Monoamines in the nervous system of the tube-wormChaetopterus variopedatus (Polychaeta): Biochemical detection and serotonin immunoreactivity

Summary The presence and distribution of biogenic monoamines in the tube-wormChaetopterus variopedatus were investigated by a radioenzymatic method and HPLC with electrochemical detection, and the cellular localization of serotonin by peroxidase-antiperoxidase (PAP) immunohistochemistry with an antibody against serotonin-formaldehyde-protein conjugate. Dopamine, norepinephrine, epinephrine, serotonin (5-HT) and some of their metabolites were detectable, dopamine and norepinephrine being present in substantially larger amounts than 5-HT and epinephrine. With few exceptions, the largest amounts of amines were localized in the most nerve-rich tissues such as tentacles, and those containing cerebral ganglia and the ventral nerve cord. Serotonin-immunoreactive unipolar neurons were widely distributed in the dorso-lateral cerebral ganglia, the neurosecretory pharyngeal ganglion and the segmental ganglia of the anterior (dorsolateral) and posterior (medioventral) nerve cords. Some nerve-fiber tracts stained in the cerebral ganglia, but the neuropiles of segmental ganglia were the most intensely reactive CNS structures. Numerous reactive fibers were also present in connectives, commissures and segmental nerves. All peripheral sensory structures included serotonin-immunoreactive cells and neurites, especially the parapodial cirri and the bristle receptors of the setae. Trunk and parapodial muscles contained reactive varicose fibers and neuronal somata. These results suggest that monoamines are abundant and widespread in these worms and that 5-HT appears to have a key sensory role.     

The influence of optic nerve section on blood metabolite levels in Carcinus maenas (Crustacea: Brachyura)

• 1.1. Blood metabolite levels were assayed in Carcinus maenas as an indicator of the functioning of the hyperglycemic hormone, HGH, secreted by the crab's eyestalk neuroendocrine tissue.
• 2.2. Bilateral eyestalk ablation eventually resulted in a hypoglycemic response after 2–3 days.
• 3.3. Bilateral optic nerve section produced a significant, long-term hypoglycemic response suggesting that release of HGH from the eyestalk sinus gland is controlled, via a promotive neural pathway, by the CNS and probably by the cerebral ganglia.
• 4.4. Injection of eyestalk extract into operated crabs consistently produced significant, short-term hyperglycemia.

     

The paramedial neurosecretory cells of the suboesophageal ganglion of the cricket,Teleogryllus commodus (Walk.)

Summary Ovariectomy, performed immediately after the final hatch, caused a reduction of stainable (neurosecretory?) material in the paramedial neurosecretory cells (PNC) (A-type) of the suboesophageal ganglion in 10 day-old females of Teleogryllus commodus (Walk.). A concomitant increase in nuclear volume and in the incorporation of ³⁵S-cysteine indicates increased synthesis of neurosecretory material. From these findings it is concluded that more stainable material is secreted in the cerebral neurohaemal organ after Ovariectomy. A functional relationship between the PNC and the ovaries is suggested.     

Egg-laying-hormone immunoreactivity in the neural ganglia and ovary of Haliotis asinina Linnaeus

Immunoreactivity against the abalone egg-laying hormone (aELH) was detected in the fine granules of type 1 and 2 neurosecretory (NS) cells, neurites in the neuropil, and blood sinuses in the connective tissue sheath of the cerebral, pleuropedal, and visceral ganglia of the tropical abalone, Haliotis asinina Linnaeus. The number of positive NS cells, and the intensity of staining in the ganglia, varied and might be related to the stage of ovarian cycle. At any stage, positive cells were most numerous in the pleuropedal, and least numerous in the visceral ganglion. In addition, several cells of the statocyst and associated nerves also exhibited the immunoreactivity. In the ovary, the most intense reactivity was detected in the follicular and granular cells adjacent to mature oocytes, in the trabeculae and the ovarian capsule. The cytoplasm of mature oocytes was also moderately stained. The results indicate that the cerebral, pleuropedal, and visceral ganglia are the main sites of aELH-producing cells. The ovary may also produce aELH locally.     

