On the fine structure and distribution of secretory cells in the supraoesophageal ganglion of the lugworm (Arenicola marina L.)

GENERAL

AND

COMPARATIVE

ENDOCRINOLOGY

31, 350-363 (1977)

On the Fine Structure and Distribution of Secretory Cells in the Supraoesophageal Ganglion of the Lugworm (Arenicola marina L.) D. I. D. HOWIE Zoology

Department.

Trinity

College,

Dlrblin.

2. Irelund

Accepted November 8. 1976 The ultrastructure of the brain in Arenicola has been investigated with special reference to secretory cells and processes. Electron-dense secretory granules (elementary granules) are found in neurones, both in the brain proper and in the neuroectodermal connectives. Based on differences in fine structure four categories of secretory neurones appear distinguishable: (i) large dorsal neurones with elementary granules < 1200 A in mean diameter, probably containing nonpeptidergic secretion; (ii) equally large neuroectodermal cells with elementary granules > 1200 8, in mean diameter, which may contain peptidergic secretion; and (iii) two types of elongate or fusiform cells, which, in some cytological details, mimic either dorsal neurones or neuroectodermal cells; they contain, respectively, elementary granules < 1200 8, and > 1200 8, in mean diameter. The similarities which exist between the elongate cells and the two categories of larger neurones leave some doubt about their separate status. The neuroectodermal cells and the elongate cells with larger granules are confined to the subectodermal nervous tissue and the connectives. The majority of the processes of these cells terminate on the perineurium of the connectives. Dorsal neurones and their processes are chiefly confined to the brain proper. From previous experiments it is known that the maturation hormone produced by the brain is found only in the posterior half of the brain. The zonation of secretory cells and processes, described here. fails to match this known variation in hormone content. No obvious storage/release site has yet been detected, and it seems likely that hormone is released via terminals scattered over the perineurium.

Various aspects of sexual maturation are known to be under humoral control in polychaet species. The literature has been thoroughly reviewed on a number of occasions (Durchon, 1962; Clark, 1965, 1969; Golding, 1972; Clark and Olive, 1973). In Arenicola, the final phase of ripening of the gametes involves the appearance of metaphase figures of the first maturation division in the eggs and, in the males, the release of spermatozoa from sperm morulae. Spawning follows automatically, and the eggs are immediately fertilisable (Howie, 1961a, b). Decerebration inhibits all these processes, ~while replacement, using brain homogenates, restores ripening of the gametes and spawning. As a result,

the existence of a “maturation” hormone has been postulated (Howie, 1963, 1966). There is also some evidence that proliferation of spermatogonia is influenced by a brain hormone (Howie and McClenaghan, 1965; Olive, 1972a,b). Despite this experimental evidence of neurohormone secretion inArenicola, there are few histological signs of secretory activity in the brain. Neurones colouring strongly with the usual stains for neurosecretion, e.g., paraldehyde fuchsin and Alcian blue, are sparse or absent (Howie, 1966). The paucity of secretory cells colouring with these or other dyes is in sharp contrast with the situation in other annelids. In the most commonly studied polychaet genus, Nereis, at least 350

Copyright All rights

Q 1977 by Academic Press. Inc. of reproduction in any form reserved

ISSN 0016-6480

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four types of secretory cells were described initially by Scharrer (1937). While not all of these have proved to be neurosecretory on subsequent examination; nevertheless, evidence of secretory activity is widespread in the brain. Golding (1967) and Dhainaut-Courtois (1968) have made ultrastructural studies of nereid brains and attempted to relate the numerous secretory cells observable with the electron microscope to the cell types recognisable with the light microscope. Secretory cells are frequent in the brains of other errant polychaets, oligochaets. and leeches (see Golding, 1972, 1974: Myhrberg, 1972; Hagadorn et ul., 1963, respectively). The brain in the lugworm has now been reinvestigated for secretory cells, in this case at the ultrastructural level. Some initial observations have been made on the distribution of both the cells and their processes to discover whether or not this corresponds in any way to the evidence from partial decerebration experiments, which have established that the maturation hormone is located only in the posterior half of the prostomium (Howie, 1966). MATERIALS

