Electron microscopic autoradiography on the localization of serotonin in the frog median eminence

Summary For the identification of the nerve fiber containing serotonin (5-hydroxytryptamine) in the frog median eminence, an electron microscopic autoradiography was performed with 5-hydroxytryptophane-³H which is the precursor of serotonin. At 1 and 24 hours after the intraperitoneal injection, most silver grains were located over the nerve fibers and endings, and a few were also found over the glia cell and the perivascular space. A large number of silver grains were located over the type 3 nerve endings (Nakai, 1971) containing small dense granules about 600–1000 Å in diameter 1 and 24 hours after the injection. Some silver grains were localized over the nerve endings containing intermediate-size dense granules 1100–1700 Å in diameter. Silver grains were also frequently observed over the nerve fibers in the inner layer of frog median eminence. There is no significant difference in the pattern of distribution of silver grains between tissues of 1 hour and 24 hours after the injection.The authors wish to thank Prof. H. Fujita for his advice and criticism.     

Serotonin-containing neurones in vertebrate retinas

Abstract: It has been established by a combination of HPLC and electrochemical detection that frog, lizard, goldfish, rabbit, and bovine retinas contain both dopamine and serotonin. Immunohistological and immunoradiographical methods show that serotonin is localised in amacrine perikarya and processes situated in the inner plexiform layers of frog, lizard, and goldfish retinas. The amount of serotonin in the mammalian retina appears to be too low for detection in neurones. The serotonin in the bovine retina is located mainly in the inner nuclear and plexiform layers, suggesting that the amine is present in the same types of cells as found for frog, lizard, and goldfish retinas. Retinas incubated in [³H]serotonin showed that radioactivity is associated with processes in the inner plexiform layer and amacrine perikarya. These results suggest that the neuronal elements that contain endogenous serotonin also have the capacity to accumulate exogenous amine and are consistent with the opinion that serotonin has a neuronal function in retinas of a variety of vertebrates.     

Effects of 5-hydroxytryptophan on serotonin in nerve endings

—Preparations of synaptosomes (P₂) from the telencephalon and from the diencephalon plus optic lobes of the pigeon and from the telencephalon of the rat were used to study the effects of 5-hydroxytryptophan (5-HTP) on (a) the levels of serotonin (5-HT) in nerve endings and (b) the release of 5-HT from nerve endings. The levels of 5-HT were significantly higher (3.21 × 0.35 nmol/g original tissue weight) in the P₂ fraction isolated from the telencephalon of pigeons given intramuscular injections of 50mg/kg of d ,l -5-HTP in comparison to control values (1.42 ± 0.07). A similar twofold increase was observed with the P₂ fraction isolated from the diencephalon plus optic lobes. In addition, the levels of 5-HTP and 5-hydroxyindoleacetic acid also increased significantly in these P₂ fractions isolated from pigeons given d ,l -5-HTP injections in comparison to values obtained for pigeons given saline injections. In vitro studies using preparations of synaptosomes (from both pigeon and rat) labelled with [³H]5-HT indicated that 0.10 mil l -5-HTP increased the release of [³H]5-HT twofold over control values. A concentration as low as 0.001 mm l -5-HTP was tested on the P₂ fraction from the telencephalon of the pigeon and was found to significantly increase the release of [³H]5-HT over control values. This effect by l -5-HTP was blocked if a decarboxylase inhibitor was added to the medium. l -5-HTP at a concentration of 1.5 mm had no apparent effect on the release of [³H]norepinephrine or [³H]dopamine from synaptosomes prepared from the telencephalon of the rat or pigeon. The results are discussed in terms of the role of serotonin in producing certain types of behavioral depressions exhibited by pigeons and rats given injections of 5-HTP.     

