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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.
for her supervision of the problem.
c.
Marsden
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 ••••••••••••••••••••••••••••••••••••••••••••
SECTION I:
BRAIN ANATOHY
••••••••••••••••••••••••••••••••••••••••••••••••••••
4
.......................................
4
Drain of Nephtys •••••••••••••••••••••••••••••••••••••••
7
INTRODUCTIOI~
General Introduction
1~e
1
...........................................
Dissections ................................................
NATERIALS AND METHOUS
11
11
Histological Work ••••••••••••••••••••••••••••••••••••••••••
12
....................................................
16
External Morphology of the Head ••••••••••••••••••••••••••••
16
Eye-Spots ••••••••••••••••••••••••••••••••••••••••••••••••••
18
Gross Morphology of the Brain ••••••••••••••••••••••••••••••
19
Cranial Nerves
20
OBSERVATIONS
.............................................
Microanatomy of the Brain
...................................
Prostomial and Brain Cavities •••••••••••••••••••••••••
Brain 11embranes ••••..•.•••••••••••••••••••••••••••.•••
...............................
............................
...............................................
.............................................
Epidermal Sensory Cells
(~neral Brain Architecture
Neurons
Neuroglia
Internai Organization of the Brain ••••••••••••••••••••
General Brain Organization •••••••••••••••••••••••
Circumoesophageal Connectives ••••••••••••••••••••
24
24
27
28
28
29
lionic Nue lei .•••••••..••..•••••••.•••..•.•••
32
34
34
35
35
DISCUSSION ••••••••••••••••••••••••••••••••••••••••••••••••••••••
47
SECTION II:
NEUROSECRETION
....................................................
General Introduction .......................................
Morphological Characteristics of Neurosecretory Cells
Histochemical Aspects of Neurosecretion ....................
Functional Significance of Neurosecretion ..................
INTRODUCTION
54
54
54
58
61
TABLE OF CONTENTS (contd.)
Neurosecretion and Diurnal Rhythms • • • • • • • • • • • • • • • • • • • • • • • • •
64
1~eurosecretory
Cells in Polychaetes • • •• • • • • • •• • • •• ••• • • • • • •
66
...........................................
69
.....................................................
70
DISCUSSION • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •
80
MATERIALS AND METHODS
OBSERVATIONS
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:
has analysed the se portions
forebrain, midbrain and hindbrain.
<~ccording
Racovitza
to the following table:
Brain Region
Organs Innervated
Nervous Centres
Fonctions
Palpa! area or
fore brain
Palps and gustatory
grooves
Palpai ganglia
Touch and
tas te
Sincipital area
or midbrain
Eyes and antennae
Optic ganglia and
antennal ganglia
Sight and
ta ste
Nuchal area or
hindbrain
Nuchal organs
Nuchal ganglia
Olfaction
(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
-5well 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
simplification of the brain.
acc~npanied
by
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 completely 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.
cerebral centres.
These have been studied more extensively than other
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
in his study.
Neeht~s
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 completely 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 prostomium 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 anterodorsal 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 posteralateral 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 lobes 11 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.
occur ventrally below the neuropile.
Few nerve cell-bodies
The neuropile is composed of two
main parts, anterior and posterior, and the posterior neuropile is subdivided 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.
finds globuli cells.
bodies.
ln the brain of sorne speci.es of Nephtys, Clark
However, they are not organized into true mushroom
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.
in
~htys,
Clark has produced evidence that the cerebro-vascular complex
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 microscopically 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
ethanol.
70~
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.
worms were placed in glass dishes containing sea water.
The living
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 microstructure of the brain.
Mallory's
These were: Bodian's (1937) Protargol method;
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 counterstain, 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
-13be 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 connective 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.
transverse sections of
The other ganglion bad been eut in seriai
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.
lips are hollow.
The mouth is ventrally situated and oval.
The
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).
eye-spots are on the dorsal surface of the prostomium.
The
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.
of each eye-spot is a round black spot.
In the centre
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.
paired antennae project from the antero-dorsal edge of the
just behind the origin of the lips.
The
prostomium~
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.
structures.
The eyes of Hermodice carunculata are cup•shaped
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.
celled layer.
The retina is a single-
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.
could only be seen to disappear between the pigmented cells.
They
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.
entirely prostomial in Hermodice carunculata.
The brain is
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.
halves.
Each part is composed of two symmetrical
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.
midbrain.
The hindbrain is smaller than the forebrain and
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 continuous 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 subepidermal 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 dorsalateral 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 configuration 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.
all arise from the hindbrain.
These nerves
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. 7 1 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
( n)
The
Arisil'!.&..-~.rom
Pal~al
the Anterior Surface of the Brain (Fig. 1)
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 descriptions by Storch and Grassé.
Marsden. *
The same discrepancy has been noted by
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 prostomial tissue.
(Fig. 4, 9B).
body.
Instead it is suspended above a cavity in the prostomium
This cavity is continuous with the coelomic cavity of the
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.
are large and chromophobic and nearly
In this inner layer the nuclei
s~rical
and are evenly spaced in a single layer.