Neurosecretory cells in the central nervous system of the adult blowfly, Phormia regina Meigen (Diptera: Calliphoridae)

• 1 Neurosecretary cells in the central nervous system of the adult blowfly, Phormia regina Meig., have been examined histologically using the parparaldehyde-fuchsin and Gomori's staining method. Six groups of the neurosecretory cells occur in each hemisphere of the brain, the medial, frontal, lateral A, lateral B, posterior I and posterior II groups. In the subesophageal ganglion, four B-cells and two A-cells are present. In the thoracico-abdominal ganglion, ten A-cells are found in the thoracic region and a total of about 50 A- and B-cells in the hind part of the abdominal region.
• 2 A comparison with the neurosecretory system of two other species of blowfly, Calliphora erythrocephala Meig., Sarcophaga bullata Parker, and the housefly, Musca domestica L., showed similar arrangements and grouping.
• 3 Neurosecretory granules have been observed along the axons originating from the medial neurosecretory cells of the brain, and the thoracico-abdominal ganglion. The granules originating from the medial groups can be traced directly to the corpus cardiacum from which they move to the aorta, crop duct and cardia through axons.
• 4 There is with advancing age a gradual increase in the size of cell bodies and nuclei of the median neurosecretory cells in both females and males of Phormia regina, and also a decrease in stainable granules. This increase in size is dependent on nutrition, with no increase in water alone, a slight increase on sugar, and a maximum increase on sugar and liver. Corresponding increases in size occur in the ovaries in connection with feeding the same substances.

     

Distribution and ultrastructure of neurosecretory cells in the cerebral ganglion of the earthworm

Neurosecretory (Nsy) cells within the cerebral ganglion of Lumbricus terrestris were classified ultrastructurally. The Nsy cells within the subesophageal ganglion, nerve cord ganglion, and the peripheral nervous system were also examined. A comparative survey of Nsy cells of four other species of oligochaetes, Eisenia feotida, octolasion cyaneum, Dendrobeona subrubicunda, and Allolophora longa, was also carried out. Seven cell types (A1, A2, A3, A4, A5, C, and SEF), distinguished by special cytological and ultrastructural features, were found within the cerebral ganglion. Distribution of these cells inside and outside the cerebral ganglion was studied in detail by light and electron microscopy. The nerve terminals of each cell type were followed into the neuropile region. Exocytosis from cell bodies appears to be the main release mechanism for the Nsy granules, whereas small Nsy vesicles are released through synapses in the neuropile. Peripheral fibers of some cell types (A1, A2, and A3) extend through the capsule to the pericapsular epithelium. It is possible that Nsy cells secrete hormones from their cell bodies and peripheral processes and that their centrally directed axons release modulators/transmitters within the neuropile.     

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.     

SEROTONIN AND FREE AMINO ACID ANALYSIS OF GANGLIA AND ISOLATED NEURONES OF APLYSIA DACTYLOMELA

—The presence of serotonin and different amino acids was investigated in the ganglia and in isolated giant neurones of Aplysia dactylomela. With a few exceptions the pattern of substances was similar in all the ganglia. Of the many identified neurones studied only one giant neurone located in each cerebral ganglion was found to contain serotonin. GABA was detected in most extracts, including those of the serotonin-containing neurone, known cholinergic, and known neurosecretory neurones. Putrescine, recently detected in extracts of nervous tissue and isolated neurones of Helix, was not detected in Aplysia nervous tissue.     