The catecholaminergic system of an annelid (Ophryotrocha puerilis,Polychaeta)

Summary The complex catecholaminergic (CA) nervous system of the polychaete Ophryotrocha puerilis is documented using glyoxylic acid induced fluorescence (GIF) and immunohistochemistry. CA-neurons are found both in the central and peripheral nervous system. In the brain, about 50 CA-neurons are present in the perikaryal layer together with numerous CA fibres. Two pairs of CA perikarya are characteristic for each ganglion of the ventral nerve cord. CA-neurites in the ventral nerve cord are mainly arranged in 4 strands paralleling the longitudinal axis of the worm. Fluorescent neurons with receptive ciliary structures are present in body appendages (antennae, palps, urites, parapodial cirri), in the body-wall, and within the oesophageal wall. Furthermore, a subepidermal nerve net of free CA nerve endings has been found. After incubation of specimens with dopamine prior to the development of GIF more fluorescent perikarya could be observed; the fluorescence was also intensified. Pre-incubation with reserpine reduced the intensity of GIF. Results of high pressure liquid chromatography and immunostaining with a polyclonal antibody against a dopamine-glutaraldehyde-complex suggest that dopamine is the major CA transmitter. It is thought that dopaminergic neurons together with ciliary receptive structures act as mechano- and/or chemoreceptors.     

Immunocytochemical demonstration of neuropeptides and serotonin in the tapeworm Diphyllobothrium dendriticum

Summary The present immunocytochemical study concerns the distribution of four neuropeptides, FMRF-amide, vasotocin, leu-enkephalin and neurotensin, and of the bioamine serotonin in the plerocercoid larva of Diphyllobothrium dendriticum. Anti-FMRF-amide and vasotocin-reactivity occurs in perikarya and nerve fibres in the CNS and PNS of this worm. The peptide-containing fibres surround and seem to innervate the musculature and to terminate beneath the basal lamina of the tegument at the inner surface of the bothridia, suggesting a neurotransmitter function. Antileu-enkephalin reaction occurs in perikarya and fibres in the main nerve cords and in the PNS. Anti-neurotensin reactive fibres were observed in the neuropile of the nerve cords. Serotonin immunoreactivity was found in neurons in the ganglionic commissure of the brain and along the main nerve cords. This study is the first immunocytochemical identification of neuropeptides and serotonin in a parasitic flatworm and the information gained may be of importance for the development of new antihelminthics.     

Dopamine: Localization of uptake in the pedal ganglion of Quadrula pustulosa (Pelecypoda)

Uptake of ³H-dopamine in the pedal ganglion of Quadrula pustulosa was localized using combined fluorescence and optical light microscopy, in addition to electron microscope autoradiography. Light microscope autoradiograms of the same sections previously prepared for fluorescence microscopy indicated most radioactivity occurred over green, long-lasting fluorescent fiber tracts and nerve cell bodies. Ultrastructural autoradiographic results showed the majority of the radioactivity was localized over synaptic vesicles. Nerve fibers containing neurotubules, and also a limited number of nerve cell bodies were labelled with ³H-dopamine. These results suggest uptake of dopamine to be specific for dopaminergic structures.     

Extrasynaptic localization of α-bungarotoxin receptors in the rat superior cervical ganglion

Binding of [¹²⁵I]α-bungarotoxin to nicotinic cholinergic receptors (α-bungarotoxin receptors) was investigated in the rat superior cervical ganglion by light and electron microscope autoradiography. Both techniques indicated that labelling, which was inhibited by d-tubocurarine, occurred around and/or over neuronal perikarya. In particular, ultrastructural autoradiography showed that the synapses were devoid of radioactivity, suggesting that α-bungarotoxin receptors in the rat superior cervical ganglion are molecules distinct from the nicotinic (postsynaptic) receptors normally involved in ganglionic transmission. By contrast, specific labelling was found in extrasynaptic areas of the neuronal membrane in contact with satellite cells (neuron-satellite cell boundary). Quantitative analysis indicated that at that level silver grains were present on both the neuronal membrane and satellite cells. Furthermore, beside neuronal perikarya, radioactivity was also found around nerve fibres, probably in relation to both the axonal and interstitial sides of the ensheathing Schwann cells. Only a few grains were clearly accumulated inside nerve fibres. Finally, significant amounts of specific radioactivity were detected in the neuronal cytoplasm, especially at the level of rough endoplasmic reticulum and Golgi apparatus. However, parallel diffusion experiments with [¹²⁵I]α-bungarotoxin and [³H]inulin (a marker for the extracellular space) provided no evidence that the toxin enters the neuronal cytoplasm. Thus, the intraneuronal (specific) labeling was probably a reflection of α-bungarotoxin binding to membrane receptors and the subsequent internalization of the toxin-receptor complex in the neurons. We conclude that in the rat superior cervical ganglion extrasynaptic nicotinic acetylcholine receptors (α-bungarotoxin receptors) may be widely located on the neuronal membrane as well as on the plasma membrane of satellite and Schwann cells. The physiological significance of this molecular architecture is discussed.     

