Immunocytochemical demonstration of the neurosecretory X-organ complex in the eyestalk of the crab Carcinus maenas

Summary An antiserum was obtained by immunizing rabbits with sinus gland extracts from Carcinus maenas. The antiserum is almost exclusively directed against neurosecretory material in the medulla terminalis X-organ (MTGXO), as demonstrated by the peroxidase—antiperoxidase (PAP) staining method in light and electron microscopic studies. Radioimmunological binding studies indicate the presence of antibodies against the crustacean hyperglycemic hormone (CHH) or the black pigment dispersing hormone (BPDH) in the antiserum. The results suggest that the neurosecretory perikarya of the MTGXO are the sites of production of CHH and/or BPDH.Supported by the Deutsche Forschungsgemeinschaft (Ke 206/2)     

Immunocytochemical identification of hyperglycemic hormone-producing cells in the eyestalk of Carcinus maenas

Summary Antiserum raised in rabbits against extracts of sinus glands from Carcinus and shown by several criteria to contain antibodies directed against the neurosecretory hyperglycemic hormone was used to locate the hormone-producing perikarya in the optic ganglia. By means of the double antibody fluorescence technique, selective staining of the large neurosecretory perikarya of the medulla terminalis ganglionic X-organ (MTGXO) and their axons is obtained. The axon endings of the sinus gland are also stained. None of the other groups of neurosecretory cells in the eyestalk shows fluorescence. Preabsorption of the antiserum with pure hyperglycemic hormone abolishes the fluorescence.Supported by the Deutsche Forschungsgemeinschaft (SFB 87, A 3; Ke 206/2). Thanks are due to E. Schmid (Ulm) for excellent technical assistance and to Prof. R. Martin and E. Weber for help and suggestions. A short version of parts of the results has been presented at theXth Conference of European Comparative Endocrinologists, Sorrento, May 1979     

Two-dimensional gel electrophoresis of neurosecretory polypeptides in crustacean eyestalk

Summary A knowledge of the precise location of neurosecretory cell bodies is a prerequisite for studying the synthesis and subsequent processing of neurosecretory polypeptides stored in axon terminals comprising the sinus gland of the crustacean eyestalk. Structural data establish that the X organ in the medulla terminalis ganglion (mtXo) of the crayfish eyestalk represents 90–95% of the cell bodies actively synthesizing neurosecretory vesicles stored in the neurohemal sinus gland (Fig. 4). These cell bodies transport rather than accumulate neurosecretory vesicles as judged by light and electron microscopy suggesting that neurohormone precursors, but not subsequently stored products, might be found there. Two-dimensional electrophoresis of sinus gland and mtXo homogenates support this hypothesis. In crayfish, lobster and blue crab, stained two-dimensional gels display a number of sinus gland-specific polypeptides whose high concentrations and low molecular weights are consistent with stored neurosecretory material (Table 1). These neuropeptides are not detected in mtXo homogenates or in non-neurosecretory neural tissue with Coomassie Blue staining. By decreasing the porosity of the second dimension, the two-dimensional gel technique has proven useful in determining the molecular weights of a variety of neurosecretory polypeptides stored in the sinus gland. The crayfish and lobster store several polypeptides of ca. 7,000 Dalton. The blue crab stores two 7,000, two 13,000 and three 20,000 Dalton sinus gland polypeptides detected in stained gels.Following a 4 h incubation in³H-labelled amino acids, predominantly labelled 19,000–21,000 Dalton polypeptides are detected in crayfish mtXo homogenates by 2-D gel autoradiography (Fig. 12). Concomitantly, three labelled polypeptides (4,000–10,000 Dalton) appear in the sinus gland (Fig. 13), suggesting that they are cleaved from 19,000–21,000 Dalton molecules. This study is the first to examine neurosecretory precursors and their putative cleavage products in the Crustacea.Abbreviations mtXo medulla terminalis X organ - NEPHGE non-equilibrium pH gradient electrophoresis - PAF paraldehyde fuchsin - SDS sodium dodecylsulfate     

Localization of crustacean hyperglycemic hormone (CHH) in the X-Organ sinus gland complex in the eyestalk of the crayfish,Astacus leptodactylus (Nordmann, 1842)

The eyestalk of Astacus leptodactylus is investigated immunocytochemically by light, fluorescence, and electron microscopy, using an antiserum raised against purified crustacean hyperglycemic hormone (CHH). CHH can be visualized in a group of neurosecretory perikarya on the medualla terminalis (medulla terminalis ganglionic X-organ: MTGX), in fibers forming part of the MTGX-sinus gland tractus, and in a considerable part of the axon terminals composing the sinus gland. Immunocytochemical combined with ultrastructural investigations led to the identification of the CHH-producing cells and the CHH-containing neurosecretory granule type.     

