Structural organization of neurosecretory cells ...

Structural organization of neurosecretory cells terminating in the sinus gland of the terrestrial isopod, Oniscus asellus, revealed by paraldehyde fuchsin and cobalt backfilling R. G. CHIANG'AND C. G. H. STEEL Department

of Biology,

York University, Dow~rt.svie~~, Ont., Ccrrltrdtr M3J IP3

Can. J. Zool. Downloaded from www.nrcresearchpress.com by Renmin University of China on 06/03/13 For personal use only.

Received July 20, 1984 CHIANG, R. G., and C. G. H. STEEL.1985. Structural organization of neurosecretory cells terminating in the sinus gland of the terrestrial isopod, 0ni.sc.u.scr.sellu.s, revealed by paraldehyde fuchsin and cobalt backfilling. Can. J. Zool. 63: 543-549. The morphology of the brain - sinus gland neurosecretory system in the terrestrial isopod, 0ni.scu.s a.sellu.s, is described with paraldehyde fuchsin and with cobalt backfilling of the sinus gland. Paraldehyde fuchsin stained the A, B, and P cells located medially in the protocerebrum and the y cells located in the optic lobe. Cobalt applied to the sinus gland delineates an axon tract that extends from the sinus gland medially along the posterior surface of the protocerebrum and descends into the protocerebrum at the level of the central protocerebral neuropile. Cobalt backfilled to the B, P, and y cells but not to the A cells. One cell group located distally to the most distal optic lobe neuropile filled with cobalt, but was not stained with paraldehyde fuchsin. It is argued that the B and P cells together comprise the equivalent of the decapod "X-organ." Varicosities, which may represent additional storage and (or) release sites for neurosecretion. appear in the axon tract over the region of the optic lobe. Extensive dendritic arborizations of the B and P cells occur along the anterior-medial side of the central protocerebral neuropile. Additional arborizations of these cells occur in the contralateral protocerebral lobe, suggesting a pathway for neural coordination of left and right sinus glands. Further observations on changes in the staining properties of the p and y cells during the moult cycle suggest the involvement of P cells with moulting and the involvement of y cells with egg development. CHIANG, R. G., et C. G. H. STEEL.1985. Structural organization of neurosecretory cells terminating in the sinus gland of the terrestrial isopod, Oniscus asellu.~,revealed by paraldehyde fuchsin and cobalt backfilling . Can. J . Zool . 63: 543 - 549. La coloration a la paraldehyde-fuchsine et le remplissage au cobalt de la glande du sinus ont permis d'etudier la morphologie du systeme neurosecreteur cerveau - glande du sinus chez I'isopode terrestre 0ni.sc.u.s cr.se1lu.s. La paraldehyde-fuschsine a colore les cellules A, B et P localisees dans la partie mddiane du protocerdbrum et les cellules y situees dans le lobe optique. Le cobalt applique a la glande du sinus met en relief une etendue des axones qui part de la glande du sinus, s'etend medialement le long de la surface posterieure du protocerebrum et descend dans le protoc@r@brumau niveau du neuropile protocerebral central. Le cobalt s'est infiltre jusque dans les cellules B, f3 et y , mais n'a pas atteint les cellules A. Un groupe de cellules situe au-dela du neuropile le plus distal du lobe optique s'est rempli de cobalt, mais ne s'est pas color@a la paraldehydefuschsine. Nous croyons que les cellules B et P constituent ensenible I'kquivalent de I'organe X des decapodes. 11 existe aussi des renflements le long du trajet de I'axone qui surplombe la region du lobe optique; ce sont peut etre des sites additionnels d'accumulation ou de liberation des neurosecretions. Les cellules B et P envoient u n important reseau de ramifications dendritiques le long du c6te anterieur median du neuropile protocerebral central. D'autres ramifications de ces cellules se retrouvent egalement dans le lobe protocerebral contralatkral, ce qui suggere I'existence d'une voie de coordination nerveuse des glandes gauche et droite du sinus. D'autres observations sur les changements des propriktes d'absorption des colorants dans les cellules P et y durant le cycle de la mue indiquent que les cellules P semblent jouer u n r61e dans le processus de la mue et que les cellules y semblent jouer un r6le dans le developpement des oeufs. [Traduit par le journal]