AND METHODS

The worms used were collected from Booterstown Strand, Dublin. Thin sections have been examined from prostomiums collected and fixed in June, July, September, and November. i.e., during the period when gametes are developing in the coelom up to spawning in the autumn. Prostomiums were removed without anaesthetic by the method described for decerebration (Howie. 1963). As the prostomium measures only 1.2 x 1.O mm. it can be fixed whole, but it was occasionally divided transversely or longitudinally into two portions prior to fixation. The material described and figured was fixed in chilled Palade’s mixture (1% osmium tetroxide in a Veronal-acetate buffer) and embedded in Araldite. Sections were stained in Reynold’s lead citrate or. sequentially, with uranyl acetate and lead citrate. Fixation was not improved when the tonicity of the fixative mixture was raised to that of sea water by using sea water as the carrier or by the addition of salt or sucrose. Fixation using various concentrations of glutaraldehyde from 2.5 to 5% (with or without sucrose added) and postosmification gave poor results.

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Fixation in 5% glutaraldehyde in sea water at pH 3 with postfixation in 2% 0~0, yielded better results. but none of the glutaraldehyde techniques is as good as the simpler method of Palade. Sections were cut on an LKB Ultratome. Thick sections were taken along with each series of thin sections for accurate location of fields examined under an electron microscope. These thick sections were stained with hot alkaline toluidine blue. The dye fades rapidly when sections are mounted but is remarkably stable in unmounted sections. Thin sections were examined principally with an Hitachi HS7 electron microscope.

RESULTS

The brain lies entirely within the prostomium, and consists of paired forebrain lobes, an unpaired midbrain, and paired posterior lobes. The last of these merge into short “nuchal” nerves running to the base of the nuchal groove. The brain and nuchal nerves are joined to the overlying ectodermis by columns of nervous tissue, or neuroectodermal connectives, referred to subsequently as “connectives.” Nerve cell bodies occupy principally the dorsal part of the brain and extend into the connectives, spreading out and mingling with the basal portions of the ectodermal cells (subectodermal nervous tissue). The neuropile occupies the ventral portion of the brain. For illustrations of the structure and histology of the brain, see Howie (1966). At the level of ultrastructure, stored secretory materials appear in many neurones in the form of electron-dense “elementary granules.” Categorisation of these cells is necessary for descriptive purposes, but I have avoided, as far as possible, using a system of cell “types” labelled with letters or numbers, as this may imply nonexistent homologies with other species. Dorsal Neurones

In the dorsal part of the brain proper, lo-20% of the cell population contains appreciable quantities of electron-dense elementary granules. This may be an overestimate, as selection of sections for critical examination is biased in favour of those containing prominent secretory cells. Cells

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containing large numbers of electron-lucent vesicles are very rare. A high proportion of the cell profiles which contain large numbers of elementary granules appears to be sections of large neurones (the dorsal neurones). Cells of this type form most of the dorsal cap of neurones, and nearly all of them contain some vesicles of variable electron density. The sections may be oval, with diameters up to 12.5 x 25.5 ,um, or roughly pyriform, with diameters up to 13.6 x 14.5 pm. This variation in shape may depend on the plane of sectioning, but it is equally possible that it reflects a genuine variation in the shape of the cells. The dorsal neurones are generally surrounded by three or four glial membranes, and in about half of them the plasma membrane is deeply indented by trophospongium (Fig. 1). These are highly complex cells, containing numerous Golgi bodies and mitochondria. However, the endoplasmic reticulum is relatively impoverished. A few cisternae are scattered through the cytoplasm, and these are often swollen to form irregular vesicles. Occasionally the cisternae are arranged concentrically. The distribution of elementary granules within the cell is always patchy. Embedded in such patches are larger membrane-limited bodies. These are generally 0.7 pm in diameter, but a few reach 1.1 pm. The largest of these inclusions often contain two electron-dense masses, with the limiting membrane indented between them, and could be formed by fusion of two of the smaller variety. These bodies may be uniformly electron dense, but occasionally they contain what look like the cores of elementary granules and fragments of other cell organelles. While the association of these inclusions with patches of elementary granules might suggest that they are storage bodies, it seems likely that some, at any rate, are autophagic lysosomes. No enzyme studies have yet been attempted on these organelles. The mean diameters (actually mean slice