Immunocytochemical localization of a substance in the eyestalk of the prawn,Palaemon serratus,reactive with an anti-FMRF-amide rabbit serum

Summary By use of a specific antiserum against the molluscan cardio-excitatory tetrapeptide FMRF-amide in combination with the PAP-method it was possible to obtain positive immunocytochemical reactions in several neurosecretory regions of the eyestalk of the prawn Palaemon serratus. FMRF-amide-like material was found in perikarya and nerve fibers of the medulla terminalis and in neurons in the lamina ganglionaris. The immunoreactivity observed in the glandular tissue located at the basal insertion of the eyestalk muscles must be ascribed to a non-specific reaction. The identification of immunopositive nerve fibers, ending on a nerve bundle in the medulla terminalis, and the fact that immunoreactive material was absent in the neurohemal sinus gland seem to indicate a neurotransmitter/neuromodulator function.     

Appearance and innervation of CHH-producing cells in the eyestalk of the crayfish Astacus leptodactylus examined after tracing with Lucifer Yellow

Summary By injection of the fluorescent dye Lucifer Yellow into individual Crustacean Hyperglycemic Hormone (CHH)-producing cells, the shape of these neurosecretory cells in the eyestalk of the crayfish Astacus leptodactylus can be traced. A highly fluorescent perikaryon gives rise to an axon that can be followed by the fluorescent label to the neurohemal region, the sinus gland. The proximal part of that axon sends out extensive branches into the neuropil of the medulla terminalis. Electron-microscopic investigations reveal synaptic input to these axonal ramifications.     

Immunocytochemical identification of structures containing putative red pigment-concentrating hormone in two species of decapod crustaceans

Summary By use of an antiserum raised against the Nterminal sequence pGlu-Leu-Asn-Phe..., common to red pigment-concentrating hormone (RPCH) of Pandalus borealis and three structurally similar insect neuropeptides, putative RPCH-immunopositive structures were revealed in the eyestalks of Carcinus maenas and Orconectes limosus and in the brain and thoracic ganglion (TG) of C. maenas. In the eyestalks, complete neurosecretory pathways were demonstrated, consisting of perikarya, axons and terminals in the neurohemal organ, the sinus gland (SG). In C. maenas approximately 20 small RPCH cells are present as a distinct group adjacent to the medulla terminalis ganglionic X-organ (MTGXO, XO). They are morphologically different from the larger XO perikarya, which contain the crustacean hyperglycemic hormone (CHH). The occurrence of both neuropeptides in distinct neurosecretory pathways was ascertained by immunologic double staining (PAP/gold) or by analysis of consecutive sections. In addition, a group of two to four larger RPCH cells is located in the proximal part of the MT. In O. limosus, RPCH cells are found in the XO. Cells corresponding to the proximal MT cells of C. maenas were not found. In both species, a few more weakly staining immunopositive perikarya were observed in clusters of cell somata of the optic ganglia. It is uncertain whether these are connected to the SG.In the brain of C. maenas, several smaller and three larger perikarya were consistently observed in the dorsal lateral cell somata adjacent to the olfactory lobes. In the optic nerve, two axons that project into the eyestalk were stained. Some axons were also observed in the ventral median neuropil of the brain. In the TG, RPCH cells were found in small numbers in median positions, i.e., in clusters of somata between the ganglia of the appendages.HPLC analysis of the red pigment-concentrating activity from the SG of C. maenas revealed that the retention time of the neuropeptide is similar but not identical to that of Pandalus borealis RPCH.     

