ce ssary for regeneration at about the same time as juvenile hormone secretion ceases. This accounts for the observation that the regenerative ability declines steadily throughout the life of the animal.
When the oocytes are nearly full sized, the worm is incapable of re generating.
Neurosecretion and Diurnal Rhythms
Certain studies on the problem of biological clocks have produced evidence of the role of neurosecretory products as endogenous controlling factors of diurnal rnythms. The existing literature dealing with this aspect of neurosecretion is quite scarce and appears to be confined to the arthropods, particularly the insects. -65-
It has been shown that the daily activity cycles of various crust aceans will persist under constant conditions. For instance, Kalmus showed that during several weeks in constant darkness, the crayfish
persistent daily rhythm has been described in Orconectes virilis
(l,laterman, 1961). In this organism and in Procambarus clarkii and
Cambaru~ diogenes, these diurnal rhythms persist at least five weeks in darkness. In all these investigations, removal of the eye-stalks abolished the diurnally rhythmic character of the locomotor activity.
Since the eye-stalks, or, more specifically, the sinus glands, do store and release certain neurohormones, the possibility of the involvement of neurosecretory cells in the control of the diurnal locomotor activity rhythms cannat be ruled out.
The most detailed investigation of the role of neurosecretory cells as a timing-mechanism has been conducted by Harker (1954, 1955, 1956,
19bO). She made a series of s.tudies on the diurnal activity rhythm of the cockroach, Periplaneta americana L. The cockroach displays a diurnal locomotor activity rhythm, with the active phase occurring at night and the quiescent phase during the day.
After extensive experimental work, Harker concluded that a complex of processes interact to produce the diurnal behavioural rhythm of the cockroach. According to her, the situation is as follows:
The timing of the active phase of the cockroach is controlled by the stimulus of a change from light to darkness.. This stimulus is received through the medium of the ocelli and is thence transmitted, by nervous pathways, to the suboesophageal ganglion where it has the effect of activating a group of neurosecretory cells located in each side of the -66-
suboesophageal ganglion, on the ventro-lateral surface of the ganglion.
The presence in the blood stream of the neurosecretory product of these cells produces the active phase of the activity cycle. There is a lag of two to four hours before locomotor activity reaches its peak, but secretion from these suboesophageal neurons begins immediately, or very soon after, the beginning of darkness.
Harker found that the ability of the suboesophageal ganglion neuro secretory cells to react in response to environmental light changes is dependent upon another endogenous factor. This factor consists of a hormone which cornes from the corpora cardiaca and enters the suboesoph ageal ganglion via the corpus allatum-suboesophageal ganglion nerve.
The presence in the suboesophageal ganglion of this hormone serves to maintain the secretory rhythm of the neurosecretory cells in response to changes in light conditions.
Harker found that the neurosecretory cells in an implanted sub oesophageal ganglion are able to maintain their diurnal secretory rhythm for a few days despite the fact that ail nervous connections have been broken. This indicated that there is always enough corpus cardiacum hormone substance pre sent in the ganglion to support the sec re tory rhythm for a time and that fading of the rhythm only occurs when this substance is exhausted.
Neurosecretory Cells in Polychaetes
According to Clark (1959), neurosecretory cells have been described in the supraoesophageal ganglia of the following polychaetes:
Aphrodite ~ul~ and Lepidonotus sguamatus, severa! Nereids, twelve species of Nephtyidae, Arenicola marina, Lanice conchilega, three
Sabellids and the Serpulid Apomatos similis. Knight (1964) has -67-
identified neurosecretory cells in the supraoesophageal ganglion of the Serpulid Spirobranchus giganteus.
Detailed studies of polychaete neurosecretory cells have centred on the Nereidae. Gabé (1954) has described three types of neuro- secretory cells in the brain of ~~s:
(a) Gells with a homogeneous acidophil cytoplasm.
(b) Fusiform cells near the posterior optic nerves, which have a reticulate cytoplasm containing fuchsinophil droplets.
(c) Large round cells containing fuchsinophilic secretory products in vacuoles.
The secretion of all these cell types is PA/S-oositive and stains with paraldehyde fuchsin. The a and b cells are stained with acid chrome haematoxylin but the c cells are not. Although all these cell types can be shown to undergo a secretory cycle, it now appears that only one of them is unequivocally neurosecretory (Clark, 1963).
Clark has made a detailed study of the neurosecretory system of the supraoesophageal ganglion of Nephtys (1959). He found that neuro- secretory cells are very numerous and occur in all regions of the brain, except in the nuchal nucleus (Nucleus nuu) and in the corpora pedunculat:a of those species which possess these structures. He estimated that 75% of the nerve cells of the supraoesophageal ganglion can be classed as neurosecretory on morphological grounds. On the basis of differences in cellular morphology and secretory cycle, Clark distinguished three types of neurosecretory ce lls in ttte supraoe sophageal ganglion of Nephtys.
