Neuropeptide tyrosine in the brain of the African lungfish,Protopterus annectens: Immunohistochemical localization and biochemical characterization

Lungfishes, which share similarities with both fishes and amphibians, represent an interesting group in which to investigate the evolutionary transition from fishes to tetrapods. In the present study, we have investigated the localization and biochemical characteristics of neuropeptide Y (NPY)-immunoreactive material in the central nervous system of the African lungfish, Protopterus annectens. NPY-immunoreactive cell bodies were found in various regions of the brain, most notably in the telencephalon (septal area, ventral striatum, and nucleus accumbens), in the diencephalon (preoptic nucleus, periventricular region of the hypothalamus, and ventral thalamus), and in the tegmentum of the mesencephalon. A strong immunoreaction was also detected in cell bodies of the nervus terminalis. Immunoreactive nerve fibers were particularly abundant in the ventral striatum, the nucleus accumbens, the diagonal band of Broca, the hypothalamus, and the mesencephalic tegmentum. Positive fibers were also seen in the median eminence and in the neural lobe of the pituitary. The NPY-immunoreactive material localized in the brain and pituitary was characterized by combining high-performance liquid chromatography (HPLC) analysis and radioimmunological quantitation. The displacement curves obtained with synthetic porcine and frog NPY and serial dilutions of brain and pituitary extracts were parallel. Reversed-phase HPLC analysis of telencephalon, diencephalon, and pituitary extracts resolved a major NPY-immunoreactive peak that coeluted with frog NPY. The similarity between the distribution of NPY-containing neurons and the biochemical characteristics of the immunoreactive peptide in the brain of lungfish and frog strongly favors a close phylogenetic relationship between dipnoans and amphibians. © 1995 Wiley-Liss, Inc.     

Topography and associations of luteinizing hormone-releasing hormone and neuropeptide Y-immunoreactive neuronal systems in the human diencephalon

Neuropeptide Y (NPY) potentiates the effect of luteinizing hormone-releasing hormone (LHRH) on luteinizing hormone secretion in several species, including human. In addition to the pituitary sites, the interactions of the NPY and LHRH systems may involve diencephalic loci. However, the morphologic basis of this putative communication has not yet been elucidated in the human brain. To discover interaction sites, the distribution and connections of LHRH and NPY-immunoreactive (IR) neuronal elements in the human hypothalamus were investigated by means of light microscopic single- and double-label immunocytochemistry. NPY-IR perikarya and fibers were found to be widely distributed in the ventral diencephalon, with high densities in the preopticoseptal, periventricular, and tuberal regions. Small neuronal cell groups were infiltrated with a dense network of varicose NPY-IR fibers in the lateral preoptic area. The LHRH-IR perikarya were located mainly in the preopticoseptal region, diagonal band of Broca, lamina terminalis, and periventricular and infundibular nuclei. A few LHRH-IR neurons and fibers were scattered in the mamillary region. The overlap between the NPY and LHRH systems was apparent in the periventricular, paraventricular, and infundibular nuclei. Double-labeling immunohistochemistry showed NPY-IR axon varicosities in contact with LHRH-IR perikarya and main dendrites. The putative innervation of LHRH neurons by NPY-IR fibers was also seen in 1-microm-thick plastic sections and with confocal laser scanning microscope, thus further supporting the functional impact of NPY-IR terminals on LHRH-IR neurons. The present findings suggest that the hypophysiotropic LHRH-synthesizing neurons may be innervated by intrahypothalamic NPY-IR fibers. Confirmation by ultrastructural analysis would demonstrate that the LHRH system in the human hypothalamus is regulated by NPY, as has been demonstrated in nonhuman species.     

Distribution and characterization of neuropeptide Y in the brain of an elasmobranch fish

Using a specific antiserum raised against synthetic neuropeptide Y (NPY), the distribution of immunoreactivity in the brain and pituitary of the elasmobranch fish Scyliorhinus canicula has been examined with the indirect fluorescence and the peroxidase-antiperoxidase methods. The highest density of NPY-immunoreactive neurons was found in the basal telencephalon and in the hypothalamus. Numerous NPY-containing perikarya were located in the entopeduncular and the preoptic nuclei, in the nucleus lobi lateralis and in the nucleus lateralis tuberis. NPY-immunopositive fibers were observed throughout the fish brain. In particular, dense networks of fibers were present in the entopeduncular and the habenular nuclei, in the nucleus tuberculi posterioris and in the lateral lobes. Scattered fibers were observed in all other parts of the brain except in the cerebellum where no NPY-immunoreactive material could be detected. A plexus of NPY-immunoreactive fibers arising from the preoptic neurosecretory complex appeared to run through the basal hypothalamus and the pituitary stalk. These fibers terminated in the intermediate lobe of the pituitary, suggesting that NPY may be involved in the control of melanotropin secretion. The NPY-immunoreactive material localized in the brain and pituitary was characterized by combining high-performance liquid chromatography (HPLC) analysis and radioimmunological detection. Brain and pituitary extracts showed a good cross-reactivity to the NPY antiserum, but serial dilutions of tissue samples did not completely parallel the standard curve. HPLC analysis resolved two major forms of immunoreactive NPY in the hypothalamus while the pars intermedia contained only authentic NPY. The widespread distribution of NPY neurons in the fish brain and pituitary suggests the involvement of NPY in a variety of physiological functions, including the neuroendocrine control of the pituitary.     