Regulation of circulating levels of the crustacean hyperglycemic hormone: evidence for a dual feedback control system

The effects of glutamate, aspartate, glycine, proline, alanine, taurine, glycerol, glucose and lactate injections on the haemolymph levels of the crustancean hyperglycemic hormone and/or glucose and lactate in the shore crab, Carcinus maenas, were investigated. Only glucose and lactate caused significant changes of hyperglycaemic hormone levels. Glucose injections resulted in a drop of both hormone and lactate, while lactate had an opposite effect, i.e. it raised both crustacean hormone and glucose levels. The results suggest that during increases in glycolytic flux, lactate may cause a release of hormone by a positive feedback mechanism. The hormone would then stimulate glycogenolysis, thus increasing glucose availability. If more glucose is released than is metabolized, excess glucose may leak from the cells and suppress crustancean hyperglycemic hormone release from the X-organ/sinus gland complex by negative feedback.Abbreviations ABTS 2,2-azino-bis (3-ethylbenzthiazoline sulphonic acid) - ANOVA one-way analysis of variance - BSA bovine serum albumin - BW body weight - CHH crustacean hyperglycemic hormone - ELISA cnzyme-liked immunosorbent assay - GIH gonadinhibiting hormone - IgG immunoglobin G - MIH moult-inhibiting hormone - MTGXO medulla terminalis X-organ - PB sodium phosphate buffer - PBS phosphate buffered saline - Pi inorganic phosphate - XO-SG X-organ-sinus gland complex     

Localization of substance P-like,leucine-enkephalin-like,methionine-enkephalin-like,and FMRFamide-like immunoreactivity in the eyestalk of the fiddler crab,Uca pugilator

Summary The occurrence and distribution of substance P (SP)-like, methionine-(Met)- and leucine-(Leu)-enkephalinlike, and FMRFamide-like immunoreactivities were determined in the neuroendocrine complex of the eyestalk of the fiddler crab, Uca pugilator, by immunocytochemistry. SP-like immunoreactivity was found in the optic peduncle, sinus gland, medulla externa, medulla interna, lamina ganglionaris, and retinular cells. Met-enkephalin-like and Leuenkephalin-like immunoreactivity was observed in most of the retinular cells, optic peduncle, sinus gland, medulla terminalis, and lamina ganglionaris. However, Met-enkephalin-like, but no Leu-enkephalin-like, immunoreactivity was seen in the medulla terminalis X-organ. FMRFamide-like immunoreactivity could be seen in all parts of the eyestalk except in the sinus gland, lamina ganglionaris, and retinular cells. FMRF-amide-like activity was especially strong in the three chiasmatic regions connecting the optic ganglia. The possibility that these four peptides may function as neuroregulators in the fiddler crab is discussed.This investigation was supported in part by Grant No. PCM-8300064 from the National Science Foundation to MF and Biomedical Research Support Grant No. 2 SO7RRO5373 SUB from the University of Kansas Medical Center to LLV     

Distribution of serotonergic neurons in the eyestalk and brain of the crab,Cancer antennarius

1. Serotonin-containing neurons were localized immunocytochemically in crab cerebral ganglia and their extensions in the eyestalk.2. Approximately 155 serotonergic cells were found in identifiable regions of the brain, the largest number being localized in the anterior cell cluster (40 reactive cells) and the bilateral anterior olfactory cell clusters (40 cells each).3. Serotonin immunoreactive cells were found in all three ganglionic divisions of the eyestalk. The medulla terminalis contains up to 15 reactive cells, of which only one occurs in the X-organ (origin of neurosecretory axons in the sinus gland nerve). The m. terminalis also contains three identifiable cells in the mediolateral border adjacent to the sinus gland nerve, of which one is a giant (up to 100 μm diameter), designated MT-1. The axon of MT-1 branches profusely after entering the m. terminalis neuropil.4. No serotonin immunoreactivity was apparent within the sinus gland, the sinus gland nerve or the organ of Bellonci.5. These findings are discussed in relation to the known serotonergic control of peptide hormone secretion by the eyestalk X-organ-sinus gland complex.     

Comparative electrophoretic studies of crustacean neurosecretory hyperglycemic and melanophore-stimulating hormones from isolated sinus glands