(slightly irregular)
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 midbrain (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.
philic cytoplasm.
nucleolus.
The fibroblasts are large fusiform cells with an acido•
They have a large spherical nucleus with a single
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
prostomial basement membrane.
of the lateral
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 intervening 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.
cuticle.
The cilia protrude through tiny pores in the
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.
blood vessels penetrating the brain tissue.
There are no
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).
also scattered throughout the neuropile (Fig. 7, 22).
They are
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.
ordinary neurons:
There appear to be two types of
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.
of ordinary neurons may exhibit fuchsinophilia.
Both types
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.
neurons of each type had two or three such nucleoli.
Sorne
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).
while the
cell~bodies
The axons have numerous collateral branches,
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
The
cell~body.
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.
large oval nucleus is centrally located.
The
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.
throughout the cytoplasm.
There are numerous very fine granules distributed
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.
width about
6~),
pyriform neurons.
hillock.
Except for the ir large size ( length about 15}1 ;
they are morphologically very similar to the ordinary
The cell-body is pyriform, with a prominent axon
The cell-body of the giant neuron is proportionally more
slender and elongated than that of the ordinary pyriform neuron.
are tiny dendrites extending from the cell-body.
There
The cytoplasm may
show any of a wide variety of staining reactions, ranging from chromophobia 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 cellbodies (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.
are diverted along other paths (Fig. 17B).
Sorne of them
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.
tracts are the palpai nerves.
These
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).
the brain.
Globuli cells are found nowhere else in
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.
axon tracts of nuclei I and II.
The peduncles of the globuli are the
On each side of the brain the peduncles
of the two globuli merge with each other to form a single ventrallydirected 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.
These are a pair of very diffuse nuclei.
(Fig. 8, 19A).
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.
dorsally towards the dorsal edge of the brain.
They run
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).
compressed nuclei.
These are a pair of very small, laterally
They are situated side by side between the bases of
the two stomatogastric nerves.
stomatogastric nerves.
The axons run laterally and enter the
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.
unite to form a discrete fiber tract.
root independently.
The axons do not
Each axon enters the connective
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
edge of nucleus
v.
nE~urons
arrayed along the outer
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.
of these nuclei do not form discrete fiber tracts.
The axons
They run ventrally
and then turn medially to enter the transverse commissure.
The neurons
are a mixture of pyriform and subspherical types.
(Fig. 19C).
XIII.
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.
connected with the neuropile.
The commissure itself is intimately
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.
and impinge upon
th~
They are directed dorsally
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
v.
below the anterior portion of nucleus
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.
towards the neuropile.
The axons are directed medially
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 the neuropile.
In other words, nucleus XVI is partially embedded
The neurons are all of the ordinary pyriform type.
-43-
This is a pair of small, compact, spherical
XVII.
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.
(Fig. 19G, 22).
XVIII.
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.
XIX.
The neurons are of the ordinary pyriform type.
(Fig. 22).
in be tween XVII and XVIII.
These are very small paired nuclei hemmed
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
these nerves leave the cerebral cortex.
medially and enter the optic nerves.
nerve~,
just before
The axons are directed dorso-
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
XXV.
(Fig. 23).
(F~.."_,_l2J..
The transition from midbrain to hindbrain
is marked by a vast and abrupt reduction of the cortical area.
hindbrain there are no nerve cell-bodies beneath the neuropile.
In the
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 somewhat laterally and which emerges from the dorsal surface of the hindbrain 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.
constitute nuclei XXVI.
These clusters
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 prostomial 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
cu lata than in Nephtys.
Hermodic~ ~­
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.
to nucleus VII.
These are very few and are confined
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
Nere
v,
while in
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.
of nuchal nerves,
th~
In Hermodice carunculata, there are four pairs
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
Hermo~}~
carunculata.
~ephtys,
but this is not present in
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.
br:1 in of
These antennal nuclei are not represented in the
~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.
have four· eye-spots, an anterior pair and a posterior pair.
Both worms
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
and Nephtx._s are constructed on similar patterns.
~.!lculata
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 nucleuspreopticus 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 neurosecretory cells are necessarily morphological (Clark, 1959).
A true
-55-
neurosecretory cell has the characteristics of a neuron plus the
characteristics of an endocrine cell.
nerve celln.
'~land-
Like an ordinary neuron, the neurosecretory cell possesses
an axon, neurofibrils and Nissl bodies.
(Clark, 1956).
ln other words, it is a
The dendrites may be lacking
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 polymorphonuclear 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 connected with their active metabolism. Thus 4 or 5
capillaries sometimes surround a large glandnerve 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 neurosecretory cells (Clark, 1959), it is well known that vacuolation also
characterizes the trophospongial phenomena which result
of neuroglial cell processes into neurons.
from the invasion
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:
its terminais and/or
the perikaryon.
(1) by movement dawn the axon to
(2) by direct diffusion through the membrane of
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 paraventricular 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 hypothalamohypophyseal tract to the neurohypophysis,
re le a se into the b lood stream.