Melanin-concentrating hormone system in the brain of the lungfish Protopterus annectens

The neurochemical anatomy of the lungfish brain is of particular interest, because many features in these animals might be representative of the common ancestor of land vertebrates. In the present study, we have investigated the localization and biochemical characteristics of melanin-concentrating hormone (MCH)-immunoreactive material in the central nervous system of the African lungfish, Protopterus annectens. The most prominent group of MCH-immunoreactive cell bodies was found in the dorsal hypothalamus. Additional groups of MCH-immunoreactive perikarya were detected in the telencephalon within the medial and dorsal pallium, the medial subpallium, and the ventral part of the lateral subpallium. Brightly immunofluorescent nerve fibers were seen in the anterior olfactory nucleus, the ventral part of the medial pallium, the medial subpallium, and the anterior preoptic area. In the diencephalon, the hypothalamus and the medial region of the dorsal thalamus exhibited a dense accumulation of fibers. MCH-immunoreactive fibers were also found in the tectum and the tegmentum of the mesencephalon and within the reticular formation of the rhombencephalon. In the pituitary, several small groups of cells of the intermediate lobe showed a bright fluorescence. Reversed-phase high-performance liquid chromatography (HPLC) analysis of diencephalon and pituitary extracts resolved a major MCH-immunoreactive peak that coeluted with synthetic salmon MCH. The distribution of MCH in the brain of P. annectens suggests that, in lungfishes, this peptide may exert neuromodulator or neurotransmitter functions. The presence of MCH-like immunoreactivity in the intermediate lobe of the pituitary indicates that, in dipnoans, MCH may also act as a typical pituitary hormone. J. Comp. Neurol. 390:41–51, 1998. © 1998 Wiley-Liss, Inc.     

Distribution of neuropeptide Y-like immunoreactivity in the hypothalamus of the adult golden hamster

The distribution of neuropeptide Y (NPY)-like immunoreactivity within the hypothalamus of the adult golden hamster was investigated with conventional immunohistochemical techniques. Neuropeptide Y immunoreactive cell bodies were found in greatest numbers in the arcuate nucleus while a few stained perikarya were seen in the internal and subependymal zones of the median eminence. Isolated perikarya were observed in the anterior commissure and supracommissural portion of the interstitial nucleus of the stria terminalis. Immunoreactive axons were located throughout the hypothalamus with the highest concentrations in the subependymal and internal zones of the median eminence, the interstitial nucleus of the stria terminalis, the medial preoptic area, and in the following nuclei: periventricular, suprachiasmatic, paraventricular, perifornical, median preoptic, and arcuate. Moderate to dense plexuses of immunoreactive fibers were observed in the anterior, lateral, and posterior hypothalamic areas and in the infundibular stalk. The supraoptic nucleus and lateral preoptic area displayed a small number of labeled axons whereas the ventromedial nucleus contained only a few fibers. NPY immunoreactive fibers were present in the optic tract and in the dorsomedial aspect of the optic chiasm. Labeled fibers penetrated the ependymal lining of the third ventricle throughout the ventral aspect of the periventricular zone. Additional fibers were observed in the pia lining the ventral aspect of the hypothalamus. This systematic analysis of hypothalamic NPY immunoreactivity in the adult golden hamster suggests that a portion of the labeled fibers display a distribution that is similar to previously described noradrenergic fibers in the hypothalamus.     

Alpha-melanocyte-stimulating hormone (alpha-MSH) in the brain of the African lungfish, Protopterus annectens: immunohistochemical localization and biochemical characterization.

The distribution of alpha-melanocyte-stimulating hormone (alpha-MSH) containing neurons and the molecular forms of alpha-MSH-related peptides exhibit substantial differences in the brains of fish and amphibians. Lungfishes, which share similarities with both fishes and tetrapods, represent a valuable group in which to investigate the neuroanatomical and neurochemical facets of evolution. In the present study, we have localized and characterized alpha-MSH-immunoreactive peptides in the central nervous system of the African lungfish Protopterus annectens. Perikarya exhibiting alpha-MSH-like immunoreactivity were observed in two distinct regions of the hypothalamus: the rostral part of the preoptic nucleus and the caudal part of the hypothalamus. In the caudal hypothalamus most alpha-MSH-immunopositive perikarya were located in both the subependymal and deepest layers of the ventral periventricular region. Scattered alpha-MSH-immunopositive cells were occasionally detected in the dorsal side of the caudal hypothalamus. The alpha-MSH-immunoreactive material localized in the brain was characterized by combining high-performance liquid chromatography (HPLC) analysis and radioimmunological detection. The displacement curves obtained with synthetic alpha-MSH and serial dilutions of brain and pituitary extracts were parallel. HPLC analysis of lungfish hypothalamic extracts showed that the major immunoreactive peak coeluted with synthetic desacetyl alpha-MSH and its sulfoxide derivative. An additional peak coeluted with synthetic sulfoxide alpha-MSH. In contrast, in the pituitary, the predominant form of alpha-MSH-like material coeluted with the N,O-diacetyl alpha-MSH standard. These results provide the first evidence for the presence of alpha-MSH-related peptides in the brain of a lungfish. The distribution of alpha-MSH neuronal systems in the lungfish is very similar to that reported in amphibians, supporting the existence of phylogenetic convergences between these two vertebrate groups.     