Summary By disc electrophoresis of sinus gland extracts fromOrconectes limosus, Pacifastacus leniusculus andCarcinus maenas, marked differences in the electrophoretic mobility were demonstrated for the hyperglycemic neurohormone (HGH) from the 3 species. In the astacuran materials, 2 peaks of activity were observed whereas inCarcinus material only 1 peak could be demonstrated with certainty. Melanophore-stimulating activity (MDH) was resolved into several peaks, but 2 predominant peaks with almost identical mobility were observed in all 3 species. Electrophoresis of sinus gland material from the above species and fromHomarus gammarus, Procambarus clarkii, Astacus leptodactylus, A. astacus, Uca pugilator, Cancer magister, Eriocheir sinensis andHemigrapsus oregonensis revealed that all astacuran HGHs fall in one group of substances with low mobility whereas some of the brachyuran hormones (fromCarcinus, Uca andCancer) moved faster. The hormones ofEriocheir andHemigrapsus have mobilities similar to those of the astacuran hormones, but, like the other brachyuran hormones, they are not hyperglycemic in Astacura. HGH was found to be associated with the strongest staining band in the pherograms from all species investigated. It amounts to at least 10% of total sinus gland protein, that is about 0.5 g/gland inOrconectes and 0.6 g/gland inCarcinus. Accumulation of neurosecretory material in the neurohaemal organ is demonstrated by comparison of sinus gland and medulla terminalis extracts. In SDS gels, a large fraction of material (containing the HGH) in the mass range of 6000–8000 daltons is found in the the sinus gland whereas this fraction is barely demonstrable in the medulla terminalis. It was investigated whether cysteine-rich carrier proteins comparable to the vertebrate neurophysins exist in the sinus gland. No positive results were obtained either by aldehyde fuchsin staining of gels or by analysis of³⁵S-labeled sinus gland material. noreactivity in gels after electrophoresis of sinus gland extracts. No positive reaction was obtained with eluates from HGH containing slices. Recent amino acid analyses of HGH (Kleinholz, 1975; Keller and Wunderer, in preparation) do not suggest any similarities between crustacean HGH and vertebrate glucagon.     

Immunohistochemical Identification of Hyperglycemic Hormone- and Molt-Inhibiting Hormone-Producing Cells in the Eyestalk of the Kuruma Prawn, Penaeus japonicus

This study deals with the localization of crustacean hyperglycemic hormone (CHH, Pej-SGPIII) and molt-inhibiting hormone (MIH, Pej-SGP-IV) in the eyestalk of the kuruma prawn Penaeus japonicus using immunohistochemistry. High-titer and highly specific antisera were raised in rabbits against synthetic Pej-SGP-III C-terminal peptide (Glu-Glu-His-Met-Ala-Ala-Met-Gln-Thr-Val-NH2) and Pej-SGP-IV C-terminal peptide (Val-Trp-Ile-Ser-Ile-Leu-Asn-Ala-Gly-Gln-OH), both of which were conjugated with bovine serum albumin by a cross linker. Eyestalks were removed from mature male prawns at the intermolt stage of the molting cycle and fixed in Bouin's solution. Serial sections stained immunohistochemically showed that neurosecretory cells of Pej-SGP-III and Pej-SGP-IV were located in the same cluster of the medulla terminalis ganglionic X-organ (MTGX), and that three kinds of neurosecretory cells, which were stained with anti-PejSGP-III antiserum and/or anti-Pej-SGP-IV antiserum were present. The number of neurosecretory cells which stained with both antisera was much fewer than that of neurosecretory cells which stained with one of the antisera only. The axon and axon terminals in the sinus gland were also stained and the staining density of the sinus gland was always deeper than that of the neurosecretory cells.     

A comparative immunocytochemical investigation of the crustacean hyperglycemic hormone (CHH) in the eyestalks of some decapod crustacea

The eyestalk of the astacideans Orconects limosus, Nephrops norvegicus, and Homarus gammarus, and the palinuran Palinurus vulgaris, was examined with an antiserum raised against purified crustacean hyperglycemic hormone (CHH) of the astacidean species Astacus leptodactylus. A distinct immunopositive reaction occurs in a group of neurosecretory cells in the medulla terminalis ganglionic X-organ (MTGX), in the MTGX-sinus gland tractus, and in a considerable part of the sinus gland. The immunoreactive sites in the eyestalk of the investigated species correspond to the site of production, storage, and release of the CHH. Preliminary investigations with this antiserum also indicate that a positive immunoreaction can be obtained in the eyestalk of other decapod crustaceans, for example, of the brachyuran Macropipus puber and the caridean Palaemon serratus.     