Histology and ultrastructure of the olfactory organ of the freshwater pulmonate Helisoma trivolvis

Summary The olfactory organ of Helisoma trivolvis is located on the surface of the body at the base of the cephalic tentacles. An evagination of skin, the olfactory plica, at the base of the tentacle extends over the olfactory organ dorsally. The epithelium of the olfactory organs contains unspecialized epithelial cells, ciliated epithelial cells, basal cells, mucous secretory cells, and sensory dendrites. The surface of the epithelium has a complex brush border of thick plasmatic processes, which branch to form several terminal microvillar twigs. Long slender cytoplasmic processes form a dense spongy layer among the plasmatic processes beneath the level of the terminal twigs. Bipolar primary sensory neurons clustered beneath the epithelium of the olfactory organ send dendrites through the epithelium to the free surface. Some sensory endings have a few short cilia, but most bear only microvilli. Cilia of sensory endings and epithelial cells extend beyond the brush border of the epithelium. Small axons arise from the perikarya of the sensory neurons and enter a branch of the olfactory nerve. HRP tracing indicates that the axons pass to the cerebral ganglion without interruption. Histochemical tests indicate that the sensory neurons are neither aminergic nor cholinergic.     

Biochemistry and ultrastructure of serotonergic nerve endings in the lobster: serotonin and octopamine are contained in different nerve endings

In this article we report that the distribution of serotonin in the lobster nervous system parallels the distribution of octopamine and that the same tissues that contain endogenous serotonin can synthesize it from tryptophan. Octopamine and serotonin are highly concentrated in a neurosecretory region of the second thoracic roots in association with a group of neurosecretory cells. The roots possess separate high-affinity uptake systems for both serotonin and tryptophan. Radioactive serotonin, accumulated in tissues during incubations with either tritiated serotonin or tritiated tryptophan, can be released, in a calcium-dependent manner, by depolarization with potassium. A detailed morphological examination of the second thoracic roots shows four distinct categories of nerve endings in the vicinity of the neurosecretory cells. Octopamine is synthesized in one of these types of endings and serotonin in another. The high-affinity uptake systems for serotonin and tryptophan are found only in association with the endings that make serotonin. These endings and all the biochemical parameters of serotonin metabolism in the roots are selectively destroyed by previous injection of animals with the neurotoxin 5,7-dihydroxytryptamine.     

Somatostatinergic nerves in the cervical spinal cord of the monkey

Somatostatinergic nerves in the spinal cord of the monkey were investigated utilizing immunohistochemistry with various antibodies against synthetic somatostatin. In contrast to earlier investigations, it is shown that somatostatinergic nerve endings occur in most of the areas of the grey matter of the spinal cord. The somatostatinergic axons are, however, characteristically distributed in three main regions: (1) Densely-packed endings are seen in lamina II of the substantia gelatinosa, forming a crescent-shaped pattern in the columna dorsalis. Somatostatin immunoreactivity is also seen in lamina I and in the Lissauer tract. (2) A fine network of fibers is observed around the central canal; the endings are concentrated on special cell bodies. Some single perikarya are also stained in this region. (3) A loose network of single fibers is found ending on perikarya of the columna lateralis or ventralis. The perikarya of the nerve axons, with the exception of those terminating in the columna dorsalis, have as yet not been identified. In order to better understand the somatostatinergic system of the spinal cord, these newly-detected somatostatinergic nerves must be studied and their exact pathways analyzed.     

