The hypothalamo-hypophysial system of the wild carp,Cyprinus carpio L.

Summary The posterior neurohypophysis (PNH)-pars intermedia complex of the wild and pond carp, Cyprinus carpio L., has been studied by light, fluorescence and electron microscopy. Gomori-positive neurosecretory fibres are abundant in the main trunk of the neurohypophysis as well as its roots penetrating the pars intermedia. Terminals of these fibres are in contact with capillaries of the general circulation and with glandular cells of the pars intermedia. Monoaminergic fibres with a weak green fluorescence, somewhat increasing after injection of nialamide into the pond carp, have largely the same distribution. Three types of neurosecretory fibres and their terminals have been recognized in the PNH-pars intermedia complex. Types-A₁ and -A₂ fibres, containing granules of 140–180 nm and 100–160 nm in diameter respectively, are peptidergic Gomori-positive. Type-A₂ fibres predominate in the PNH. The least frequent monoaminergic type-B fibres have granules of 60–100 nm in diameter. Numerous peptidergic and few monoaminergic neurosecretory terminals make contact with the capillaries located within the roots of the PNH as well as at the border between them and the pars intermedia. Both peptidergic and monoaminergic terminals make direct synaptoid contacts with the gland cells or end close to connective tissue septa, basal lamina or pituicytes. The PAS-positive gland cells and to a lesser degree the leadhaematoxylin-positive gland cells show these relationships with neurosecretory terminals. The question concerning the mode of interaction between peptidergic and monoaminergic structures in the dual control of the gland cells of the pars intermedia of teleosts is discussed.     

Electron microscopical study on the development of the nerve supply of the pituitary pars intermedia of the mouse

Summary The development of the nerve supply of the pituitary pars intermedia (PI) of C3H mice was studied by electron microscopy. Nerve fibres and terminal structures, most probably adrenergic, first appear in the newborn. The adult innervation pattern is achieved by the end of the first postnatal week.In the adult animal two types of nerve terminals were distinguished; type A (peptidergic or neurosecretory) and type B (adrenergic). The peptidergic fibres were scarce and exhibited no synapse-like contacts. It is suggested that they are of secondary importance in a direct nervous hypothalamic control of PI function. Type B terminals were found throughout the PI. They formed synapse-like contacts with the glandular cells, indicating that the primary innervation is exerted by adrenergic neurons.An autonomous differentiation of the glandular cells and in the adult a combined direct nervous and neurohumoral control of PI function is suggested.This investigation was supported by grant No B 2180-026 from the Swedish Natural Science Research Council. The skilful technical assistance of Mrs Ulla Wennerberg is gratefully acknowledged     

The hypothalamo-hypophysial system in acipenseridae

Summary The percentage of peptidergic (A₁ and A₂) and adrenergic (B) neurosecretory terminals was studied in the neurohypophysis of sexually mature female sturgeons. Neurosecretory terminals of the A₂ type prevail in the neurohypophysis, whereas A₁ and B terminals are rare. The activity of these types of terminals was established (1) during upstream migration, (2) shortly after spawning, and (3) three to six weeks after spawning. Terminals of B type are the most active elements during all the periods studied. These elements become strongly activated in sturgeons during upstream migration, i.e., earlier than the peptidergic neurosecretory terminals. Peptidergic terminals, especially elements of type A₂, become synchronously and strongly activated in fish shortly after spawning. In the late postspawning period neurosecretory terminals of all three types become synchronously inactive, persisting in a quiescent state in comparison to the two previous periods. The appearance of neurosecretory material discharged into the intercellular clefts by exocytosis correlates on the whole with the activity level of the A₁ and A₂ terminals in each individual studied. A functional correlation exists between the activity of the peptidergic and adrenergic neurosecretory terminals in the neurohypophysis. The data obtained are discussed with reference to a concept regarding spawning in some fish species as a physiological stress (Polenov et al., 1976). A possible dual control (peptide and monoamine neurohormones) over the function of visceral organs and glandular cells of the intermediate lobe of the hypophysis is also suggested (Polenov, 1970, 1975, 1978; Polenov and Belenky, 1973).Dedicated to the memory of Professor Wolfgang Bargmann, a great scientist and a generous friend     

Peptidergic and monoaminergic innervation of the gonads in the holothurian Cucumaria japonica

In the holothurian gonad structure of the peptidergic and monoaminergic systems has been described. Axons of their cells form tissue and hemal terminals. Epithelial cells and smooth myocytes of the gonadal wall get direct innervation, having contacts with the axonal terminals that are separated by the cleft 50-75 nm in the diameter. It is possible that neuropeptides and biogenic monoamines are transported to the germ and follicular cells of the germinative gonadal zone via hemolymph of the hemal sinus.     