~1ere
they are storerl prior to
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.
secretory substance is
tr~nsported
The neuro-
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.
following factors:
The situation is complicated by the
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 staining 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 histological 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.
blue and Gabé 1 s paraldehyde fuchsin.
These are alcian
Adams and Sloper (1956) showed
that in vertebrates, alcian blue, after performic acid oxidation, stains
neurosecretory material specifically.
been confirmed in invertebrates.
So far, this observation bas not
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 neurosecretory.
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 investigation, the criteria for neurosecretory cells are necessarily morphological 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 similarities.
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 cornpanent 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, i t 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 postulated 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 paraventricular 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 regeneration.
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.
day the ganglion again contains very little hormone.
contain
i1
By the fifth
Intact worms also
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.
immature Nereids and Nephtyids
The supraoe sophageal ganglion of
secretes a hormone that inhibits sexua]
-64-
maturation of the worms (Durchon, 1952; Cl.n:k, 1956).
knovm as the juvenile hormone.
This hormone is
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 regenerating.
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 crustaceans 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).
Cambaru~
In this organism and in Procambarus clarkii and
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 neurosecretory 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 suboesophageal 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 suboesophageal 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
(c)
Late afternoon, 4:00 - 5:00
(d)
Midnight, 12 midnight- 1:00 a.m.
p.m~
p~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).
time of day \vhen this specimen was fixed is not kno,m.
Unfortunately, the
-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
cell" as i t is used in the following account.
11
neurosecretory
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, i t 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,
vacuole s.
th·~
neurosecretory material does not occur within cytoplasmic
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
The cytoplasm shows no fuchsin-
Afternoo? (Fig. 26, 33):
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.
chromophobic and inconspicuous.
The axons are still
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 connective tissue lining.
Sorne of the granules in the
depression are aggregated to form composite clusters
ranging from
2-6~
in diameter.
This is apparently the final stage in
4.
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/Shematoxylin presented a staining pattern which was entirely different
from that of ganglia stained with paraldehyde fuchsin.
two PA/S-positive sites in the brain.
XIV.
There were only
These were nucleus VI and nucleus
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 circumoesophageal 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 circumoesophageal 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 midbrain.
The only anatomically specialized feature in this
area is a thickening of the syncytial lining of the infracerebral 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
secretory cells (Clark, 1959).
ccm~on
feature of neuro-
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
Nucleus XIV is entirely neurosecretory.
Neeh~.
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.
neurosecretory material is
relea~ed
There is a suggestion that sorne
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 circumoesophageal 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
clock.
the paradigm of the internai biological
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 transplantations 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.
techniques.
Histological studie s should coincide with all the experimental
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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-91-
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
brain
basal granules
brain membrnne
bipolar neuron
blood vessel
b. (B.)
b.g.
b.m.
b.n.
b.v.
:
=
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
fore brain
fb.e.
fihril of eridermal sensory cell
fuchsinophilic cytoplasm
f.c.
fuchsinophilic granule
f.g.
fuchsinophilic ordinary pyriform neuron
f.o.o.n.
FB.
g.c.
g.p.n.
=
hb. (HB.)
globuli cells
giant pyriform neuron
=
hindbrain
i.c.t.m.b. (I.C.T.N.B.)
=
inner connective tissue membrane of brain
-92-
L.
lip
l.d.b.c.
l.h.hb.
L.P.T.
M.
MB.
ms.b.
lining of dorsal brain cavity
left half of hindbrain
longitudinal pedal tract
mouth
midbrain
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
peristomium
PER.
P.G)
first perl~l ganglion
P.N. = palpal nerve
P.N.l = pedrl ncrve to first parapodium
posterior optic nerve
P.O.N.
PP.
palp
paired antennae
PR.A.
prostomium
PRO.
r.h.hb. = right half of hindbrain
root of stomatognstric system
r. s. s. (R.S.S.)
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 fuchsinHalmi's trichrome.
Photomicrograph .
-98-
Fi g . 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 fixation, Gabé 1 s paraldehyde fuchsinHalmi1s trichrome. Photomicrograph .
-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 membranes 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<Jne s
and the lining of the coe lomic snace bene ath the brain.
free-hand drawing based on histological sections.
-104-
Fig . 12.
Frontal section through the brain at the level
of the intra-cerebral canal. Bouin fixation,
Bodian 's Protargol. Photomicrograph.
--ci.
Fig . 13.
cu.
Ciliateù sensory cell in the dorsal prostomial
epidermis. Bouin fix a 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-
Fi g . 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
Fig. 17.
B
A, Transverse section through the midbrain showing the
origin of the circumo~sophageal connectives.
B, Diagrammatic representation of the corpora pedunculata 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&nglion 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 transverse 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. Photomi crograph .
-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 fuchsinHalmi '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.
Fig. 27.
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.
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.
Photomicro graph .
-119-
Fig . 30.
Secretory giant pyriform neurons
in nucleus VI. Bouin fixation,
PA/S (following diastase)-hematoxylin. 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 fuchsinophilic granules in the syncytial pericapsular 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 cytoplasrn, 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.