Introduction Physiological properties of neurosecretory cells (NSCs) have been studied extensively in crustaceans, as these animals possess relatively simple nervous systems and their NSCs are readily accessible for experimental investigations (Kleinholz and Keller 1979; Cooke 1981; Cooke and Sullivan 1982; Stuenkel 1983). However, mechanisms by which sensory influences regarding moulting and moult inhibition are integrated by the nervous system to elicit a neurohormonal response have yet to be investigated in crustaceans, partially owing to the difficulty in determining what nervous stimuli initiate such endocrine events. This disadvantage, which applies primarily to decapods, can be avoided by examining terrestrial isopods; in these animals, the stimulus to moult initiation is known (Steel 1980). Further, the appearance of calcium deposits on the sternites allows the stages of the moult cycle to be determined accurately and the onset of premoult can be detected well in advance of apolysis of the integument (Steel 1982), which has often been used as the earliest morphological sign of 'Author to whom all correspondence should be addressed.

premoult in other crustaceans (see Passano 1960). To study the mechanisms by which the nervous system regulates activity of NSCs in terrestrial isopods, it is necessary to know the morphology of the NSCs controlling processes such as moulting and reproduction. It has been shown that NSCs regulating these processes terminate in the sinus gland (SG) (Reidenbach 1965; Charmantier and Trilles 1975), a neurohaemal organ attached to the optic lobe in the head (see Gabe 1966). However, the morphology of the brain-SG neurosecretory system in isopods remains poorly understood despite several investigations describing this system (Matsumoto 1959; Gabe 1966; Messner 1966; Vitez 1970; Warburg and Rosenburg 1978; Martin 19720, 19726; Juchault and Kouigan 1975; Chataigner et al. 1978; Martin 1982; Zahid et al. 1982; Martin et al. 1983). The number and location of NSCs terminating in the SG is debatable (e.g., Martin 1972a, 19726, 1982) and the dendritic arborizations of these NSCs have yet to be described. In this present work, we have used paraldehyde fuchsin (PAF) and cobalt backfilling to ascertain the morphology of the brain-SG system in the terrestrial isopod, Oniscus asellus. The combination of these two procedures has allowed us to locate neurosecretory cell bodies in the protocerebrum which

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send processes to the SG and also reveals the ramifications of their dendritic processes within the brain where the central nervous system can modulate their electrical activity. These results provide the clearest description of the morphology of the neurosecretory system in the head of a crustacean and are important for understanding how the nervous system coordinates the release of hormones from NSCs in isopods.


Materials and methods Adult isopods, Onisc-uscr.sellu.s, were used throughout this study and were taken from populations maintained in the laboratory under one of the following two conditions (Steel 1980). ( i ) A large colony of isopods was confined in a single glass-top container filled with pieces of wood upon which the animals were found in the wild. This colony was held at 4OC in a 8 h light : 16 h dark (8L: 16D) day length cycle. These conditions effectively kept the animals in a prolonged intermoult stage. (ii) A second colony was reared in culture dishes. These animals were maintained at 21 t 1°C in a 16 h light : 8 h dark (16L:8D) day length cycle which promoted moulting and reproduction. he-moult stages were classified according to previously described criteria (Steel 1982). For histological analysis, 5- 10 heads from animals at selected stages of the moult cycle were fixed in a modified aqueous Bouin's fixative, embedded in paraffin, and serial sections were cut at 5 pm and stained with PAF (Steel 1977). The number and distribution of stainable cells was reconstructed for each preparation from the serial sections and the intensity of staining for each cell was assessed on an arbitrary seven point scale. For cobalt backfilling, intact animals were inimobilized with Plasticine, with the anterior side of the head orientated in an upward direction. The SG of one side was exposed by removing a small portion of the exoskeleton of the head. The SG was drawn into a glass capillary, 50-60 p m inside diameter, containing 0.5 M CoCII. Cells were allowed to fill extracellularly at room temperature for 15-20 h and the cobalt was precipitated with ammoniuni sulphide (Pitman et crl. 1972). Specimens were fixed for I0 min in a solution of acetic acid - alcohol ( 1 :4), dehydrated in alcohol, intensified according to Davis ( 1982). and cleared with methyl benzoate.