HOWIE

diameters) of the elementary granules in individual dorsal neurones, calculated from 60 or more measurements of granule profiles touching random lines drawn through each cell, range from 600 to 1300 A (Fig. 2). The degree of electron density of the granules is variable. Subjectively, it appears to increase with the number of granules stored. The limiting membrane is distinct and crenellated. Both proposed modes of granule formation have been observed in the dorsal neurones, i.e., the packaging of electron-dense material preformed in the Golgi lamellae or the budding off of small clear vesicles which grow in size and density of the core as they migrate away from the Golgi zone. While certain cytological features of the dorsal neurones are constant, e.g., the association of elementary granules and lysosomes, there is considerable variation in other respects. Attempts have been made to segregate the population into more homogeneous subgroups, with respect to mean granule diameters, on the basis of such features as the presence or absence of trophospongium or variable cell profile, but without success. Numerical data in relation to these and other cells are summarised in Table 1. Nelrroectodermnl

Secretory

Cells

Counts in thin sections show that as many as 41% of the cells in the connectives associated with the mid- and hindbrain can contain appreciable quantities of elementary granules. Again, these data must be treated with caution. A small proportion of the cells counted was dorsal neurones. The majority were either elongate cells (see below) or cells belonging to a second category of large secretory cells, the neuroectoderma1 secretory cells, found characteristically, and possibly only, in the connectives. Neuroectodermal cells are more prominent in connectives associated with the mid- and hindbrain, but they also occur at the level of the forebrain. At one stage of this inves-

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FIG. 1. Large dorsal neurone (x 10,000); G, Golgi body; T, trophospongium; ysoson les indicated by arrowheads.

EG, elementary gr

D. I. D. HOWIE

FIG. 2. Mean diameters of elementary granules in various categories of cells.

tigation, it was believed that they were the dominant secretory cell of the connectives; more recent observations suggest that they are greatly outnumbered by the elongate cells. The neuroectodermal cells, like the dorsal neurones, are large (Table l), and their profiles vary in shape from oval to pyriform. In other respects, they differ substantially from the dorsal neurones. These differences may be summarised as follows. 1. The neuroectodermal cells become more evenly charged with elementary gran-

ules to the extent that the granules may occupy the whole cytoplasm apart from isolated patches of endoplasmic reticulum and Golgi bodies (Figs. 3 and 4). 2. The elementary granules are larger and more uniformly electron dense, and the bounding membranes are difficult to discern. The range of mean granule diameters is from 1200 to 1900 1\ (Fig. 2). Comparison of the populations of mean granule diameters in neuroectodermal cells and dorsal neurones confirms that these are significantly different at the 9% level (Tukey procedure). 3. Lysosomes and/or storage bodies are rare and are not specially associated with the elementary granules. 4. In the neuroectodermal cells, the endoplasmic reticulum is prominent and rough. The cisternae are concentrically arranged and often appear to spiral outwards from a “centre.” This may contain the debris of various cell inclusions. 5. The cytoplasmic matrix is generally denser than in the case of the dorsal neurones (Fig. 4). 6. Golgi bodies and mitochondria, though present, are less frequent. 7. Glial membranes are less prominent. The neuroectodermal cells display the mode of formation of elementary granules in which small electron-lucent vesicles are first budded off from the Golgi bodies. Only clear vesicles can be seen in the immediate vicinity of the Golgi bodies (Fig. 3).

TABLE I NUMERICAL DATA RELATING TO SECRETORYCELLS IN THE BRAIN Cells Maximum slice diameter

Granules

Secretory cells

Parallel to long axis (w)

Parallel to short axis (pm)

Nuclei Mean slice diameters (w)

Dorsal neurones Neuroectodermal cells Elongate 1 Elongate 2

12.5 8.2 4.6 3.4

13.6 13.0 5.0 4.1

3.8 3.4 2.9 2.5

x x x x

25.5 20.0 13.9 15.4

x 14.5 x 13.0 x 6.0 x 6.1

x x x x

5.4 5.4 4.2 4.3

Range of mean slice diameters (A)

Mean all cells (A)

600/ 1400 1200/1900 110011800 60011200

1010 1660 1430 970

SECRETORY

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3. 3. Neuroectodermal cell (X 18.000). ER. endoplasmic reticulum; N, tangential section of nucleus.