He designates them as A, B and G cells. The neurosecretory substance of the B and C cells stains with paraldehyde fuchsin, PA/S and acid chrome haematoxylin, while that of the A cells does not. On the other hand, the -68-
secretory substance of the A cells is stained by phloxin while that of the B and C cells is not. Axonal transmission of the secretion was seen in the B and C cells, but never in the A cells. -69-
MATERIALS AND .t-JETHODS FOR THE HORK ON NEUROSECRETION
Twelve specimens were used for the work on neurosecretion. They were all medium-sized worms (about 10 cm. long) collected in Barbados in the summer of 1963 during the months of June and July. All specimens were fixed in Bouin's fluid, embedded in paraffin and eut in transverse seri al sections at 7~10)-l and stained with Gab~ 's par aldehyde fuchsin and counterstained H'i th Ha1mi 's trichrome stain.
Three specimens were fixed at each of the following times of day:
(a) Early morning, 6:00 - 7:00 a.m.
(b) Midday, 12 Noon - 1:00 p.m~
(c) Late afternoon, 4:00 - 5:00 p~m.
(d) Midnight, 12 midnight- 1:00 a.m.
The early morning, miday and late afternoon specimens were taken directly from the sea and immersed in the fixative. The midnight specimens were taken from a population of worms maintained in one of the exhibition tanks in the laboratory at the Bellairs Research Institute.
These are large, 3-foot deep marine aquaria set into the wall. They are open above to the sky except for a covering of transparent plastic material. Consequently, specimens in them are exposed to very natural light conditions.
In addition to this material, the author also referred to the supraoesophageal ganglion which had been stained in PA/S and hematoxylin by Harsden (see "Haterials and Methodsn, Section I). Unfortunately, the time of day \vhen this specimen was fixed is not kno,m. -70-
OBSERVATIONS FOH THE HORK ON t-;1WROSECRETION
Again it should be stressed that morphological and stain reaction evidence is not conclusive for unequivocal designation of neurosecretory cells (Clark, 1963). Nevertheless, these characteristics are of im portance, since they indicate which cells are to be further investigated for neurosecretory activity.
It is necessary to qualify the meaning of the term 11 neurosecretory cell" as it is used in the following account. The cells so designated in this description are neurons which show the morphological and staining characteristics of neurosecretory cells. The final proof that they are neurosecretory can be made only by demonstrating that they are the source of hormones. This description provides a basis for further and more decisive investigations of a physiological nature.
In studying neurosecretion, a total of twelve supraoesophageal ganglia were stained by the paraldehyde fuchsin-Halmi technique. Three of these ganglia had been fixed in early aJO., three at noon, three in late p .. m. and three at midnight. In each of these supraoesophageal gi1nglia, it was found that 50-80% of the nerve cells stained positively with the paraldehyde fuchsin. In other v1ords, 50-80% of the cerebral nerve cells can be classified as neurosecretory on the basis of staining reaction.
Tn most cases, fuchsinophilic neurons occurred in all the ganglionic nuclei except nuclei I and II (the corpora pedunculata) in the forebrain.
The globuli cells were the only nerve cell type which never exhibited fuchsinophilia. The greatest concentration of fuchsinophilic cells occurred in nuclei VI and XIV, where all the neurons displayed an affinity for paraldehyde fuchsin. -71-
All the preparations studied indicated that, in Hermodice carunculata, th·~ neurosecretory material does not occur within cytoplasmic vacuole s. In ste ad the neurosecretory ce ll s were char ac terized by fine fuchsinophilic granules in a chromophobic, acidophilic or fuchsinophilic cytoplasm (Fig. 25), or, in some cases, simply by an agranular cytoplasm staining uniformly in mauve or purple with paraldehyde fuchsin.
In the dorsal and lateral cortex of the midbrain, some of the fuchsinophilic cells apparently lack dendrites. This is a peculiar feature in some neurosecretory cells which has been noted also by Clark
(1956).
All the neurons of nucleus XIV stained with paraldehyde fuchsin.
They were the only fuchsinophilic cells in the brain showing evidence of a definite secretory cycle. Cells in other sites presented a variety of appearances, depending upon the concentration of fuchsinophilic granules and the chromophobia or degree of acidophilia or fuchsinophilia of the ground cytoplasm. However, no secretory cycles could be discerned.
The secretory cycle of the cells of nucleus XIV was studied. All the neurons of nucleus XIV are of the ordinary subspherical type and possess dendrites. Their axons run dorsally and impinge upon the con nective tissue membrane which lines the floor of the dorsal brain depression. In sorne cases the axons can be seen to penetra te this connective tissue lining and terminate within the cavity of the dorsal brain depression (Fig. 28). 'l'he cells appear to undergo a diurnal secretory cycle which is synchronous throughout the entire nucleus.