Ultrastructural identification of noradrenergic nerve terminals and vasopressin-containing neurons of the paraventricular nucleus in the same thin section

Since many peptidergic cell groups receive a diverse and complex monoaminergic innervation, we have developed a double-label procedure to visualize a peptide and a catecholamine in the same ultrathin section. Radiolabeled norepinephrine (NE) is applied locally and its reuptake into NE terminals is demonstrated by ultrastructural radioautography. Controls in this and other studies demonstrate that the NE labels only NE (and possibly epinephrine) terminals and not dopaminergic or serotonergic terminals. In the same tissue, vasopressin is localized by immunocytochemistry on unembedded sections that are subsequently embedded in epoxy resins for thin sectioning. The procedure as described here shows that NE terminals in the periventricular zone of the paraventricular nucleus of the hypothalamus innervate both vasopressin-positive and vasopressin-negative structures. This technique is useful in determining the chemical connectivity of the hypothalamus.     

Ultrastructural immunocytochemical characterization of isotocin,vasotocin and neurophysin neurons in the magnocellular preoptic nucleus of the goldfish

Summary We describe the ultrastructural localization of isotocin, vasotocin and neurophysin in the magnocellular preoptic nucleus of the goldfish. With the aid of immunocytochemical techniques, we see staining both in classical neurosecretory granules and in diffuse agranular form throughout somata and processes. Signs of cellular and synaptic interactions between chemically identified neurons include axon terminals which contain vasotocin immunoreactivity and membrane specializations (puncta adhaerentia) between adjacent somata. Our investigations provide an anatomical basis for neuroendocrine and neurotransmitter-like functions of peptidergic neurons in the teleost preoptic nucleus.     

Immunogold labelling of serotonin-like and FMRFamide-like immunoreactive material in neurohaemal areas on abdominal nerves of Rhodnius prolixus

The ultrastructure of neurohaemal areas on abdominal nerves of the blood-sucking bug Rhodnius prolixus was investigated. Four types of axon terminals were found, distinguished by the morphology of their neurosecretory granules. By use of post-embedding immunogold labelling, granules in Type I axon terminals were shown to contain serotonin-like immunoreactive material, and granules in Type II axon terminals were shown to contain FMRFamide-like immunoreactive material. There was no colocalization of these materials. It is suggested that Type III terminals contain peptidergic diuretic hormone, which has previously been reported to be present in electron-dense neurosecretory granules in this neurohaemal area. The identity of material in Type IV terminals is unknown.     

Ultrastructural changes in isolated peptidergic nerve terminals induced by digitonin permeabilization and K+ stimulation in the sinus gland of the crab,Cardisoma carnifex

Summary The preparation of isolated peptidergic nerve terminals from the sinus gland (a neurohemal organ) of the crab (Cardisoma carnifex) is described. In this species the nerve endings can have diameters up to 30 m. They release neurosecretory material as judged by the decrease in the volumetric density of granules upon depolarization with potassium. Similar results were obtained after permeabilization of the nerve terminals with digitonin, but only in the presence of micromolar concentrations of calcium. This preparation should prove useful in correlating electrical events with other cellular processes involved in stimulus-secretion coupling.     

Peptidergic and aminergic neurotransmitters of the exocrine pancreas of the Houbara bustard (Chlamydotis undulata)

The immunochemical distribution of peptidergic and aminergic neurotransmitters in the exocrine pancreas of the Houbara bustard, Chlamydotis undulata, was determined. Immunoreactivity to choline acetyltransferase (ChAT), vasoactive intestinal polypeptide (VIP), and galanin (Gal) occurred mainly as varicose terminals in the walls of capillaries around the acini and arterioles within the connective tissue. Neuronal cell bodies immunoreactive to ChAT were infrequently observed. Neuropeptide Y (NPY), pancreatic polypeptide (PP), and somatostatin (Som) were observed mainly in intra-acinar cell bodies but nerve fibers immunoreactive to these neuropeptides were also seen along the basal surfaces of the acini. Immunoreactivity to NPY and PP was also discernible in cells of the pancreatic ducts. In addition, NPY occurred as varicose terminals in vessels around the ducts. SP occurred rarely in interacinar ganglia. The distribution of tyrosine hydroxylase (TH) was similar to that of ChAT and, in addition, the occasional TH immunoreactive intra-acinar neuronal cell body was observed. Neuronal nitric oxide synthase (nNOS) occurred in neuronal cell bodies among the acinar cells as well as nerve fibers along the bases of the acini. The potential roles of these peptidergic and aminergic neurotransmitters in the neurohormonal control of pancreatic secretion are discussed.     