BRAIN

G, Golgi body and associated

vesi-

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FIG. 4. Neuroectodermal

Elongate

Cells

cell (NC) and dorsal neurone (DN) for comparison (~21,000).

diameter may exceed 15.0 pm, while the shorter axis may be < 6.0 pm. The nucleus, The remaining secre,tory cells, isolated so too, is markedly elongate. The cells can far, are distinctly fusiform or elongate. In contain numerous electron-dense granules sections parallel to the long axis the greater (Fig. 5), but in sections parallel to the short

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Elongate

axis, and passing through the nucleus, the nucleus occupies almost the whole of the section, so that few elementary granules can be observed. Like the large secretory neurones, the elongate cells are probably divisible into two categories. The first type (Elongate 1) has mean granule diameters varying between 1100 and 1800 A (Table l), and the cytoplasm contains relatively few

THE

cell.

LUGWORM

Type

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2 (X 19.000).

other organelles. They appear to be confined to the connectives and are most readily located among the bases of the ectodermal cells. The second type (Elongate 2) has been observed less frequently but appears to have a wider distribution. They are found alongside the Type 1 cells in the connectives but also have been located deep in the

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brain proper among the dorsal neurones. The Type 2 cells are furnished with a glial sheath of several membranes (Fig. 5). Relative to the Type 1 cells, they appear more complex, with numerous mitochondria. The mean diameters of the elementary granules range chiefly between 800 and 1200 A, i.e., smaller than those of the Type 1 cells at the 99% level of significance (Tukey procedure). Elongate cells can be found in both anterior and posterior parts of the brain. In many respects (other than shape), the elongate Type 1 cells mimic neuroectoderma1 cells, and the Type 2 cells mimic dorsal neurones. Comparison of the populations of mean granule diameters shows that in the first pair the populations are significantly different, whereas in the second pair they are not (at the 5% level), again applying the Tukey procedure. Secretory Granules in Nerve Fibres and Terminals Tracts of nerve fibres make up much of the brain tissue. The principal tracts form the neuropile. In addition, vertical and oblique fibre tracts transect the neuropile. These occur chiefly in the midbrain. Finally, loose bundles of fibres occur in the connectives. Some of these penetrate down into the brain proper. The fibre tracts have been scrutinised to locate axons containing secretory granules. It was hoped that the population of granules in each of the main tracts would be sufficiently characteristic in size and appearance to yield information on the general distribution of axons associated with the cell “types.” In the same way, profiles of the perineurium have been searched to locate secretory cell terminals, and a comparison has been made between the granules in terminals in the connectives and those in terminals situated in the brain proper. Sections containing vertical or oblique fibre tracts are of special interest because of the possibility that these tracts might be

HOWIE

homologous with the “neurosecretory neuropile” described by Golding and others in various errant polychaets (see Golding, 1974). However, in investigations to date, what at the light microscope level had been taken to be bundles of nerve fibres has proved under the electron microscope to be made up largely of glial cell processes, containing dense aggregations of fibrils. In fact, very few secretion-containing axons occur in the vertical and oblique fibre tracts. Figure 6 shows mean diameters of secretory inclusions found in the fibres forming the neuropile and the tracts in the connectives. A very few fibres contain only small electron-lucent vesicles (mean diameters between 400 and 600 A). Most of the secretion-bearing axons, whether in the neuropile or in the connectives, contain

FIG. 6. Mean diameters of vesicles and elementary granules in nerve fibres and terminals in various parts of the nervous tissue within the prostomium. Superimposed (dashed line) is the histogram of mean granule diameters in dorsal neurones.