Neurosecretory centers from the eyestalk of Siriella armata M. Edw. (Crustacea: Mysidacea): ultrastructural variations during normal and experimentally inhibited molt cycle

Summary The ultrastructure of the medulla interna-medulla externa X-organ (MI-ME Xo)-sinus gland (SG) complex in the eyestalk of Siriella armata is described during the normal and the experimentally inhibited molt cycle. In the normal SG, four types of neurosecretory axon terminals, each containing distinguishable neurosecretory granules, can be described. Thus, type-2 granules are synthesized by G1 neurons forming the MI-ME Xo. The cell bodies and axonal endings of these cells in the sinus gland have been examined at the following molt stages: intermolt (stage C4), premolt (D0 and D2), and postmolt (A1, A2 and B). Changes in ultrastructure of the G1 cells have been monitored and correlated to inhibitions of the molt-and reproductive cycle produced by electrocauterization of the MI-ME Xo. The results obtained suggest that the neurosecretion from the G1 cells exerts a positive influence on molt and brood preparation. The occurrence of a distal group of G1 cells whose axons terminate at a different site from the SG suggests that the neural factors of the MI-ME Xo are diverse and control different physiological activities.     

Immunolocalization of allatostatin-like neuropeptides and their putative receptor in eyestalks of the tiger prawn, Penaeus monodon

Allatostatin (AST)-like immunoreactivity (IR) was localized in the eyestalk of Penaeus monodon by immunohistochemistry using four anti-AST antibodies. Depending on the antisera, AST-like immunoreactivity was detected in neuronal bodies of the lamina ganglionalis, cell bodies anterior to the medulla externa and cell bodies on the anterior and posterior of the medulla terminalis. Neuronal processes in neuropiles of the medulla externa, medulla terminalis, sinus gland and nerve fibers in the optic nerve were also recognized. No IR in cell bodies or in nerve fibers was found in the medulla interna. Strong AST-like immunoreactivity was found in hundreds of cells of the X organ. The localization of AST-like peptides suggests that they function as neurotransmitters and/or neuromodulators. Antiserum to the Drosophila AST receptor (Dar-2) recognized a single protein in P. monodon eyestalk protein extracts that was identical in size to that found in Drosophila protein extracts. Using this antiserum the putative P. monodon AST receptor was localized to the sinus gland in both juvenile and adult eyestalks. To our knowledge this is the first demonstration of a neuropeptide receptor localized to the crustacean sinus gland. This suggests that ASTs may function directly on the sinus gland as a neuromodulator. In juvenile eyestalks, the putative AST receptor was also localized to neuronal X organ cells of the medulla terminalis in males but not in females. The significance of this sex-specific receptor localization is unclear but emphasizes that ASTs function within the nervous system of the eyestalk.     

Immunocytochemical study of crustacean hyperglycemic hormone in the eyestalks of the prawn Palaemon serratus (Pennant) and some other Palaemonidae,in relation to variations in the blood glucose level

With the use of rabbit antisera against crustacean hyperglycemic hormone (CHH), it is possible to describe a distinct immunopositive reaction in a group of neurosecretory cells in the medulla terminalis ganglionic X-organ₂ (MTGX₂), in the MTGX-sinus gland tract, and in a considerable part of the sinus gland from several species of prawns belonging to the Palaemonidae. By introductory studies on the CHH system in Palaemon serratus, we can postulate a sequence in the activity cycle of the CHH-producing cells on the basis of differences in staining intensity of the immunoreaction and such morphometric parameters as cellular and nuclear diameter. By studying the CHH-producing system in combination with variations in the glucose level of the blood, an “inverse relationship” is observed between the number of immunoreactive cells and the blood glucose level during different periods of the year as well as during different stages of the molting cycle. A “shift in phase” of this correlation during the diurnal cycle suggests that several rhythmical phenomena may play a role in the regulation of glycemia in Crustacea.     

Immunocytochemical demonstration of the neurosecretory systems containing putative moult-inhibiting hormone and hyperglycemic hormone in the eyestalk of brachyuran crustaceans

Summary By use of antisera raised against purified moultinhibiting (MIH) and crustacean hyperglycemic hormone (CHH) from Carcinus maenas, complete and distinct neurosecretory pathways for both hormones were demonstrated with the PAP and immunofluorescence technique. By double staining, employing a combination of silver-enhanced immunogold labelling and PAP, both antigens could be visualized in the same section. Immunoreactive structures were studied in Carcinus maenas, Liocarcinus puber, Cancer pagurus, Uca pugilator and Maja squinado. They were only observed in the X-organ sinus gland (SG) system of the eyestalks and consisted of MIH-positive perikarya, which were dispersed among the more numerous CHH-positive perikarya of the medulla terminalis X-organ (XO). The MIH-positive neurons form branching collateral plexuses adjacent to the XO and axons that are arranged around the CHH-positive central axon bundle of the principal XO-SG tract. In the SG, MIH-positive axon profiles and terminals, clustered around hemolymph lacunae, are distributed between the more abundant CHH-positive axon profiles and terminals. Colocalisation of MIH and CHH was never observed. The gross morphology of both neurosecretory systems was similar in all species examined, however, in U. pugilator and M. squinado immunostaining for MIH was relatively faint unless higher concentrations of antiserum were used. Possible reasons for this phenomenon as well as observed moult cycle-related differences in immunostaining are discussed.     