Results Puruldehyde fuchsin The most prominent part of the sinus gland of terrestrial isopods is a bulbous protrusion from the ventrolateral surface of the optic lobe. In living preparations, this protrusion has a brilliant blue white appearance owing to a striking Tyndall blue effect, attributable to the refractile properties of neurosecretory granules. In serial sections stained with PAF, this protrusion is apparent as an aggregation of deeply staining "droplets" approximately 5 pm in diameter (Fig. 1). These droplets are continuous with the PAF-positive axons in the optic lobe, which extend to neurosecretory cell perikarya located in the more proximal regions of the protocerebrum. We conclude that the droplets represent the terminals of these axons. They are surrounded by a very fine sheath and thus are near the haemolymph interface. This arrangement of neurosecretory terminals closely juxtaposed to the haemolymph is the classical indication that this structure is indeed a neurohaemal organ. The distribution of PAF-positive NSCs was compiled by reconstruction from serial sections and is summarized in Fig. 2. Four anatomically distinct groups of PAF-positive perikarya were identified and have been named according to the terminology originally introduced for Armudillidium by Matsumoto ( 1959). Axon tracts containing PAF-positive material could be traced from three of these four groups (B, P, and y cells) to terminals in the SG (Fig. 2). Thus, the SG contains terminals

FIG. I. Paraldehyde fuchsin stained sagittal section of the sinus gland and optic lobe of Onisc.us cr.sel1u.s illustrating axons (arrow) of deeply staining neurosecretory cells terminating in the sinus gland. Scale bar = 20 pm.

from at least three anatomically distinct groups of NSCs. The P cells are the largest and most densely staining cells. These cells are polygonal (average diameter, 18-20 pm) and are located in the anterior protocerebrum close to the midline. A prominent bundle of PAF-stainable axons arises from these cells and descends into the central protocerebral neuropile. Numerous streaks of PAF-positive material were found in this region of neuropile in the locations where arborizations of these axons were seen when the cells were filled with cobalt (see below). In this region, the axons of the cells are joined by those from the B cells, whose perikarya are located posterior to the p cells. The B cell perikarya are spherical and average about 15 pm in diameter. This combined tract of axons stains very intensely with PAF and is readily traced through the optic lobe to the SG. A third group of PAF-positive neurons is located in the central protocerebum (A cells, Fig. 2), but axonal processes from these cells could not be traced. These are large, teardropshaped cells (average diameter, 20-30 pm) in which the PAFpositive material is relatively sparce but is characteristically arranged in concentric whorls around the nucleus. The fourth group of cells revealed by PAF staining is located in the optic lobe adjacent to the medulla externa. These cells ( y cells, Fig. 2) are small ( I I pm diameter) but numerous, with up to 15 cells visible in nonbreeding intermoult animals. Numerous fine axons emanate from this group and join the tract of axons from the central protocerebrum shortly before it enters the SG. Numerous very fine strands of PAF-positive material were seen within the lamina ganglionaris. These appear to connect to y cell axons and are interpreted as arborizations of these cells within the lamina. The presence of arborizations of y cell axons in this region suggests that photic input may be an important regulator of y cell activity. Cobulr buckfilling The axon tract delineated by cobalt proceeds towards the midline from the SG along the posterior surface of the protocerebrum (Fig. 3). As its descends into the brain at the level of the central protocerebral neuropile, it divides to send branches laterally to two groups of cells bodies located near the dorsal

y

cells


\

B cells

fj c e l l s \

Central Prot o c e r e b r a l Neuro~ile

Medulla Externa

/

A cells FIG. 2. Dorsal plan view of the brain of 0ni.sr~u.srr.sellu.s illustrating the number and distribution of ncurosccrctory cells as determined from paraldehyde fuchsin stained serial sections. The axons of thc B. P, and y cells appear to terminate in thc sinus gland, whereas those of the A cells could not be traced.