This secretory cycle appears to be as follows: -72-
1. Le~e Afternoo? (Fig. 26, 33): The cytoplasm shows no fuchsin-
ophilia. The entire cytoplasm is stained darkly with fast
green indicating an acidophilic condition. The axons are
chromophobic and very difficult to detect. There are a few
very fine fuchsinophilic granules (diameter about .5~ ) in
the distal ends of those axons which protrude into the brain
depression. A few such granules are found within the brain
depression, and also adhering to the connective tissue lining
of the depression, and within the lining itself.
2. The perikaryon is stained uniformly
pale purple with the paraldehyde fuchsin. Scattered throughout
the cytoplasm are numerous fine dark purple fuchsinophilic
granules about .5)-J- in diameter. These granules also adhere
to the outside of the nuclear membrane. The axons are still
chromophobic and inconspicuous. Fuchsinophilic granules in
the distal ends of axons, in the dorsal brain depression and
in the connective tissue lining of the depression are all
extremely scarce.
3. Early Morning (fig. 28, 33): The ground cytoplasm is acidophilic
in the proximal portions of the neurons and fuchsinophilic
in the distal portions. The fuchsinophilic granules are
concentrated towards the axon hillock. These granules occur
in the proximal portions of the axons, thereby rendering the
axons very conspicuous. In sorne of the neurons, fuchsinophilic
granules could be spotted along the entire length of the axons.
Hany tiny fuchsinophilic granules (diameter about .5?) may be -73-
found inside the brain depression, within its connective
tissue lining and also adhering to the inside of the con
nective tissue lining. Sorne of the granules in the
depression are aggregated to form composite clusters
ranging from 2-6~ in diameter.
4. This is apparently the final stage in
the secretory cycle. The cytoplasm of the neurons is
acidophi lie and fine ly vacuolated. The only traces of
fuclasinophilia are a few streaks adhering to the cell
membranes and a few fine granules in sorne of the axons.
Fuchsinophilic granules are very abundant within the
dorsal brain depression. Here many of them form con
spicuous clusters 2-6JA in diameter.
The single supraoe sophageal ganglion which was stained in PA/S hematoxylin presented a staining pattern which was entirely different from that of ganglia stained with paraldehyde fuchsin. There were only two PA/S-positive sites in the brain. These were nucleus VI and nucleus
XIV. The giant pyriform neurons of nucleus VI were PA/S-positive. The ground cytoplasm of the perikaryon and axon hillock was weakly positive and stained pale mauve. Fine, strongly positive, clark purple granules
\vere found in the ground cytoplasm and along the axons as far as the entrance of the axons into the dorsal connective roots (Fig. 30).
All the neurons of nucleus XIV had a PA/S-positive ground cytoplasm staining uniformly pale pinkish mauve.
The Fate of the Neurosecretory Material
The axons of most of the neurosecretory ce lls of Hermodice ~ cu ata run into the neuropile. In the case of the neurosecretory cells -74-
of nuclei VI and VIII, the axons run into the dorsal root of the cir cumoesophageal connectives (Fig. 19F). The axons from the neurosecretory nucleus XIV terminate in the dorsal brain cavity or on the connective tissue lining of this cavity (Fig. 28).
In the case of the neurosecretory cells entering the neuropile, fuchsinophilic material has often been observed in the axons near the cell-bodies, and, less frequently, along the entire course of the axons as far as their point of entrance into the neuropile. Many fine fuchsinophilic granules were found scattered throughout the neuorpile and also inside the longitudinal canal running through the neuropile.
Larger fuchsinophilic droplets ranging from 5-7)-J. in diameter were also encountered in this canal.
Secretory products have been detected in the axons of nuclei VI and VIII as far as their entrance into the dorsal roots of the circum oesophageal connectives (Fig. 14). Granules of fuchsinophilic material were found in sorne of the axons of the circumoesophageal connectives, and also strung out along the outer edge of the axons. Evidently these granules are the products of neurosecretory cells in nuclei VI and VIII. Sorne of these granules were PA/S-positive as well as fuchsinophilic, an indication that they are secretory products of the giant neurons in nucleus VI.
The neurosecretory products of the neurons of nucleus XIV were detected along the entire course of the axons (Fig. 28). Fuchsin ophilic granules of the same appearance as the secretory products of these cells were found in the connective tissue lining of the dorsal brain cavity and inside the dorsal brain cavity itself. In the dorsal brain cavity, these granules could be seen adhering to the axon terminais of nucleus XIV neurons, floating free in the lumen and also adhering to the outer walls of the blood vessels. On rare occasions similar fuchsinophilic granules were seen within the blood vessels in the dorsal brain cavity. These findings strongly suggest an elimination route for the neurosecretory products of nucleus XIV by entrance into the blood vascular system in the region of the dorsal brain cavity.