The Organum vasculosum laminae terminalis

Summary The Organum vasculosum laminae terminalis (OVLT) of the duck is lined innerly by specialized ependymal cells (tanycytes) and outwardly by a well-developed superficial vascular network, the capillaries of which often show a fenestrated endothelium. The OVLT also includes glial cells, internal non-fenestrated capillaries, bundles of fine nerve fibers and three groups of axonal swellings. One type contains granulations of 1000–1400 Å in diameter as well as 300–500 Å clear vesicles. The second type exhibits granulations and dense core vesicles of 500–800 Å in diameter along with small electron-lucent vesicles having diameters of 300–400 Å. In the third type, exclusively clear vesicles 300–600 Å in diameter are found. Asymmetrical synapses on dendrites and neuronal perikarya are found at every level of the organ. In the most external zone, the interposition of tanycyte endings sometimes allows neurosecretory axons to reach the parenchymal basement membrane (basal lamina).When tritiated molecules (amino acids or monoamines) are administered either in vitro by incubation or in vivo by intraventricular injections, radioautographic grains are observed over the tanycyte perikarya. Although this labeling is observed at every time point following the administration of the tracers, within three minutes only ³H-GABA appears to be concentrated in the cytoplasmic processes of the tanycytes. ³H-noradrenaline and ³H-serotonin are taken up and retained by some axons of the second type described above. Noradrenergic fibers are primarily localized in the inner zone of the OVLT where they display axodendritic synaptic contacts. Serotonergic fibers appear sparsely distributed in the OVLT but are more numerous in the lateral edges of the organ where synaptic differentiations on dendrites or on dendritic spines are also observed.It is concluded that the duck OVLT probably displays a neuroendocrine activity. Uptake and selective transport of exogenous molecules by tanycytes are also suggested by the present radioautographic observations. Finally, monoaminergic innervation is discussed at the OVLT level with special reference to the occurrence of serotonergic synapses.Supported by the Département de Biologie du C.E.A., and the I.N.S.E.R.M. (C.R.A.T., 74.1.438.45)     

Electron microscopic autoradiography for demonstration of pineal serotonin in rat

Summary The fine localization of rat pineal serotonin has been studied by means of electron microscopic autoradiography. Two hours after the intravenous injection of tritium labelled 5-hydroxytryptophan, the location of large number of silver tangled threads is seen in the sympathetic nerve terminals. There is also a less specific accumulation of the silver grains in the pinealocytes, some appearing in the cytoplasmic organelles and some in the nucleus.In quantitative terms, 43% of the total count of silver grains were in the nerve endings whereas pinealocytes, which comprise a much larger volume of the section, contain a proportionally much smaller number of silver particles (53%). Furthermore the perivascular spaces, which comprise a larger percentage in volume of the section than the nerve endings has nevertheless only about 4% of the grains counted.Although the precise localization of the silver grains is obscure, the reaction of the granulated vesicles in the nerve terminals to the double fixation used, is similar to that shown by the extremely dense material in vesicles of platelets, which was demonstrated to contain serotonin. The results therefore suggest that the silver grains appearing in the nerve terminals two hours after the administration of 5-hydroxytryptophan are in the serotonin binding site in the axon terminals, containing the granulated vesicles.     

Localization of aldosterone and corticosterone in the central nervous system,assessed by quantitative autoradiography