Control of the pars intermedia of the lizard,Anolis carolinensis

Summary The distribution of the tracer substance horseradish peroxidase (HRP, Mw 40,000) in the neuro-intermediate lobe of the lizard, Anolis carolinensis, was studied at various time intervals (13 min to 24 h) after vascular injection. HRP rapidly entered the extracellular lumen of the neural lobe, but did not penetrate into the third ventricle. The tracer was found in micropinocytotic vesicles (MPVs) of ependymal cells within 13 min after injection. The number of cellular inclusions containing HRP increased during the period of observation (24 h). The tracer was sparsely taken up by aminergic and peptidergic nerve terminals of the external layer. After transection of the hypophysial stalk, numerous dense, labelled droplets were found in the peptidergic terminals, and the number of labelled inclusions in ependymal cells increased.MPVs were frequently found in extensions of stellate cells of the intermediate lobe, and endocytotic vacuoles (EVs) developed especially in the perikaryon. HRP was also found in large cisternae of the secretory cells, appearing predominantly towards the perivascular septum (PVS). These cisternae were found to communicate with the extracellular lumen, probably representing a system of the extracellular space extending into the secretory cell. After transection of the hypophysial stalk, there was an increase in the number of small EVs in secretory cells of the intermediate lobe.The results are discussed in terms of MSH-release regulation and possible participation of the extracellular lumen, glial and stellate cells in the transport of regulating factors and secretory material.Supported by grants from the Swedish Natural Science Research Council (to Dr. Patrick Meurling) and the Royal Physiographic Society of LundThe author is indebted to Mrs. Lena Sandell and Miss Inger Norling for excellent technical assistance and photographic aid; and to Dr. Rolf Libelius and Dr. Ingmar Lundquist for generous advice and stimulating discussions concerning the tracer technique     

The hypothalamo-hypophysial system in acipenseridae

Summary The distribution of adrenergic and peptidergic (Gomori-positive) structures of the hypophysial neuro-intermediate complex in Acipenseridae has been studied by means of light, fluorescence, and electron microscopy. Adrenergic fibres (B-fibres) and their terminals have been detected in the neurohypophysis of these fishes. The terminal swellings of B-fibres as well as the terminals of the neurosecretory peptidergic fibres (A₁ and A₂) make contact with the basement membrane of the connective tissue layer separating the neurohypophysis from the intermediate lobe. Capillaries are situated within this layer and, therefore, the main part of the fibre terminals is in contact with the pericapillary space. The release of catecholamines from the adrenergic terminals into the capillaries connected with the general circulation is supposed. The diffusion of catecholamines through the connective tissue layer into the parenchyma of the intermediate lobe is also suggested. Hence, the glandular activity of the intermediate lobe seems to be under the dual control of adrenergic and peptidergic elements of the hypothalamus.The authors wish to express their deep appreciation to G. M. Persov, Dr. Sc. Biol., Head of the Laboratory of Experimental Ichthyology, Petershof Biological Institute of the University of Leningrad, for the material supplied for fluorescence microscopy, and to Mr. G. V. Sabinin for photographic services.     

Coexistence and corelease of cholinergic and peptidergic transmitters in frog sympathetic ganglia

Both acetylcholine (ACh) and a peptide that resembles luteinizing hormone-releasing hormone (LHRH) serve as transmitters in sympathetic ganglia of the bullfrog. Although ACh is contained and released from both preganglionic B fibers, which form synaptic contacts with only B cells in the ganglia, and preganglionic C fibers, which are in synaptic contact with C cells only, the LHRH-like peptide is contained and released exclusively from preganglionic C fibers. The same preganglionic C fibers appear to supply both ACh and the LHRH-like peptide because the thresholds for the cholinergic fast excitatory postsynaptic potential (EPSP) correlate well with the thresholds for the peptidergic late slow EPSP recorded in the same C cell. Further, anatomical studies showed that almost all nerve terminals on C cells contained the LHRH-like peptide. Some of these same terminals must also contain and release. ACh, mediating the cholinergic fast EPSPs with millisecond synaptic delays. Therefore at least some, if not all, terminals of preganglionic C fibers contain and release both cholinergic and peptidergic transmitters.     