The ultrastructure of the sinus gland of Carcinus maenas (Crustacea: Decapoda)

Summary The sinus gland of Carcinus maenas consists of the swollen axonal endings of the neurosecretory cells of the major ganglia and acts as a storage release centre for the membrane bound neurosecretory material. These neurosecretory granules fall into five different types based on size and electron density. Their contents are released by exocytosis of the primary granules or smaller units budded from the primary granules.I thank Professor E. Naylor for his constant advice and Professor E. W. Knight-Jones, Department of Zoology, University College, Swansea, for the provision of laboratory facilities. I am grateful to the Science Research Council for the financial support. Finally, I thank the Electron Microscope Unit, Southampton General Hospital, where the work was completed.     

The ultrastructure of the sinus gland of Gammarus oceanicus (Crustacea: Amphipoda)

Summary The sinus gland of Gammarus oceanicus, like that of other crustaceans, is composed of three elements: neurosecretory axons, glial cells and stromal sheath. Five neurosecretory axon types are identified on the basis of granule diameter, shape, and electron density, and axon matrix density. Exocytosis appears to be the major release mechanism of neurosecretory material. The preterminal regions of neurosecretory axons contain axoplasmic reticulum and neurotubules. Their arrangement in the axon and relationship with one another suggest a transport function. Multilamellar bodies are found in the terminal regions of neurosecretory axons. They arise from mitochondria and may be involved in granulolysis.The technical assistance of G.A. Bance, statistical assistance of D. MacCharles and D.W. Hagen, and financial support provided by the University of New Brunswick Research Fund to K.H. are gratefully acknowledged     

Immunocytochemical localization of pigment-dispersing hormone (PDH) and its coexistence with FMRFamide-immunoreactive material in the eyestalks of the decapod crustaceans Carcinus maenas and Orconectes limosus

Summary By use of a new antiserum, raised against synthetic pigment-dispersing hormone (PDH) from Uca pugilator, immunoreactive structures were studied at the light-microscopic level in the eyestalk ganglia of Carcinus maenas and Orconectes limosus. PDH-reactivity was mainly found in two types of neurons that were located between the medulla interna (MI) and the medulla terminalis (MT) in both species. Several additional perikarya were located in the distal part of the MI in O. limosus. In C. maenas, two to three PDH-positive perikarya were found in the region of the X-organ (XO) in the MT. Processes from single and clustered cells could be traced into all medullae of the eyestalk. Axons from the immunoreactive perikarya running between MI and MT form a larger tract that traverses the MT. Fibers from this tract give rise to extensive arborizations and plexuses throughout the proximal MT. A plexus containing very fine fibers is located at the surface of the MT in a position distal to the XO-area of C. maenas only. The proximal plexus also receives PDH-positive fibers through the optic nerve. PDH-perikarya in the cerebral ganglion may also project into the more distal regions of the eyestalk. Distal projections of the perikarya between the MI and MT consist of several branches. Most of these are directed toward the MI and ME (medulla externa) wherein they form highly organized, layered plexuses. One branch was traced into the principal neurohemal organ, the sinus gland (SG). In the SG, the tract gives off arborizations and neurosecretory terminals. It then proceeds in a proximal direction out of the SG, adjacent to the MT. Its further course could not be elucidated. The lamina ganglionaris (LG) receives PDH-fibers from the ME and fine processes from small perikarya located in close association with the LG in the distal part of the first optic chiasma. The architecture of PDH-positive elements was similar in both C. maenas and O. limosus. The distribution of these structures suggests that PDH is not only a neurohormone but may, in addition, have a role as a neurotransmitter or modulator. Immunostaining of successive sections with an FMRF-amide antiserum revealed co-localization of FMRFamideand PDH-immunoreactivities in most, but not all PDH-containing perikarya and fibers. The axonal branch leading to the SG and the SG proper were devoid of FMRFamide immunoreactivity.