In the brain of Hennodice carunculata there is no evidence of a cerebro-vascular complex of the sort found in Nereids and Nephtyids.
Ho1-vever, beneath the posterior regions of the midbrain, fuchsinophilic granules were demonstrated in the syncytial pericapsular membrane of the brain, in the coelomic cavity beneath the brain, and in the thickened syncytial lining of this cavity (Fig. 31). Sorne of them were seen adhering to the outer walls of blood vessels running through this lining. On rare occasions fuchsinophilic granules were seen inside these blood vessels. The fuchsinophilic granules in the pericapsular membrane often occurred in dense clusters about 6jl( in diameter (Fig. 32). -76-
CONCLUSIONS DRAHN FRON THE HORK ON NEUROSECRETION
The following conclusions may be drawn from the findings of the histological study of neurosecretion in Hermodice ~cu]ata using
Gabé•s paraldehyde fuchsin and PA/S techniques.
1. A large proportion of the neurons in the cerebral cortex of
Hermodice carunculata display the staining properties
characteristic of neurosecretory cells. Such neurons occur
in all the ganglionic nuclei except nuclei I and II, which
constitute the corpora pedunculata. Nuclei VI and XIV are
unique in that they are composed entirely of neurosecretory
ce 11 s.
2. The neurosecretory cells of Hermodice carunculata are not
morphologically different from the ordinary cerebral neurons
except for the fact that sorne of them lack dendrites. They
differ from the ordinary neurons in their staining affinities.
Neurosecretory cells are found within each category of neuron
in the brain, with the exception of the globuli cells which
never show evidence of secretory activity.
3. The neurosecretory product of the neurons of nuclei VI and
XIV contains PA/S-positive substances (other than glycogen)
which are simultaneously present in the ground cytoplasm
(and also in the axoplasm in the case of nucleus VI neurons).
4. The intracellular neurosecretory products in the brain do not
occur in vacuoles. They appear always to take the form of
very fine fuchsinophilic granules, about .5)A in diameter, in
the cytoplasm. -77-
5. The axons of most of the cerebral neurosecretory cells run
into the neuropile. The axons from nue lei VI and VIII run
into the dorsal root of the circumoesophageal connectives,
while the axons from nucleus XIV apparently penetrate the
lining of the dorsal brain cavity and terminate inside this
cavity.
6. Evidence of axonal transmission of neurosecretory products
has been found in the case of neurosecretory neurons entering
the neuropile and the dorsal roots of the circumoesophageal
connective s. Extrace llu lar fuchsinophi lie granules occur in
the neuropile and in the circumoesophageal connectives but
are not present in the cortex. This is a further indication
that neurosecretory products leave the cells by migration
down the axon, rather than by direct diffusion through the
cell membrane. In the circumoesophageal connectives, extra
cellular neurosecretory granules occur strung out in beaded
fashion along the edges of axonal routes. This alignment
suggests that they have been exuded from the axon before
passing as far as the axon terminais.
7. Although there is no specialized cerebro-vascular complex
beneath the supraoesophageal ganglion, there is a close
proximity between the ganglion and the blood-vascular
system in the region below the posterior part of the mid
brain. The only anatomically specialized feature in this
area is a thickening of the syncytial lining of the infra
cerebral coelomic space. Many small blood vessels, derived -78-
from the dorsal longitudinal vessel, run through this
thickened lining. The paraldehyde fuchsin technique has
provided evidence that this area provides a site for the
passage of neurosecretory products from the brain into the
blood stream. It appears that neurosecretory products may
be accumulated and stored in the pericapsular membrane prior
to release into the blood-vascular system.
8. In the case of nucleus XIV one envisages a nearly uninterrupted
route for the neurosecretory products from the cell-bodies to
the blood vascular system.
9. There is an indication that some neurosecretory products pass
from the neuropile into the longitudinal brain canal where they
are accumulated and stored.
10. The subspherical neurons of nucleus XIV are all neurosecretory.
There is suggestive evidence that they undergo a diurnal
secretory cycle which is synchronous throughout the entire
nucleus. The apparent secretory cycle involves the accumulation
of fine fuchsinophilic granules throughout the cell-body,
especially in the region of the nuclear membrane, followed by
the elimination of these granules from the cell via the axon.
TI1ere is evidence of a precursor substance which is located
in the ground cytoplasm and which changes from an acidophil
to a fuchsinophil condition as the cycle proceeds. The
percursor material is never depleted during the secretory
cycle. Extracellular evidence of the cyclic secretory activity
of nucleus XIV is manifested in the cyclic variation in the -79-
quantity of fuchsinophilic material deposited in the dorsal
brain cavity and in its connective tissue lining and blood
vessels.