midline and ventrally to arborizations on the medial-anterior side of the central protocerebral neuropile. The cobalt-filled tract travels laterally from these arborizations. traverses the midline. and forms another region of arborizations in the contralateral side of the protocerebrurn. Cell bodies are confined to the ipsilateral side and varicosities occur along the axon tract extending over the surface of the optic lobe. These features are described in detail below and are summarized in Fig. 10. In total, four groups of cell bodies were filled by cobalt and three of the groups correspond to cells identified by the PAF staining above. Two groups are located at the dorsal surface of the protocerebrum near the midline (Fig. 4); their locations correspond to the B and P cells. The posterior group (B cells) consists of up to four cell bodies which tend to be clumped together. Axons arising from these cell bodies form a single axon tract and individual axons were not resolved (Fig. 5 ) . Cell bodies of the P cells are more dispersed, enabling individual axons to be resolved. These axons join those of the B cells to form the main axon tract to the sinus gland. No cells in the location of the A cells were found to fill with cobalt. The other two groups of cell bodies that were filled with cobalt are located in the optic lobe. One group is situated on the anterior side of the optic lobe distal to the lamina ganglionaris (Fig. 6). This group contains approximately 10- 12 closely spaced spherical cell bodies with diameters ranging from 8 to 10 pm. No collaterals or connections to the main axon tract could be resolved. These cells were not selectively stained with PAF. The second group of cobalt-filled cell bodies in the optic lobe is located in the vicinity of the medulla externa; the cells are dispersed around the anterior surface of the optic lobe at this point. These cells correspond in location, number, and cell size to the cells observed with PAF staining. In some cases. very small axon processes from these cells were seen to meander between the medulla externa and lamina ganglionaris towards the main axon tract going to the SG. However, axon processes, observed within the lamina ganglionaris in PAF-stained material, are too fine to be resolved with cobalt backfilling. Two regions of arborizations were revealed by cobalt backfilling. One region is associated with the ipsilateral central protocerebral neuropile and the other with the contralateral central protocerebral neuropile. These regions are connected to one another by a cobalt-filled tract. The diameter of this connecting tract varies along its length, but is always constricted at the midline (Fig. 7). Both regions of arborizations extend

over the medial anterior side of their associated neuropiles indicating that the contralateral arborizations are formed in the region where arborizations from the contralateral NSCs are situated. In neither case do the arborizations appear to penetrate deeply into the neuropile. The ipsilateral arborizations are far more extensive than those on the contralateral side, although the extent to which cobalt fills the arborizations may be limited by diffusion. In at least one preparation (Fig. 8 ) , the ipsilateral arborizations can be seen to originate from both the B and P cells. Varicosities in cobalt-filled axons are also evident in the portion of the axon tract which extends along the posterior side of the optic lobe. As seen in Fig. 9, these varicosities appear as irregularities in the diameter of the axon or bulbous structures attached to the axons by a short branch. In animals mainfor several months. the varicosities are tained in intermo~~lt often so extensive that individual axons cannot be resolved. However, in animals undergoing moulting and reproduction, varicosities are not as extensive and individual processes could be resolved along the surface of the optic lobe. In most cases, varicosities are restricted to the more distal regions of the axon tract and axons in the more medial regions of the protocerebrum lack varicosities (e.g., Figs. 3. 4).

Cvc*lic.alI-hungc.s in PA F-positivc~cscdls The occurrence of cyclical changes in PAF-positive NSCs was investigated by comparing the appearance of the cells in animals fixed at various stages of the moult cycle and of egg development. Gross changes in cytological appearance were seen in both P and y cells, but no changes were detected in A or B cells. The cells stain intensely with PAF in intermoult and in early premoult animals (stages 1 and 2 of Steel 1982); but, with the onset of apolysis, the P cells commence a progressive decrease in staining intensity, becoming very pale by the time of posterior ecdysis. There is a parallel progressive decrease in the density of staining of neurosecretory terminals in the SG during premoult. as described earlier by Gabe (1952). The axonal tract between the p cell perikarya and the SG likewise decreases in prominence. These observations collectively indicate depletion of stainable neurosecretion from the P cells during premoult and imply that release of secretion from the P cells is closely correlated with moulting. This sequence of changes is unchanged in animals undergoing both moulting and egg development (in 2I0C, 16L: 8D). Thus, no changes in P


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FIG. 3. Posterior view of the protocerebrum illustrating the axon tract delineated by cobalt applied to the left sinus gland. Varicosites (V) are located over the optic lobe and arborizations (A) are located on the central protocerebral neuropile in both sides of the head. Cell bodies (CB) are out of the plane of focus. m , midline; scale bar = 0.1 mm. FIG.4. Dorsal view of the left lobe of the protocerebrum showing the main axon tract coursing over the posterior surface of the protocerebrum (between arrowheads). The tract curves anteriorly into the protocerebrum and divides to go to two separate groups of cell bodies (CB) located dorsally near the midline. A, arborizations; scale mark = 20 Fm. FIG. 5. Dorsal view of the right lobe of the protocerebrum illustrating axons from cell bodies of the B cells converging to form an axon tract that connects the B cells to the main axon tract (at arrow) going to the sinus gland on the right side of the head. Scale bar = 20 Fm. FIG. 6. Anterior view of the right optic lobe showing cobalt-filled cell bodies located on the anterior side of the optic lobe distal to the lamina ganglionaris (LG). Scale bar = 20 Fm. FIG. 7 . Anterior view of the preparation in Fig. 3 illustrating arborizations associated with the contralateral central ptotocerebral neuropile. CB, cell body of B cell; m, midline; scale bar = 20 Fm.