c-nv.----x’ 3PLKEl”KI

--* LCLLJ

’ ” “I1IY

a mixture of electron-lucent vesicles and elementary granules (mean diameters, taken together, between 600 and 1200 A, see Fig. 7). The granules have a crenated membrane. A further category of axons contain large, dense elementary granules (means > 1200 A). These axons form a very small part of the neuropile, but a substantially greater proportion of the fibre population in the connectives. Not surprisingly, in view of the absence of secretion-bearing fibres from the vertical and oblique fibre tracts, there is no special accumulation of secretory endings where these tracts meet the perineurium. Indeed, no large aggregation of secretory terminals which might form a storage/release site has been located anywhere in the brain. Instead, small groups of terminals are scattered on the inner surface of the perineurium both of the brain and of the connectives. The relative frequency of the different sizes and types of secretory inclusions found in the fibre tracts repeats itself

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in the terminals. Endings containing only electron-lucent vesicles are even less frequent. The most common endings contain a mixture of electron-lucent vesicles and elementary granules with mean diameters < 1200 A. Terminals containing large dense granules (> 1200 A in diameter) are found both in the brain and in the connectives, but they form a much higher proportion of the endings in the latter (Fig. 6). Small groups of endings, packed with mitochondria, are characteristic of the surface of the nervous tissue. Some occur on their own, others in association with terminals containing elementary granules. In consequence, such endings are very common. The mitochondria are similar to those found elsewhere in the nervous tissue and ectodermis, i.e., they tend to be small in diameter, but elongate, with short, fingerlike cristae. Presumably, these endings are the “secretory end-feet” (S.E.F.) described by Golding (1974), but secretory granules or vesicles have not been ob-

FIG. 7. Elementary granules and vesicles: A, in fibres within the connectives and B, in the neuropile. Fibre with large dense granules (D); mixed fibre (M); and small transparent vesicles, at arrowhead (~20,000).

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served therein. The frequency of the mitochondria is sufficiently high to obscure any other inclusions which may be present. The perineurium surrounding the brain is thick and fibrous, especially ventrally. However, it becomes increasingly attenuated as the connectives reach out to meet the ectodermis. It is important to record that where connectives meet the ectodermis, the nervous tissue spreads out, forming a subectodermal layer, again lined with thin perineurium. No distinction is attempted, in this paper, between this subectodermal nervous tissue and the cells and processes in the connectives. Between the connectives, blood capillaries are found attached to the coelomic side of the perineurium, usually by a single cell. No special accumulation of secretory endings has yet been discovered in the vicinity of such points of attachment. Very occasionally, fibres containing elementary granules have been seen to penetrate some way through the perineurium, but never to do so completely. DISCUSSION Clark’s (1965) opinion that a high proportion of annelid neurones are capable of some form of secretory activity has been supported by ultrastructural studies on representatives of each of the major classes 1967; (Hagadorn et al., 1963; Golding, Dhainaut-Courtois, 1968; Myhrberg, 1972), and now on the lugworm. Despite the lack of staining with neurosecretory stains, previously reported in this species (Howie, 1966), up to 20% of the neurones in the dorsal part of the brain contain substantial numbers of elementary granules. More surprising is the high proportion of secretory neurones discovered in the connectives and at the base of the ectoderms. This observation may, after all, lend support to the earlier histological results obtained by Arvy (1954) which led her to report the presence cells in A. of numerous “neurosecretory” marina.

The principal

cells of the dorsal region of

HOWIE

the brain are the dorsal neurones. They are large, complex cells whose most obvious feature, when charged with secretion, is the spatial association of lysosomes or storage bodies with patches of elementary granules. The ultrastructural images of these cells correspond well with the large neurones, seen under the light microscope (Howie, 1966). Dorsal neurones also occur in the larger connectives, but appear to be replaced as the dominant secretory cell type by neuroectodermal cells whose most striking feature is the concentric cisternae of rough endoplasmic reticulum. More recent work (unpublished) casts some doubt on the high frequency of neuroectodermal cells recorded during these initial observations. The two types of cells are separable on a variety of other cytological criteria, but the most significant of these are the size and appearance of the elementary granules. In shape and in the relative volume of nucleus to cytoplasm, the elongate or fusiform cells differ markedly from the foregoing categories of large cells. The fact that they are found most commonly immediately below the ectodermis also suggests a functional difference between them and the large secretory neurones. On the other hand, the elongate Type 1 mimic neuroectodermal cells, and the elongate Type 2 mimic dorsal neurones both in regard to distribution (as between the brain proper and the connectives) and in cytological features. For example, there are close similarities in the size and appearance of the elementary granules (Fig. 2), although, in fact, the mean granule diameters of the elongate Type 1 cells and the neuroectodermal cells are significantly different on statistical analysis. Extensive viewing indicates that there tends to be continuous variation of many of the cytological features of the cells in the brain. Under these circumstances and in the absence of additional cytochemical or physiological evidence, the validity of the cell “types” proposed here must remain tentative. On the basis of accumulated evidence