Neuroendocrine control of gonadogenesis in regenerating Lineus lacteus (Heteronemertea)

Previous studies have shown that a gonad-inhibiting hormone (GIH) secreted by the cerebral ganglia controls reproduction in nemertean worms. In order to determine whether the cerebral hormone also controls regenerative growth, as it does in other marine worms such as Nereis diversicolor (Polychaeta), we compared gonadogenesis with enterogenesis in regenerating Lineus lacteus under the influence of, and withdrawn from the influence of, GIH. Experimental evidence is presented for a high specificity and a striking efficiency of GIH on sexual development. Over 3 months, regenerating decerebrate worms differentiated mature gonads whereas regenerating worms with cerebral ganglia remained sexually undifferentiated. On the other hand, regenerative enterogenesis occurred equally well in worms with or without a brain.We also tried to delineate the influence of temperature and light on neuroendocrine activity of regenerating L. lacteus. In L. lacteus we compared sexual development in regenerating worms and normal worms co-exposed over one year, (i) either to light or dark, and (ii) either to a winter temperature (12 °C) or a summer temperature (20 °C). When continually exposed to a winter temperature, both normal and regenerating worms differentiated mature gonads from 8th to 12th month regardless of the light-dark alternative. On the other hand, worms exposed to a summer temperature reached sexual maturity at the end of year only when they were also continually exposed to dark. Summer temperature plus light permanently inhibited the sexual development, i.e. resulted in continuous release of GIH.     

A study of the cerebral region of the visual system in the pulmonata

The distribution of central axons of receptor cells of the eyes and the locations of neurons sending axons into the optic nerves were studied in the cerebral ganglia of the pulmonate mollusksLymnaea stagnalis andHelix sp. by the method of axonal transport of cobalt chloride injected via the optic nerves. Afferent fibers of these nerves form terminal ramifications (chiefly dorsally) in the middle part of the cerebral ganglion. Some of them pass through the commissure to the symmetrical region of the opposite cerebral ganglion. Neurons innervating the eyes are located in several regions of both cerebral ganglia. InLymnaea they are distributed near the point of entry of the optic nerve, in the region of the commissure, the mesocerebrum, and the posterior part of the ganglion. InHelix these neurons are found in the same regions except in the posterior part of the ganglion. In electrophysiological experiments responses of neurons in these parts of the cerebral ganglion to adequate stimulation of the eye were recorded. Differences in the character of responses and also the presence of neurons indifferent to stimulation of the eye are evidence of the functional heterogeneity of these areas. This suggests that morphologically separate visual centers do not exist in the cerebral ganglion of the Pulmonata. Neurons giving specific responses to stimulation of the eye and evidently belonging to different levels of the visual system (afferent or efferent divisions) are closely connected both with each other and with cells of other functional systems.A. A. Ukhtomskii Physiological Research Institute, A. A. Zhdanov Leningrad State University. Translated from Neirofiziologiya, Vol. 14, No. 2, pp. 179–184, March–April, 1982.     

Effects of electrocoagulation of cerebral neurosecretory cells and implantation of corpora allata on oöcyte development in Locusta migratoria

The implantation of active corpora allata into intact Locusta females during growth accelerates pre-vitellogenic oöcyte growth and vitellogenesis. Localised stimulation of yolk deposition follows the implantation of active corpora allata between the ovarioles demonstrating a gonadotrophic rôle for the corpus allatum hormone. Electrocoagulation of the median neurosecretory cells of the brain prevents vitellogenesis whilst pre-vitellogenic oöcyte growth occurs normally. Implantation of active corpora allata into females with ablated cerebral neurosecretory cells promotes vitellogenesis in a proportion of test animals although mature oöcytes are never produced.It is suggested that the rôle of the median neurosecretory cells during egg development in Locusta is primarily concerned with the activation and maintenance of activity of the corpora allata. The corpus allatum hormone acts both metabolically and gonadotrophically.     

Neurosecretory cells in the central nervous system of the giant garden slug,limax maximumus

The neurosecretory system of the giant garden slug Limax maximus was studied using the alcian blue/alcian yellow (AB/AY) staining technique for neurosecretion. Stainable cells could be identified in the paired cerebral, pleural, parietal, and buccal ganglia, and in the visceral ganglion. The cells occur as single cells or in groups of up to 100, with diameters ranging between 10 and 70 μm. Axon tracts could only be traced for a small number of cells; neurohemal areas were not conclusively identified. The morphological similarities of the neurosecretory system of L. maximus is compared with that of other investigated stylommatophoran slugs.     