11. The secretory cycle of the neurons of nucleus XIV has a
circadian periodicity, with the maximal accumulation of
intracellular secretory products occurring at midnight and
the maximal quantity of released extracellular material
appearing at noon. It is postulated that these neurosecretory
cells participate in the production of the diurnal activity
rhythm of Hermodice carunculata. -80-
DISCUSSION OF THE HORK ON NEURüSECRETION
In view of the fact that neurosecretory cells have already been found in the supraoesophageal ganglia of many annelids, including severa! polychaete families, the presence of neurosecretory cells in the brain of Hermodice carunculata was not unexpected. This is the first report of cerebral neurosecretory cells in the Amphinomidae.
The cerebral cortex of Hermodice carunculata contains about the same proportion of neurosecretory cells as that of Nephtys, and the pattern of distribution of neurosecretory cells is similar in the two worms. Secretory neurons do not occur in the nuchal nucleus (nucleus U) of Nephtys, but are present in the nuchal nuclei (nuclei XXV and XXVI) of Hermodice carunculata. In the brain of Nereis, the neurosecretory cells are more localized, being concentrated in the dorso-posterior regions of the brain, near the posterior optic nerves (Defretin, 1955).
Neurosecretory cells tend to be larger than ordinary neurons (Bliss,
Durand and \lelsh, 1954), and this is the case in Nereids and Nephtyids and sedentary polychaetes. However, in Hermodice carunculata, the size and morphology of all the neurons within each category 1s very uniform, and the secretory neurons are no exception. The only distinctive morpho logical feature was the lack of dendrites in sorne of the secretory neurons of the pyriform and giant pyriform type. ~~is peculiarity has been found in the neurosecretory cells in other organisms (Clark, 1956).
In general, a vacuolated cytoplasm is a ccm~on feature of neuro secretory cells (Clark, 1959). However, in Hermodice carunculata, the neurosecretory products are not included in vacuoles, but always appear in the cytoplasm itself. In this feature, the neurosecretory cells of -81 ..
Hermodice carunculata resemble the type A neurosecretory cells of Neeh~.
Nucleus XIV is entirely neurosecretory. The cells of this nucleus were the only neurons in the brain where a secretory cycle could be discerned.
This study of neurosecretion is of a preliminary nature and involves a small number of specimens. The evidence is slender but suggestive that a diurnal periodicity exists in nucleus XIV. The secretory cycle of these neurons differs from the secretory cycles which have been described in the brains of other polychaetes. In Stage 2 of their secretory cycle (Fig. 27), the se cells present an appearance reminiscent of that of fully-charged secretory neurons of the supraoptic and paraventricular nuclei of monkeys and man (Palay, 1953).
Hanstrorn (1954) reports that, in annelid neurosecretory cells, discharge of the secretory products occurs bath by passage dawn the axon and by diffusion through the cell membrane. In Hermodice carunculata, however, there was no evidence of the latter mode of discharge.
There is no structurally soecialized cerebro-vascular complex as found in the Nephtyid and Nereid brain. There is a suggestion that sorne neurosecretory material is relea~ed and accumulated at the base of the posterior region of the midbrain, where the lining of the infra-cerebral coelomic cavity is thickened. Fuchsinophilic granules may be found in the blood vessels running through this lining and it is possible that they are of neurosecretory origin. This association between the brain and the blood-vascular system is structurally very simple and is of interest since it conceivably represents an incipient cerbro-vascular complex.
However, this association does not afford the sole pathway by which neurosecretory products are released from the brain. The circum oesophageal connectives serve as pathways for neurosecretory material produced in nuclei VI and VIII. This pathway is not unexpected -82-
in view of the fact that Clark (1958c) found that, in Nephtys, the neurosecretory cells in nuclei A, B and G are associated with the roots of the circumoesophageal connectives, although he did not detect neurosecretory material in the connectives. Herlant-Ueewis (1955, 1956) found that axons of sorne of the b-cells in Eisenia enter the circum oesophageal connectives, and also that in Lumbricus and Allolobophora a stream of granules secreted by a-cells in the supraeosophageal ganglion travels in the connectives to the suboesophageal ganglion when the nerve cord is sectioned and the gonads are removed. There is evidence that the neurosecretory products of nucleus XIV of Hermodice carunculata are transmitted directly to the dorsal brain cavity, where they are accumulated and released into the blood stream. Clark (1959) has found evidence that the neurosecretory products released into the blood vessels of the cerebro-vascular complex of Nephtys are subsequently ingested by amoebocytes in the blood. In Hermodice carunculata, thcre is no indication of amoebocytic phagocytosis of fuchsinophilic granules, either in the blood vessels in the dorsal brain cavity or in the blood vessels in the thickened syncytial lining of the infra-cerebral coelomic cavity.
The apparent diurnal periodicity in the neurons of nucleus XIV is an indication that they may be implicated in the control of the diurnal activity rhythm of the worm.