cells related to egg development are apparent. Conversely, the y cells undergo changes associated with egg development but not with moulting. These cells remain densely stained throughout moult cycles in which no egg development occurs; how-

ever, when vitellogenesis is stimulated by transfer of regularly moulting animals to 16L: 8D, changes in y cell staining ensue. Specifically, an obvious decrease in intensity of staining of these cells is seen in early premoult. This is the time when

CHIANG A N D STEEL


B cells

FIG. 8 . Camera lucida drawing of a cobalt preparation in which both the B and P cells were seen to give rise to arborizations in the neuropile.

vitellogenesis is first discernible in the ovaries during a maternal m o ~ ~(for l t details, see Steel 1980). By the time of apolysis, very few y cells can be detected. Thus, it appears that release of secretion from y cells occurs in "long" days but not "short" days (8L: 16D) and is associated with the onset of vitellogenesis. The arborizations of y cell axons in the lamina ganglionaris described above would be strategically located for the reception of such photoperiodic cues. During the later stages of premoult (from apolysis through ecdysis), secretion reaccumulate~in the y cells such that their appearance at anterior ecdysis is restored to that seen in intermoult.

Discussion There have been several previous attempts to analyze the organization of the brain - SG neurosecretory system of isopod crustaceans (see references in Introduction). These studies utilized a variety of histological and ultrastructural methods to classify neurons presumed to be neurosecretory into various types and made different assumptions about which cells might terminate in the sinus gland. Considerable confusion has remained in the literature since the earliest studies even though the problems of diagnosis of cells as neurosecretory in isopods have been discussed in depth (Gabe 1966). To exploit the advantages of isopods as models for the analysis of neurosecretory phenomena described by Chiang and Steel (1984), it is first necessary to clarify the organization of the system. We have approached this problem by combining, for the first time, the two techniques of selective staining with PAF and backfilling the sinus gland with colbalt. Thus, we are confident that cells which are both cytologically secretory and can be filled with cobalt applied to terminals in the SG are neurosecretory cells associated with the brain-SG neurosecretory system. These criteria are satisfied by three anatomically distinct groups of NSCs in the brain of Oniscus (see Fig. 10). These groups correspond in location and appearance to those originally named in Armadillidium as B, P, and y cells (Matsumoto 1959). All three groups contain PAF-positive secretory product which can be traced in serial sections to the SG and all fill with cobalt applied to the SG terminals. On the other hand, one group of PAF-positive neurons in the central protocerebrum which corresponds to the A cells of earlier workers does not fill with cobalt; if these cells are neurosecretory, their release site must be located elsewhere than in the SG assuming that all axons are large enough to transport enough cobalt to be detected. A second group of neurons very close to the SG filled with cobalt (Fig. 5), but was not cytologically distinctive. These cells may be neurosecretory since their axons terminate