Distribution of biogenic amines in the slug,Limax maximus

Summary The distribution of monoamines inLimax maximus was studied by the histochemical fluorescent method of Falck and Hillarp. The number of 5-HT-containing and catecholamine-containing perikarya in the central nervous system is small compared with the non-fluorescent perikarya. However, all the ganglia except the proto-cerebral ganglia have some amine-containing neurons. There are relatively larger numbers of fluorescent cells in the cerebral, visceral, pedal and right parietal ganglia than in the other ganglia. A single, giant 5-HT-containing neuron was observed in each meta-cerebral ganglion.Monoamine neurons are localised in a number of peripheral tissues (heart, integument, tentacles, penis retractor muscle, sole of foot, kidney, alimentary canal, reproductive organs and tentacular, pharyngeal and cephalic retractor muscles). Neurons containing catecholamine are mostly associated with sensory structures such as the statocysts, the retina of the eye and the integument of the tentacles, whereas 5-HT-containing nerve fibres are mainly observed in muscle tissues.We wish to thank the Wellcome Trust for financial support.     

On the neurosecretory system of Dendrobaena atheca Cernosvitov

Four kinds of neurosecretory cells A, B, U and C are distinguished in the central nervous system of Dendrobaena atheca Cernosvitov. A cells, which show different morphological characteristics under different physiological states and during their cyclic changes, are the most active neurosecretory cells. They form the outer layer of the cortical cell zone in the cerebral ganglion. B cells are large and medium sized and are distributed in all parts of the central nervous system. U cells are found only in the sub-pharyngeal ganglion while C cells are distributed in the sub-pharyngeal as well as in the ventral nerve cord ganglion. The number and secretory activity of C cells decrease in caudal direction. Further, Gomori-positive cells are also observed in the ganglia of the vegetative nervous system. A rudimentary neurohaemal organ, the storage zone, has been observed in the cerebral ganglion and there appears to be another neurohaemal area in the ventral nerve cord ganglion. The storage zone is formed by the terminal ends of the axons of A cells. The chrome alum haematoxylin phloxin (CHP) and aldehyde fuchsin (AF) positive substances in the form of granules are found in this area. The cerebral ganglion is richly supplied by blood capillaries. The distal end of the axons of B cells are swollen like a bulb while in some cases the axons are united to form an axonal tract. Extra-cellular material is abundant in different parts of the nervous system. In all cell types, the perinuclear zone is the first to show activity in the secretory cycle. It appears that the nucleus may be involved in the elaboration of the neurosecretory material in the cells.     

Electron microscopical and histochemical observations on the relation between medio-dorsal bodies and neurosecretory cells in the basommatophoran snails Lymnaea stagnalis,Ancylus fluviatilis,Australorbis glabratus and Planorbarius corneus

Summary In Basommatophora medio-dorsal bodies (MDB) are closely attached to the cerebral ganglia, in which, just underneath the bodies, groups of Gomori-positive neurosecretory cells (MDC) occur. It has been suggested that the MDB-cerebral ganglion complex should be regarded as a neuro-endocrine association.In the present study the morphological relation between MDB and the ganglion is histochemically and ultrastructurally investigated in Lymnaea stagnalis, Ancylus fluviatilis, Australorbis glabratus and Planorbarius corneus.Histochemical tests showed the paraldehyde-fuchsin positive material of fibers in the MDB to be different from the neurosecretory material (NSM) in the MDC. At the ultrastructural level no penetration of nerve cell processes through the perineurium, separating the MDB from the ganglion, into the medulla of the MDB was observed. However, excepting for Lymnaea, the perineurium at these places shows particular differentiations. In the medulla of the MDB granule laden profiles (granule ø 700–900 Å) occur. They appeared to be processes of MDB cells.From these results it is concluded that the medulla of the MDB should not be regarded as a neurosecretory neuropile. Apparently, the MDB-cerebral ganglion complex is no neuroendocrine association. Probably the MDB is an endocrine organ. The small electron dense granules of the profiles in the medulla were also found in the MDB cell bodies. They are thought to represent a secretion product. The close morphological relation between MDB and cerebral ganglion may be connected with the origin of the MDB cells from perineural elements.     