Peptidergic synaptic transmission in sympathetic ganglia

Biologically active peptides have been localized in neuronal cell bodies, axons, and synaptic boutons of sympathetic ganglia; some of these peptides may be neurotransmitters. For example, substances immunologically similar to substance P and luteinizing hormone-releasing hormone appear to be released from nerve terminals in sympathetic ganglia. In each case, the postsynaptic action of the peptide lasts for several minutes and is accompanied by a combination of decreases and increases in the membrane conductance that are voltage dependent. These peripheral peptidergic synapses may be models for peptidergic transmission in the central nervous system where detailed analysis is more difficult.     

An immunocytochemical study of intrapancreatic ganglia, nerve fibres and neuroglandular junctions in Brockmann bodies of the tompot blenny (Blennius gattoruggine), a marine teleost

The innervation of the Brockmann bodies in the teleost fish, Blennius gattoruggine, was studied using immunocytochemical techniques at both the light and electron microscopy levels. Islet innervation consisted of intrapancreatic ganglia, generally localized inside the rim of the exocrine tissue of the Brockmann bodies, in proximity to the islet, nerve fibres and nerve terminals with synaptic complexes. The intrapancreatic ganglia were of variable size, with different numbers of ganglionic cells, that appeared unipolar in section. The cell bodies showed immunoreactivity to galanin, oxytocin, peptide tyrosine tyrosine and glucagon. The extrinsic and intrinsic nerve fibres passed through the exocrine parenchyma and crossed the connectival septa and islet connectival sheath, penetrating into the islets, where they became increasingly thinner. They terminated on the endocrine cells with dilated nerve terminals. At least three types of terminals were detected, depending on the different vesicle content: peptidergic, cholinergic or adrenergic. They presented specialized synaptic structures, the neuroglandular junctions, some of which contained neurosecretory granules immunogold labelled by galanin antiserum. This new finding confirms the role of galanin as a neurotransmitter. This rich supply of innervation may be important in the regulation and integration of islet secretion.     

Neuropeptides in the median eminence

The presence and possible sources of more than 30 neuropeptides in the median eminence are summarized. The median eminence is the brain area which contains neuropeptides in the highest number and in the highest concentrations in the central nervous system. This area constitutes the final common pathway for signals from the brain to the pituitary. Many peptidergic fibers enter the median eminence and terminate around the pericapillary space and release their neuropeptides into hypophysial portal blood vessels. Other peptidergic fibers traverse the median eminence and terminate in the posterior pituitary. According to their origin, fibers in the median eminence can be classified as intra- or extrahypothalamic fibers. The neuropeptide-containing fibers in the median eminence are mainly intrahypothalamic, they reach the median eminence through either the lateral retrochiasmatic area or the tuberoinfundibular tract. Depending on the site of their action, neuropeptides may be either neurohormones acting on the anterior pituitary cells or neurotransmitters affecting the release of substances from other nerve terminals within the median eminence.     

Colocalized FMRFamide-related neuropeptides in the nervous system of the colorado potato beetle,Leptinotarsa decemlineata (say) (Coleoptera : Chrysomelidae) demonstrated immunohistochemically with mono- and polyclonal antibodies

The distribution of—still chemically undefined—peptide epitopes in subclasses of peptidergic neurons in the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera : Chrysomelidae) was studied in preparation for peptide isolation by immunological methods. Two monoclonal antibodies, MAC-13 and MAC-3, were selected for their capacity to reveal selected neuron populations by immunohistochemical methods. They partially mimic the immunostaining pattern of a polyclonal anti-FMRFamide antiserum. Immunoreactive neurons, containing FMRFamide-related antigens, were demonstrated with high specificity in the CNS as well as in the visceral and peripheral nervous system. Their total number is large, compared with other subclasses of peptidergic neurons, approximately 230 (peripheral neurons not included). Axons of these neurons run either to the corpora cardiaca for release of peptides into the bloodstream, or to distant target organs, such as the digestive tract, reproductive organs, and somatic muscles. Large, isolated peripheral neurons occur in the vicinity of the ventral nerve cord and release products through terminals located in or on the sheath of nerves emanating from the thoracic ganglia. Different antigens may be colocalized in neuron subclass-specific combinations and then always in the same secretory granules. These observations make clear that groups of peptidergic neurons, considered earlier as a homogeneous population, are much more diversified than hitherto suspected. We may anticipate the occurrence, in subsequent isolation procedures, of at least 3 classes of FMRFamide-related peptides. Compartments in the central nervous system containing high concentrations of single or mixed antigens have been identified.     