Nuclear localization of tritiated aldosterone in the CNS was studied in rats by numerical evaluation of silver grains, deposited over neuronal cell nuclei in thawmounted autoradiograms, and compared with the localization obtained after prior administration of a 100-fold excess of radioinert aldosterone, corticosterone or 18-hydroxy-11-deoxycorticosterone (18-OH-DOC). Corticosterone and 18-OH_DOC completely prevented nuclear localization in most regions examined. However, in contrast to pretreatment with aldosterone, pretreatment with corticosterone and 18-OH-DOC did not completely prevent the concentration of radio-activity in the cell nuclei of the indusium griseum. Traces of radioactivity were, furthermore, retained in areas CA₁ and CA₂ and the dentate gyrus in rats exposed to corticosterone, but not to 18-OH-DOC, prior to [³H]aldosterone. A similar profile of silver grain distribution to that noted with aldosterone was found for corticosterone except that with tritiated corticosterone the most intense concentration of radioactivity occurred in hippocampal areas CA₁ and CA₂ and not in the indusium griseum. Prior administration of excess deoxycorticosterone acetate abolished nuclear accumulation of tritiated corticosterone. Dihydrotestosterone, on the other hand, failed to compete with tritiated corticosterone at a dose 200-fold in excess of the tritiated steroid.We conclude that (1) a receptor readily shared by aldosterone, corticosterone, 18-OH-DOC and DOC, but not by dihydrotestosterone, is widely distributed throughout the CNS, (2) a receptor shared by aldosterone and 18-OH-DOC, but not by corticosterone may be present in hippocampal areas CA₁ and CA₂, (3) that both these as well as the receptor accepting dihydrotestosterone can be located within the same cell.Dedicated to K. A. C. Elliott on his 80th birthday.     

Transport axonal de protéines marquées dans le motoneurone géant de l'écrevisse

Isolated abdomens of crayfish were maintained in vitro and lysine (-³H) was intracellularly injected into one of the giant motoneuron of the ventral cord ganglia by iontophoresis. Membrane potentials ranging between -60 and -70 mV were recorded all along the experiment. Light microscope radioautographs showed an intense reaction over the injected nerve cell body and the initial segment of its axon; most of the surrounding tissues were free of radioactivity when the diffusion of the injected lysine (-³H) was prevented by adding cold lysine to the bathing medium. Some exchange of label was however noted with electrically coupled axons and glial sheaths. No radioactive protein could be traced in the numerous nerve endings of the neuromuscular junction. Nonetheless when a ligature was placed on the nerve root, the amount of accumulated radioactivity was increased from 3 to 6 h. in the axon of the injected motoneuron only. Electron microscope radioautographs indicate that the fast transported proteins were mainly associated with the endoplasmic reticulum and the axonal membrane. It is concluded that the visualization of the nerve endings was limited by the dispersion of the label into the numerous thin terminal branchs of the axon; however the combination of iontophoretic injection and radioautography permits to trace endogenous protein along the axon and to study molecular exchanges with other cells.     

DECARBOXYLATION OF EXOGENOUS l-5-HYDROXYTRYPTOPHAN AFTER DESTRUCTION OF THE CEREBRAL RAPHE SYSTEM

Abstract— l -5-Hydroxytryptophan ( l -5-HTP) was administered intravenously to rats (12 mg/kg) after inhibition of the peripheral aromatic l -amino acid decarboxylase with l -α-hydrazino-α-methyl dopa (MK 486). The accumulation of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid in the cerebral cortex was measured 1, 2 and 4 h after injection of 5-HTP with automated assay techniques. Besides controls two groups of rats were studied: rats after inhibition of tryptophan-5-hydroxylase with p -chlorophenylalanine (pcpa) and subjects with a chronic lesion in the area of the raphe nucleus. The net accumulation of both measured 5-hydroxyindoles was diminished in rat cerebral cortex after degeneration of 5-HT containing nerve endings, compared with control animals and pcpa-treated rats. These results indicate that the formation of 5-HT in the cerebral cortex from exogenous l -5-HTP, after inhibition of the peripheral aromatic amino acid decarboxylase, occurs predominantly in 5-HT containing nerve endings possibly by a specific 5-HTP-decarboxylating enzyme.     

Light-microscopic demonstration of methylene blue accumulation sites in mouse brain after supravital staining.

Mouse brains were stained supravitally with methylene blue and studied in paraffin sections by light microscopy. In the perikarya, the dye was found to bind primarily to the nuclei; only slight staining of the cytoplasm was observed. Dye accumulations within nerve fibers were found in the nodes of Ranvier and in the varicosities of the unmyelinated endings. Specific dye binding in dendrites corresponded mainly to beads and spines. The accumulation sites in terminal neuronal processes appeared to be closely related to the plasma membrane. These morphological data would explain the neurophysiologically proven interaction of the dye with calcium-binding sites in membranes.     