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in the SG, but it has not been possible to confirm their secretory nature with the present techniques. Two of the three groups of neurosecretory cells which we accept as terminating in the SG are located in the middorsal region of the protocerebrum (B and P cells, Fig. 10). Both these groups of NSCs give rise to axons which pass into the central protocerebral neuropile to form extensive arborizations where synaptic interaction with other neurons presumably occurs. Branches of these axons also traverse the midline and ramify in a closely similar region of the contralateral protocerebral neuropile. Thus, arborizations cf the cells from both left and right sides of the brain occur close to each other, suggesting a neuroanatomical pathway by which the activity of NSCs of the two sides of the brain could be coupled. Indeed, in simultaneous electrical recordings from both left and right SGs, we have observed that bursts of electrical activity from one SG occur synchronously with bursts of electrical activity from the other SG (Chiang and Steel 1985). The third group of PAF-positive NSCs which terminates in the SG of 0niscu.s is located in the optic lobe and correspond to the y cells of Matsumoto (1959) (Fig. 10). These are small cells with fine axons forming arborizations in the lamina ganglionaris. Which of these three groups of NSC is equivalent to the classical "X-organ" (organ of Hanstrom) described in decapods'?This question is complicated by apparent pleiomorphism within the order Isopoda, resulting in "a series of confused statements and contradictory assertions in the literature" reviewed by Gabe (1966). Even within the Oniscidea no agreement exists (Matsumoto 1959; Messner 1966). Brief consideration of the homologies of neuropile regions in the brain is necessary. The classical "X-organ" is located in a prolongation of the central protocerebral neuropile into the eyestalk (the medulla terminalis); in isopods, the eyes are sessile and hence, no distinct medulla terminalis is present. In its absence, the equivalent neuropile region would be in the central protocerebrum. Hence, it is probable that the B cells and P cells of the protocerebrum represent the equivalent of the classical "X-organ." This conclusion is supported by several additional facts. First, these cells are the main source of neurosecretory axons terminating in the SG of Oniscus; similarly, most of the terminals in the decapod sinus gland derive from the "X-organ" (see Cooke and Sullivan 1982; Andrew 1983). Second, the "X-organ" also appears to comprise two distinct cell types (Durand 1956; Andrew et al. 1978). Third, numerous experiments involving ablation of the central protocerebrum of isopods (see references in Steel 1980) produce malfunctions of moulting and reproduction comparable to the effects of ablation of the "X-organ" of decapods (see also Chiang and Stee1 1984). Finally, the present finding of cyclical cytological changes in the p cells during the moult cycle implies a role of these cells in moulting, a key function of the "X-organ." Cytological changes in P cells related to moulting have also been reported in other isopods (Messner 1966; Martin 1 9 7 2 ~ ) . The y cells of the optic lobe in Oniscus do not have an obvious counterpart in the eyestalk system. However, these cells were found to undergo cyclical changes in relation to egg development, which is regulated by day length in Oniscus (McQueen and Steel 1980), and to possess arborizations in a location appropriate for reception of photoperiodic cues. It is therefore possible that these cells play a role in reproduction. 'They may have been destroyed inadvertently in the experiments referenced above involving ablation of the central proto-


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FIG.9. Posterior view of the optic lobe illustrating varicosities formed by neurosecretory axons backfilled with cobalt applied to the sinus gland (SG). Varicosities appear as irregularities in axon diameter (arrowheads) or bulbous structures attached to the axon by a narrow branch (arrow). Scale bar = 50 pm.

Protocerebrum

Optic Lobe

FIG. 10. Schematic diagram depicting the extent of the brain - sinus gland neurosecretory system revealed by cobalt backfilling. The most distal group of cobalt-filled cell bodies was not detected with paraldehyde fuchsin.

cerebrum. NSCs in a comparable location adjacent to the medulla externa are known in decapods (Bellon-Humbert et al. 1981), but their functions are not clear. Varicosities are a prominent feature of the distal portions of neurosecretory axons in Oniscus. These contain PAF-positive material and may represent a preterminal storage site for neurosecretory material. Some of them occur at the end of axon branches (Fig. 9), resulting in structures closely resembling the terminals in the SG. It is therefore possible that release of stored neurosecretion may also occur from these segments of the axons as well from the SG itself. Terrestrial isopods have been advocated on theoretical grounds as potential models for the investigation of neurosecretion (Chiang and Steel 1984). The above analysis of the organization of the brain-SG neurosecretory system of Oniscus reveals a number of practical features indicating the suitability of this system for experimental studies of neurosecretion. First, the terminals on the SG derive from at least three anatomically distinct groups of cell bodies; this arrangement will facilitate the attribution of particular functions to particular cells and contrasts with decapod systems, such as that of the crayfish, in which all the cell bodies are said to occur primarily, but not exclusively, in one large group (Jaros 1978; Andrew 1983).

For example, the present cytological observations suggest the involvement of separate groups of NSCs in moulting and reproduction. The existence of discrete areas of arborization from different groups of cells in different areas of neuropile may facilitate identification of synaptic inputs to these cells. Further, the compact size of the SG makes this gland amenable to complete reconstruction at the ultrastructural level (R. G. Chiang and C. G. H. Steel unpublished)' and to analysis of the electrical activity associated with hormone release, since the entire gland can be contained within a single recording electrode. Acknowledgement This research is supported by grant No. A6669 from the Natural Sciences and Engineering Research Council of Canada. ANDREW,R . D. 1983. Neurosecretory pathways supplying the neurohemal organs in crustacea. In Neurohemal organs of arthropods. 'Chiang, R. G., and C. G. H. Steel. Ultrastructure and distribution of identified neurosecretory terminals in the sinus gland of the terrestrial isopod, Oniscus asellus.


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