It would be impossible, at this stage, to postulate the nature of the role of the neurosecretory products of nucleus XIV in the production of the dirunal activity rhythm. Harker's studies on the diurnal activity rhythm of the cockroach have illustrated the complexity of endogenous controlling systems. According to Harker (1960), the presence of at -83-
least two timing-mechanisms, each of which can act autonomously but can modify the other, comprise the paradigm of the internai biological clock. Furthermore, she points out that nearly all circadian rhythms appear to be set by the light conditions of the environment.
This preliminary study of neurosecretion in Hennodice carunculata has indicated the presence of diurnal rhythmicity in one group (nucleus
XIV) of cerebral neurosecretory cells. A more intensive study, involving the use of a much larger number of worms, is necessary in order to test the reproducibility of the results,.
It remains to be demonstrated conclusive ly th at nucleus XIV is involved in the production of the diurnal activity rhythm. If this were confirmed, it 'lr!Ould be of interest to investigate the mechanism by which this cellular rhythmicity is translate cl into the overall behavior of the vwrm. It is suggested that adoption of the experimental approach used by Harker (1954, 1955, 1956, 1960) would be fruitful in these investigations. Such an approach would include cauterizing operations to determine the effect of destruction of nucleus XIV neurons and surgical techniques to study the effect of brain removals and trans plantations at various intervals throughout the diurnal cycle. It would be desirable to use similar techniques on worms which have been maintained for various lengths of time in conditions of constant light and constant darkne ss. Histological studie s should coincide with all the experimental techniques. It would also be requisite to search for other endogenous factors which may be involved in the production of the diurnal activity rhythm. -84-
SlW'MARY
The brain of Hermodice carunculata (Pallas) has been studied.
Its gross morphology, microanatomy and neurosecretory system are
described.
The externa 1 morpho logy of the brain shows an archaic form which
is in keeping with the primitive position assigned to the Amphinomidae
among polychaete families. Internally, the brain is organized on a
pattern similar to that of Nephtys and Nereis. Twenty-four paired and
two unpaired ganglionic nuclei were identified. There are four paired
nuclei in the forebrain, eighteen paired and two single nuclei in the
midbrain and two paired nuclei in the hindbrain. The brain doe s not
appear to be structually as complex as that of Nereis, whereas it does
show a higher degree of organization than that of Nephtys.
The brain contains a l~rge proportion of neurons showing the
histological characteristics of neurosecretory cells. It appears that
the cerebral neurosecretory cells discharge their products solely by
axonal transmission. The secretory products from neurons in two pair
of ganglionic nuclei were found to be released from the brain via the
circumoesophageal connectives. Although there is no specialized
cerebro-vascular complex in Hermodice carunculata, there is a close
association between the brain and the blood-vascular system in the
ventral region of the posterior portion of the midbrain. There is
sorne evidence that neurosecretory material is released and accumulated
in this area and thence passed into the blood-vascular system.
In the postero-dorsal region of the midbrain, there is one group of neurons (nucleus XIV) which appears to undergo a synchronous diurnal -85-
secretory cycle. It is suggested that these cells may be involved in the production of the diurnal activity rhythm displayed by this werm.
The secretory products of these neurons appear to be released into blood vesse]s running through a prostomial cavity situated above the brain. -86-
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l:Œi:''ERENCE LETTJ:o:RS FOR ILLUSTRATIONS