Immunocytochemical demonstration of peptidergic neurons in the central and peripheral nervous systems of the flatworm Microstomum lineare with antiserum to FMRF-amide

Summary The central nervous system (CNS) and the peripheral nervous system (PNS) of the flatworm Microstomum lineare were studied by means of the peroxidase-antiperoxidase (PAP) immunocytochemical method, with the use of antisera to the molluscan cardioactive peptide FMRF-amide. FMRF-amide immunoreactive perikarya and nerve fibres are observed in the CNS and the PNS. In the CNS, immunoreactive perikarya and nerve fibres occur in the brain, in the epithelial lining and the mesenchymal surroundings of the ciliated pits, and positive fibres in the longitudinal nerve cords. In the PNS, immunoreactive fibre bundles with variocosities occur in the pharyngeal nerve ring, in symmetrical groups of perikarya on each side of the pharynx, and in the mouth area. Positive perikarya and meandering nerve fibres appear in the intestinal wall. A few immunoreactive cells and short nerve processes are observed at the male copulatory organ and on both sides of the vagina. Some immunoreactive peptidergic cells do not correspond to cells previously identified by histological techniques for neurosecretory cells. The distribution of immunoreactivity suggests that the FMRF-amide-like substance in CNS and PNS in this worm has roles similar to those of the brain-gut peptides in vertebrates. The status of FMRF-amide-like peptides as representatives of an evolutionarily old family of peptides is confirmed by the positive immunoreaction to anti-FMRF-amide in this primitive microturbellarian.     

PROTEIN SYNTHESIS IN SYNAPTOSOMAL FRACTIONS : Ultrastructural Radioautographic Study

A quantitative ultrastructural radioautographic study of in vitro protein synthesis has been carried out in rat synaptosomal fractions incubated with tritiated leucine or a tritiated amino acid mixture. Analysis of grain density distribution demonstrated that presynaptic endings are labeled. 30–50% of the developed grains, representing tritiated amino acids incorporated into proteins, were related to presynaptic endings which accounted for 75–77% of the total processes. 34–45% of the grains were related to processes containing ribosomes which accounted for only 4–7% of the total processes. The relative specific activity of these ribosome-containing processes, some of which could be identified as postsynaptic elements, was up to ten times higher than that of the presynaptic ending. These findings indicate that protein synthesis takes place in vitro in presynaptic terminals although to a significantly lesser degree than that occurring in ribosome-containing processes, which, with other nonpresynaptic processes, are at the present time unavoidable contaminants of synaptosomal fractions. Presynaptic endings that in radioautographs contained no mitochondria were labeled. Also, presynaptic endings were labeled after incubation in the presence of chloramphenical which inhibited 20% of the protein synthesis of the synaptosomal fraction. It is concluded that besides mitochondrial protein synthesis, another protein synthesizing system operates in presynaptic endings in vitro.     

Post-natal development of the pineal organ in the hamsters Phodopus sungorus and Mesocricetus auratus