Brain-Body Interactions in Nereis. Deactivation of the Cerebral Neuroendocrine System by Ganglion Transplantation

Summary

A hormone secreted by the cerebral ganglion of Nereis promotes growth and exerts an inhibitory influence in sexual maturation. During the final stages of life, there is a sharp decline in the rate of endocrine activity. The ganglion of an immature specimen can be deactivated by exposure to the milieu of the maturing animal, although a prolonged period of time (several months at least) is required to accomplish this effect. The brain of a specimen approaching maturity is more readily deactivated and this process is not dependent on maintenance of the normal nervous connections. The deactivation process can be blocked, even after its initiation, by transplantation of the ganglion into an immature host. The process by which the population of oocytes becomes homogeneous during the penultimate stage of maturation is not dependent on the concurrent decline in endocrine activity. Posterior regeneration is not directly inhibited by the presence in the body of maturing oocytes.     

Leucokinin and diuretic hormone immunoreactivity of neurons in the tobacco hornworm,Manduca sexta,and co-localization of this immunoreactivity in lateral neurosecretory cells of abdominal ganglia

Because leucokinins stimulate diuresis in some insects, we wished to identify the neurosecretory cells in Manduca sexta that might be a source of leucokinin-like neurohormones. Immunostaining was done at various stages of development, using an antiserum to leucokinin IV. Bilateral pairs of neurosecretory cells in abdominal ganglia 3–7 of larvae and adults are immunoreactive; these cells project via the ipsilateral ventral nerves to the neurohemal transverse nerves. The immunoreactivity and size of these lateral cells greatly increases in the pharate adult, and this change appears to be related to a period of intensive diuresis occurring a few days before adult eclosion. Relationships of these neurons to cells that are immunoreactive to a M. sexta diuretic hormone were also investigated. Diuretic hormone and leucokinin immunoreactivity are co-localized in the lateral neurosecretory cells and their neurohemal projections. A median pair of leucokinin-immunoreactive, and a lateral pair of diuretic hormone-immunoreactive neurons in the larval terminal abdominal ganglion project to neurohemal release sites within the cryptonephridium. The immunoreactivity of these cells is lost as the cryptonephridium is eliminated during metamorphosis. This loss appears to be related to the change from the larval to adult pattern of diuresis.     

Ganglionic distribution of inputs and outputs of C-PR, a neuron involved in the generation of a food-induced arousal state inAplysia

Cerebral neuron C-PR is thought to play an important role in the appetitive phase of feeding behavior ofAplysia. Here, we describe the organization of input and output pathways of C-PR. Intracellular dye fills of C-PR revealed extensive arborization of processes within the cerebral and the pedal ganglia. Numerous varicosities of varying sizes may provide points of synaptic inputs and outputs.Blocking polysynaptic transmission in the cerebral ganglion eliminated the sensory inputs to C-PR from stimuli applied to the rhinophores or tentacles, indicating that this input is probably mediated by cerebral interneurons. Identified cerebral mechanoafferent sensory neurons polysynaptically excite C-PR. Stimulation of the eyes and rhinophores with light depresses C-PR spike activity, and this effect also appears to be mediated by cerebral interneurons.C-PR has bilateral synaptic actions on numerous pedal ganglion neurons, and also has effects on cerebral neurons, including the MCC, Bn cells, CBIs and the contralateral C-PR. Although the somata of these cerebral neurons are physically close to C-PR, experiments using high divalent cation-containing solutions and cutting of various connectives indicated that the effects of C-PR on other cerebral ganglion neurons (specifically Bn cells and the MCC) are mediated by interneurons that project back to the cerebral ganglion via the pedal and pleural connectives. The indirect pathways of C-PR to other cerebral neurons may help to ensure that consummatory motor programs are not activated until the appropriate appetitive motor programs, mediated by the pedal ganglia, have begun to be expressed.     

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