The reference letters for the line drawings are in upper case while the reference letters for the photomicrographs are in lower case. The ganglionic nuclei are denoted by Roman numerals. a. axon a.c. acidophilic cytoplasm n.e. anterior eye a.f.g. = aggregate of fuchsinophilic granules a.h. axon hillock A.N.P. = antennal nerve to paired antennae ~.n.s. (A.N.S.) = antennal nerve to single antenna A.O .. N. = anterior optic nerve a.t. axon tract b. (B.) brain b.g. basal granules b.m. : brain membrnne b.n. = bipolar neuron b.v. blood vessel c. ;;::. cortex car. (CAR.) = caruncle c.e. (c.e.) - circumoesophageal connective C.CAR.. cavity of caruncle ch. (CH.) = chiasma ci. cilia c.l. (C .L.) cavi ty of lip c.n. (C.N.) = caruncular nerve coel. sp. (COEL. SP.) coelomic sp~ce cu. cuticle d.b.c. (D.H.C.) : dorsal brain cavity d.r.c.c. (D.R.c .. c.) = dorsal root of circumoesophageal connective
E. eve e.n. (E.N.) = nerve to lateral prostomial epidermis e. s.e. epidermal sensory cell
FB. fore brain fb.e. fihril of eridermal sensory cell f.c. fuchsinophilic cytoplasm f.g. fuchsinophilic granule f.o.o.n. fuchsinophilic ordinary pyriform neuron g.c. globuli cells g.p.n. = giant pyriform neuron hb. (HB.) = hindbrain i.c.t.m.b. (I.C.T.N.B.) = inner connective tissue membrane of brain -92-
L. lip l.d.b.c. lining of dorsal brain cavity l.h.hb. left half of hindbrain L.P.T. longitudinal pedal tract
M. mouth MB. midbrain ms.b. muscle band n. ::: nucleus n.b. neuro~lial border n.c. neuroglial cell n.f. neuroglial fiber ng. = neurogli? np. (NP.) neuropile n.tr. nerve tract o.p.n. ordinary pyriform neuron o.s.m.b. (O.S.M.B.) = outer syncytial membrane of brain o.s.n. = ordinary subspherical neuron p. peduncle p.e. (P.E.) = posterior eye PER. peristomium P.G) first perl~l ganglion P.N. = palpal nerve P.N.l = pedrl ncrve to first parapodium P.O.N. posterior optic nerve PP. palp PR.A. paired antennae PRO. prostomium r.h.hb. = right half of hindbrain r. s. s. (R.S.S.) root of stomatognstric system
S.A. = single antenna s.e. (S.C.) - strand of cytoplasm s.-e. b.m. (S.-E.B.M.) = sub-epiderntal ba seme nt membrane SET. = setiger s.n. (S.N.) stomatogastric nerve t.c. tubular canal t.c.m.b. (T.C.i1.B.) transverse commissure of midbrain T.N. = tegumentary nerve t.s.m. = thickened syncyti~l membrane v. vacuole V.CIR. = ventr2l cirrus v.r.c.c. (V.R.C.C.) = ventral root of circumoesophageal connective -93-
~~~~~:....-- PER. ~~~~7-.:;--2 ND. SET.
_..;...,..,~~~---- 3 RD. SET.
Fig. 1. Dorsal view of the anterior region of Hermodice carunculata. Free-hand drawing made ".vith the aid of a dissecting microscope. -94-
p
__;;;~---,t.,,#-~--~ ND. SET.
CIR.
Fig. 2. Ventral view of the anterior region of Hermodice carunculata. Free-hand drawing made with the aid of a dissecting microscope. -95-
Fig. 3. External morphology of the supraoesophageal ganglion, left lateral view. Pree-band drawing based on dissections and histological sections. -96-
Fig. 4. Transverse section through the prostomium at the level of the posterior eye-spots. Bouin fixation, Gabé's paraldehyde fuchsin-Halmi's trichrome. Photomicrograph . -97-
Fig. 5. Chiasma formed by the pair of cranial nerves to the single antenna. Bouin fixation, Gabé•s paraldehyae fuchsin Halmi's trichrome. Photomicrograph. -98-
Fig. 6. Transverse section through the midbrain showing the lateral epidermal nerve. Bouin fixation, Bodian's Protargol. Photomicrograph. -99-
Fig. 7. Oblique transverse section through the midbrain showing a circumoesophageal connective. Due to the oblique plane of sectionning, the stomatogastric root is also seen. Bouin fix ation, Gabé 1 s paraldehyde fuchsin Halmi1s trichrome. Photomicro graph . -100-
Fig. 8. Transverse section through the prostomium at the level of the forebrain. Bouin fixation, Gabé 1 s paraldehyde fuchsin-Halmi's trichrome. Photomicrograph. -101-
Fig. 9. Trflnsverse sections tbrough the prostomium showing the appearance of the brain mem branes and the coelomic spaces around the brain: A, at the level of the forebrain. B, at the level of the posterior end of the midbrain. Free-hand drawing based on histologicAl sections. -102-
Fig. 10. Transverse section through the rnidbrain at the level of the anterior eye-spots. Bouin fixation, Gabé's paraldehyde fuchsin-Halrni's trichrome. Photornicrograph. -103-
~7'--f#--I.CT.M.B. /--~.---a S. M. B.
"""-----C0 EL. SP.
s.e.