Summary In Phodopus sungorus, as in other mammals, the pineal organ forms an important link in the transduction of photoperiodic information to the endocrine system. The sympathetic innervation, via the superior cervical ganglion, controls the metabolism of serotonin and melatonin in the pineal, which in turn is involved in the control of the gonads. In the present study, the post-natal development of this system was investigated. Specimens 1, 5, 10, and 15 days post partum (p.p.) and adults were treated with monoamine-oxidase-inhibitor and perfused under ether anesthesia via the aorta with a buffer containing glyoxylic acid, formaldehyde and Mg⁺⁺. The brains were then dissected out and treated according to Falck-Hillarp for fluorescence microscopy and microspectrofluorometry. Day 1: The nervi conarii had reached the pineal capsule, but only in a few cases was the pineal organ invaded and then only by a few fibers. Day 5: A rich green-fluorescing net of fibers was present in the entire organ, stalk and lamina intercalaris. No 5-HT fluorescence was observable. Day 10: Similar to the stage at 5 days a rich green-fluorescing nerve fiber net was observed throughout the pineal and a yellow fluorescence in the pineal perikarya. Day 15: The general appearance resembles the adult. The nerve fibers are masked by the intense yellow fluorescence of the pineal perikarya. Fading of the latter, however, allows the catecholamine fluorescence to be seen. Golden hamsters at an age of 15 days p.p. show a similar appearance to Phodopus at an age of 15 days. Microspectrofluorometric determinations indicated the catecholamine to be noradrenaline, and confirmed a 5-HT/5-HTP origin of the yellow fluorescence appearing between day 5 and day 10. The amount of 5-HT/5-HTP was considerably less at day 10 than at day 15 or in adults. Sympathectomy by extirpation of the superior cervical ganglion abolished the catecholamine fluorescence completely in the pineal body, stalk and lamina intercalaris.Supported by grants from the Swedish Natural Science Research Council (to P. Meurling and Th. van Veen), and the Royal Physiographic Society of Lund     

Patterns and projections of crustacean cardioactive-peptide-immunoreactice neurones of the terminal ganglion of crayfish

Three distinct clusters of crustacean cardioactive-peptide-immunoreactive neurones occur in the terminal abdominal ganglion of the crayfish species Orconectes limosus, Astacus leptodactylus, Astacus astacus and Procambarus clarkii, as revealed by immunocytochemistry of whole-mount preparations and sections. They exhibit similar topology and projection patterns in all four studied species. An anterior ventral lateral and a posterior lateral cluster contain one small, strongly stained perikaryon and two large, less intensely stained perikarya, each showing contralateral projections. A posterior medial lateral cluster of up to six cells also contains these two types of perikarya. Whereas the small type perikarya belong to putative interneurones, the large type perikarya give rise to extensive neurohaemal plexuses in perineural sheaths of the third roots of the fifth abdominal ganglia, the connectives, the dorsal telson nerves, the ganglion itself, its roots and arteriolar supply. Thin fibres from these plexuses reach newly discovered putative neurohaemal areas around the hindgut and anus via the intestinal and the anal nerves, and directly innervate the phasic telson musculature. A comparison with earlier investigations of motoneurones and segmentation indicates that these three cell groups containing putative neurosecretory neurones may be members of at least three neuromeres in this ganglion. Crustacean cardioactive peptide released from these neurones may participate in the neurohumoral and modulatory control of different neuronal and muscle targets, thereby exceeding its previously established hindgut and heart excitatory effects.Abbreviations AG abdominal ganglion - adpl arteria dorsalis pleica - Ala arreria lateralis abdominalis - Asub arteria subneuralis - CCAP crustacean cardioactive peptide - CNS central nervous system - IR immunoreactive - LG lateral giant axon - LTr lateral tract - MDT medial dorsal tract - MG medial giant axon - M Tr medial tract - mcan musculus compressor ani - mfltp museulus flexor telsonos posterior - nan nervus ani (AG6 N5) - nant nervus anterior (AG6 N1, N2) - nia nervus intestinal anterior - nin nervus intestinalis (AG6 N7) - nip nervus intestinalis posterior - nteld nervus telsonos dorsalis (AG6 N6) - nielv nervus telsonos ventralis (AG6 N4) - nur nervus uropedalis (AG6 N3) - nven nervus ventralis (AG5 N3) - PIR peri-intestinal ring - PTF posterior telson flexor - VLT ventral lateral tract - VMT ventral medial tract - VNC ventral nerve cord - VIF ventral telson flexor - AVLC, PLC, PMLC anterior ventral lateral, posterior lateral, posterior medial lateral CCAP-immunoreactive cell cluster - A6AVC, A7AVC anterior ventral commissures - A7DCI dorsal commissure I - A7PVC posterior ventral commissure - A7SCII sensory commissure II - A7VCII, A7VCIII ventral commissures II and III of the sixth (A6) and seventh (A7) abdominal neuromer