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--_ _ ^ *_. IN

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from a variety of vertebrate and invertewhich are claimed to be the “a” cells of brate species, Knowles (1965) has proposed Scharrer. This possibility is being further two types of neurosecretion. Type A, pep- examined. Finally, the large and rather ditide neurosecretion, is characterised by verse dorsal neurones of the lugworm bear elementary granules > 1000 8, in diameter, similarities to many of the cells figured both in which the electron-dense contents fill the by Golding and by Dhainaut-Courtois vesicle. Type B, nonpeptide neurosecrewhich they describe as “ordinary neution, is represented by elementary gran- rones.” ules, with an irregular bounding memSince Bern (1962) first drew attention to brane, whose electron-dense contents do the care necessary in establishing whether not fill the vesicle, and whose diameter is < potential neurosecretory cells are indeed 1000 A. Subsequent investigations have not neurosecretory or not, it has been accepted challenged the validity of this classification, that important criteria, which should be which, in more extended form, is repeated applied, are whether the axons of the cells by Golding (1974). Applying these rules, in question form a distinct neurosecretory neuroectodermal cells and the elongate neuropile and whether they terminate in a Type 1 in the lugworm are potentially storage/release organ. peptide-secreting neurosecretory cells. The Unfortunately, these criteria cannot be dorsal neurones and elongate Type 2, on applied in the lugworm. Whereas dense sethe other hand, are probably nonpeptidercretory neuropile associated with an ingic and contain amine granules. fracerebral gland occurs in Nereis and Given the uncertain validity of the cell Nephtys and in Polynoidae, Aphroditidae, types described here, it is premature to at- and Sigalionidae (Dhainaut-Courtois, 1966; tempt to establish homologies between cell Golding et al., 1968; Baskin, 1970; Golding, types in animals as diverse as Arenicola 1970, 1973; Zahid and Golding, 1975), no and Nereis which, with Nephtys, is the only analogous structures have yet been located other polychaet genus in which the fine in the lugworm. Under these circumstructure of secretory perikarya has stances, and on the evidence so far availbeen examined (Golding, 1967; Dhainautable, it seems probable that the neurohorCourtois, 1968; Zahid and Golding, 1975). mones known to be present in the lugworm It is tempting, nevertheless, to draw atten- brain (Howie, 1963, 1966; Howie and tion to the similarity at the ultrastructural McClenaghan, 1965; Olive, 1972a, b) are level between the neuroectodermal cells in released from the small groups of axon terthe lugworm and the Type III cells of minals located around the perineurium. As Nereis described by Dhainaut-Courtois. in the case of the perikarya, these terminals Both display prominent concentric rough and axons show some degree of zonation endoplasmic reticulum and contain gran- (Fig. 6). Terminals containing large, dense ules with poorly defined membranes, granules form a substantial proportion of these granules being the largest in the brain. those located on the thin perineurium of the Golding (1974) believes that these are connectives but are exceedingly scarce on Scharrer’s (1937) Type “b” neurosecretory the surface of the brain proper. This cells. Dhainaut-Courtois, herself, states suggests that, in most cases, the neuroecthat the Type III cells had not been de- todermal and elongate Type 1 cells have scribed previously. She believes them to be short axons, terminating locally on the surneurosecretory, but the terminals of their face of the connectives. The mean granule axons could not be located. The elongate diameters in the neuropile and in terminals Type I cells in the lugworm bear some simi- on the perineurium of the brain, on the larity to Golding’s fusiform cells, and the other hand, show a close fit with the Type 1 cells of Dhainaut-Courtois, both of granule sizes of the dorsal neurones. From