Duality of Secretory Inclusions in Neurones — Ultrastructure of the Corresponding Sites of Release in Invertebrate Nervous Systems

Central nerve terminals have been examined for ultrastructural signs of release of neurochemical mediators in the annelids Nereis diversicolor, Harmothoe imbricata and Lumbricus terrestris. Two categories of presumptive secretory inclusions are readily distinguished. Exocytosis of ‘storage granules’ is widespread in the neuropile, and involves probable peptidergic terminals as well as more conventional terminals. Plasma membranes at such sites of release are apparently unmodified. In contrast, ‘synaptic vesicles’ are aggregated adjacent to membrane thickenings and specialized clefts, and signs of their fusion with the presynaptic membranes have been observed rarely. The presence of coated pits surmounting omega profiles involving storage granules may indicate that membrane is retrieved in the form of microvesicles from the site of exocytosis. Coated pits associated with synapses have only been observed in areas of membrane adjacent to presumed sites of vesicle exocytosis. The incidence of dual sites of release, often relating to individual terminals, may be indicative of the segregated storage and independent secretion of distinct active principles. Materials released by granule exocytosis may have the role of neuromodulators.     

Computer-assisted image analysis of the distributions of peptidergic terminals in the central nucleus of the amygdala: A preliminary study

When viewed under dark-field illumination, peptidergic terminals in sections stained by the Sternberger PAP immunocytochemical method are seen as individual points of light. Under high magnification, the degree of brightness of various areas of immunoreactive terminals is seen to be a function of the density of terminals in these areas. By analyzying the relative brightness of the immunostained central nucleus of the amygdala (CNA) with an EyeCom II PDP-$\text{11}\text{34}$ image analysis system, we have obtained a relative evaluation of the density distribution of neurotensin (NT)-, substance P (SP), VIP-, angiotensin II (AII), m-enkephalin (m-ENK) and somatostatin (SS)-immunoreactive terminals in terms of normal morphology and following a brain lesion. The EyeCom II system divides the presented image into 307200 picture elements (pixels) and assigns one of 256 grey values to the average brightness with each pixel. We have aggregated the grey level frequencies into 5 levels where level 1 corresponds to the highest terminal density and level 5 to the lowest density. At level 1, only NT- and VIP-immunoreactive terminals occupy a significant percentage of the cross-sectional area of the CNA (20%). About 15% of the area of the CNA has VIP terminals with level 5 density. The distributions of the top 20% of the terminal density range of NT, SP, AII and VIP support a classical medial/lateral division of the nucleus. The distribution of the same range of SS- and ENK terminals suggests a dorsoventral division of the CNA. A preliminary study indicates that comparison of grey level frequency histograms generated by image analysis from homologous lesioned and unlesioned sections of the CNA can yield useful information regarding post-lesion changes in the distribution of immunoreactive terminals.     

Interaction of opioid peptides and the dopaminergic system in the myenteric plexus of the guniea pig ileum

The effects of treatment with dopamine agonists and 6-hydroxydopamine on the release of opioid peptides from the myenteric plexus of guinea-pig ileum were examined. Apomorphine or bromocriptine treatment at doses that act on dopamine autoreceptors to inhibit dopamine release resulted in a significant elevation of the release of opioid peptides. 6-hydroxydopamine treatment, which produces a lesion of catecholaminergic nerve terminals also resulted in an increase in opioid peptide release. These findings indicate that interruption of dopaminergic transmission in the myenteric plexus produces an increase in the release of opioid peptides and suggest an inhibitory modulation of opioid peptidergic neurons by dopamine systems in the myenteric plexus of the guinea-pig ileum.     

Dual peptidergic innervation of the blowfly hindgut: a light- and electron microscopic study of FMRFamide and proctolin immunoreactive fibers.

1. The innervation of the hindgut, rectal valve, rectum and rectal papillae of the adult blowfly, Calliphora erythrocephala, was studied by means of light and electron microscopic immunocytochemistry, using antibodies against the neuropeptides proctolin and FMRFamide. 2. Branches from the abdominal nerves reaching the posterior portion of the gut were found to contain mostly neurosecretory type axons and to innervate the muscle coat of all hindgut structures studied. 3. Some of the axons found in these nerve branches innervating the gut display proctolin- others FMRFamide-like immunoreactivity. Both types of peptidergic axons were found to have abundant terminals in the muscle coat of the hindgut, rectum and rectal valve and in the medulla of the rectal papillae. 4. It is clear that two separate peptidergic systems derived from the abdominal ganglion are supplying the hindgut structures, and, possibly, they use proctolin- and FMRFamide-like peptides respectively as their transmitters or modulators.