Fig. 11. Transverse section through the posterior end of the rnidbrain shm·ling the structure of the brain rnembr Fig. 12. Frontal section through the brain at the level of the intra-cerebral canal. Bouin fixation, Bodian 's Protargol. Photomicrograph. --ci. cu. Fig. 13. Ciliateù sensory cell in the dorsal prostomial epidermis. Bouin fixa tion, Bodian's Protargol. Photomicrograph. -105- Fig. 14. Types of cells in the Brain of Hermodice ~~c_u)~.· A, ordinary pyriforœ neuron with ovoid nucleus. B, ordinary pyriform neuron with bean-shaped nucleus. C, ordinary subspherical neuron. D, globuli cell. E, bipolar neuron. F, giant neuron. G, neuroglial cell. Camera lucida drawings made from histological sections. -106- Fig . 15. Bipo1ar neurons in nucleu VII. Bouin fixation, Gabé's par~lde hyde fuchsin-Halmi's trichrome. Photomierograph. -107- Fig. 16. Transverse commissure of the midbrain. Bouin fixation, Bodian's Protargo1. Photomicrograph. -108- A B Fig. 17. A, Transverse section through the midbrain showing the origin of the circumo~sophageal connectives. B, Diagrammatic representation of the corpora pedun culata in the left side of the brain, left lateral vie w. Free-hand drawings based on histological sections. -109- 51------t 61-----T Fig. 18. Composite map of the suprnoesophageal g&ng lion sl,owing the positions of the ganglionic nuclei. The dorsal and lateral nuclei are shown on the right band si de; the ventral 2nd ventro-lateral nuclei are shown on the left hand side. The horizontal lines on the left lland side indicate the positions of the transverse sections shown in Fig. 19. Free-hand drawing based on histologies! sections. -110- Nllf.\-+---IV rr~"\\lio..-'1---S.l'l. S-E.BM. c.s G. 7 Fig. 19, A-H. Transverse sections through the supraoesophageal ganglion showing the positions of the g~nglionic nuclei. The positions in the brain of thP trans verse sections are indicated by numbers which correspond to the numbered lines in Fig. 18. Free-hand drawings based on histological sections. -lll- Fig. 20. Portion of a transverse section through the midbrain showing nucleus V on one side of the brain. Bouin fixation, Gabé 1 s paraldehyde fuchsin-Halmi's trichrome. Photomicrograph. -112- Fig. 21. Transverse secti~n of the midbrain showing a portion of nucleus XI. Bouin fixation, Gabé's paraldehyde fuchsin- Halmi ' s trichrome. Photomicrograph. -113- Fig. 22. Transverse section through the midbrain showing sorne lateral and ventro-lateral nuclei on one side of the brain. Bouin fixation, Gabé's paraldehyde fuchsin-Halmi's trichrome. Photomicrograph. -114- Fig. 23. Transverse section through the anterior portion of the hindbrain. Bouin fixation, Gab~'s paraldehyde fuchsin-Ha1mi's trichrome. Photomicrograph. -115- Fig. 24. Transverse section through the posterior portion of the hindbrain. Bouin fixation, Gabé's paraldehyde fuchsin Halmi 's trichrome. Photomicrograph. -116~ Fig. 25. A fuchsinophilic ordinary pyriform neuron in nucleus XII. The ce11 nucleus is situated in the axon hillock. Bouin fixation, Gabé's para1dehyde fuchsin-Halrni 1 s trichrome. Photornicrograph. -117- Fig. 26. Nucleus XIV subspherical neurons in Stage 1 of their secretory cycle. Fixed in Bouin's at 5:00p.m., Gabé's paraldehyde fuchsin-Halmi's trichrome. Photomicrograph. Fig. 27. Nucleus XIV neurons in Stage 2 of their secretory cycle. Fixed in Bouin's at 12 midnight, Gabé's paraldehyde fuchsin-Halmi's trichrome. Photomicrograph. -118- Fig. 28. Nucleus XIV neurons in Stage 3 of their secretory cycle. Fixed in Bouin's at 6:30 a.m., Gabé's paraldehyde fuchsin-Halmi's trichrome. Photomicrograph. Fig. 29. Nucleus XIV neurons in Stage 4 of their secretory cycle. Fixed in Bouin's at 12 noon, Gabé's paraldehyde fuchsin-Halmi's trichrome. Ph otomicrograph . -119- Fig. 30. Secretory giant pyriform neurons in nucleus VI. Bouin fixation, PA/S (following diastase)-hema toxylin. Photomicrograph. -120- Fig. 31. Transverse section through the posterior region of the midbrain showing the thickened syncytia1 membrane 1ining the infra-cerebral coelomic space. Bouin fixation, Gabé's paraldehyde fuchsin-Halmi 1 s trichrome. Photomicrograph. Fig. 32. Transverse section through the posterior region of the midbrain showing an aggregate of fuch sinophilic granules in the syncytial pericap sular membrane of the brain. Bouin fixation, Gabi 1 s paraldehyde fuchsin-Halmi's trichrome. Photomicrograph. -121- 1. LAT.E P.t.t. 2.. MICNIGHT 3. EARLY A.M. Fig. JJ. Neurons of nucleus XIV at different stages in their secretory cycle. The sparse heavy stippling indicates acidophilic ground cyto plasrn, the fine dense stippling indicates fuchsinophi lie ground cytoplasm and the coarse black dots and streaks represent fuchsinophilic neurosecretory products. Camera lucida drawings made from histological sections 'vhich had be en fixed in Bouin t s and stained in Gab~'s paraldehyde fuchsin•Halmi's trichrome.