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this it may be deduced that terminals on the surface of the brain are composed, in the main, of processes of dorsal neurones. In experiments involving progressively greater ablation of the prostomium (unpublished) it has been found that small fragments containing only the nuchal nerves and part of the hindbrain can maintain spawning, whereas the midbrain and forebrain fail to do so. Postmortem examination of the smallest fragments capable of maintaining spawning reveals that the only element of the blood supply to the prostomium invariably remaining intact in such fragments is the network of capillaries investing the connectives dorsal to the nuchal nerves and hindbrain. If the maturation hormone is blood borne, then it would seem that it must be transported by this capillary network, having been released from the small groups of terminals on the connectives. In the current work it has been shown that these terminals contain a high proportion of large, possible peptidergic granules derived from elongate Type 1 cells and neuroectodermal cells. Unfortunately the remaining evidence presented in this paper fails to support this hypothesis. The neuroectodermal and elongate Type 1 cells, though confined to the connectives, are not limited to the posterior part of the brain. Furthermore, no close microanatomical relationship exists between the scattered secretory endings in the connectives and the investing capillary network. Uncertainties, therefore, remain as to the site of production and release of maturation hormone. The strong possibility remains that hormone can escape from the brain from thinly scattered terminals, and without the necessity of a specialised storage/release site.

REFERENCES Arvy, L. (1954). Contribution a I’etude de la neurosecretion chez les annelides polychetes sedentaires. Bull. Lub. Mar. Dinurd. 40. 16-24. Baskin, D. G. (1970). Studies on the infracerebral gland of the polychaete annelid, Nrreis lirnnicolu. in relation to reproduction, salinity and regeneration. G~tz. Camp. Endocrinol. 15, 352360. Bern, H. A. (1962). The properties of neurosecretory ceils. Gen. Camp. Endocrinol. SuppI. 1. 1 l7132. Clark, R. B. (1965). Endocrinology and the reproductive biology of polychaetes. Oceanogr. Mar. Biol. Amu.

I wish to thank MrS. Claire Chambers (nee McClenaghan) and Mr. F. Walker for their assistance and Mr. McAirt for statistical advice.

3, 21 l-255.

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Clark, R. B., and Olive, P. J. W. (1973). Recent advances in polychaete endocrinology and reproductive biology. Oceunogr. Mar. Biol. Anntr. Rev. 11, 175-222. Dhainaut-Courtois, N. (1966). Le complexe c&ebrovasculaire de Nereis pelagica L. (Annelide Polychete). Donnees histologiques et ultrastructurales. C. R. Ac,ud. Sci. Paris 262, 2048-2051. Dhainaut-Courtois. N. (1968). Etude histologique et ultrastructurale des cellules nerveuses du ganglion cerebral de Nereis pelagica L. (Annelide Polychete). Comparaison entre les types cellules I-VI et ceux decrits auterieurement chez les Nereidae. Gen. Camp. Endocrinol. 11, 414-443. Durchon. M. ( 1962). Integration of reproductive functions. 1. Neurosecretion and hormonal control of reproduction in Annelida. Gen. Camp. Endocrinal. Suppl. 1, 227-340. Golding. D. W. (1967). The diversity of secretory neurones in the brain of Nrrris. Z. Zel(forsch. 82, 321-344. Golding, D. W. (1970). The infracerebral gland in Nephfys-a possible neuroendocrine complex. Grrz. Camp. Endocrinol. 14, I 14- 126. Golding. D. W. (1972). Studies in the comparative neuroendocrinology of polychaet reproduction. Gen.

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Golding, D. W. (1973). Neuroendocrine phenomena in aphroditid and related polychaetes. I. The morphology and cytology of the infracerebral gland and cerebral neurosecretory system. Actu zoo/. Srockholni 54, 101-120. Golding, D. W. (1974). A survey of neuroendocrine phenomena in non-arthropod invertebrates. Biol. Rev.

ACKNOWLEDGMENTS

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Clark, R. B. (1969). Endocrine influences in annelids.

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Golding, D. W.. Baskin, D. G.. and Bern. H. A. ( 1968). The infracerebral gland-A possible neuroendocrine complex in Nereis. J. Morphol. 124, 187-216. Hagadorn, I. R., Bern, H. A.. and Nishioka. R. S.

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