Immunohistochemical localization of gamma-aminobutyric acid in the rat pituitary gland and related hypothalamic regions

The distribution of gamma-aminobutyric acid (GABA) containing neurons in the rat pituitary gland and related hypothalamic areas was immunohistochemically investigated using antibodies raised against GABA conjugated to bovine serum albumin by glutaraldehyde. A dense network of GABA-like immunoreactive fine varicose nerve fibers was observed within the posterior and intermediate lobes of the pituitary gland, surrounding endocrine cells and capillaries, but not in the anterior lobe. In the pituitary stalk, the dense varicose fibers ran along the anterior wall of the posterior lobe into the posterior and intermediate lobes. A small number of GABA-like immunoreactive cell bodies were evident in the intermediate lobe. GABA-like immunoreactive fibers occurred at low to high density in most parts of the hypothalamus. GABA-like immunoreactive neurons were observed in some regions related to the pituitary gland (such as periventricular nucleus, paraventricular nucleus, arcuate nucleus and accessory magnocellular nucleus). These results provide morphological evidence for the presence of GABAergic neurons in the rat hypothalamo-pituitary system.     

Immunohistochemical localization of γ-aminobutyric acid in the rat pituitary gland and related hypothalamic regions

The distribution on γ-aminobutyric acid (GABA) containing neurons in the rat pituitary gland and related hypothalamic areas was immunohistochemically investigaed using antibodies raised against GABA conjugated to bovine serum albumin by glutaraldehyde. A dense network of GABA-like immunoreactive fine varicose nerve fibers was observed within the posterior and intermediate lobes of the pituitary gland, surrounding endocrine cells and capillaries, but not in the anterior lobe. In the pituitary stalk, the dense varicose fibers ran along the anterior wall of the posterior lobe into the posterior and intermediate lobes. A small number of GABA-like immunoreactive cell bodies were evident in the intermediate lobe. GABA-like immunoreactive fibers occurred at low to high density in most parts of the hypothalamus. GABA-like immunoreactive neurons were observed in some regions related to the pituitary gland (such as periventricular nucleus, paraventricular nucleus, arcuate nucleus and accessory magnocellular nucleus). These results provide morphological evidence for the presence of GABAergic neurons in the rat hypothalamo-pituitary system.     

Distribution of galanin-like immunoreactivity in the brain of Rana esculenta and Xenopus laevis.

The immunocytochemical distribution of galanin-containing perikarya and nerve terminals in the brain of Rana esculenta and Xenopus laevis was determined with antisera directed toward either porcine or rat galanin. The pattern of galanin-like immunoreactivity appeared to be identical with antisera directed toward either target antigen. The distribution of galanin-like immunoreactivity was similar in Rana esculenta and Xenopus laevis except for the absence of a distinct laminar distribution of immunoreactivity in the optic tectum of Xenopus laevis. Galanin-containing perikarya were located in all major subdivisions of the brain except the metencephalon. In the telencephalon, immunoreactive perikarya were detected in the pars medialis of the amygdala and the preoptic area. In the diencephalon, immunoreactive perikarya were detected in the caudal half of the suprachiasmatic nucleus, the nucleus of the periventricular organ, the ventral hypothalamus, and the median eminence. In the mesencephalon, immunoreactive perikarya were detected near the midline of the rostroventral tegmentum, in the torus semicircularis and, occasionally, in lamina A and layer 6 of the optic tectum. In the myelencephalon, labelled perikarya were detected only in the caudal half of the nucleus of the solitary tract. Immunoreactive nerve fibers of varying density were observed in all subdivisions of the brain with the densest accumulations of fibers occurring in the pars lateralis of the amygdala and the preoptic area. Dense accumulations of nerve fibers were also found in the lateral septum, the medial forebrain bundle, the periventricular region of the diencephalon, the ventral hypothalamus, the median eminence, the mesencephalic central gray, the laminar nucleus of the torus semicircularis, several laminae of the optic tectum, the interpeduncular nucleus, the isthmic nucleus, the central gray of the rhombencephalon, and the dorsolateral caudal medulla. The extensive system of galanin-containing perikarya and nerve fibers in the brain of representatives of two families of anurans showed many similarities to the distribution of galanin-containing perikarya and nerve fibers previously described for the mammalian brain.     

GABA-ergic innervation of thyrotropin-releasing hormone-synthesizing neurons in the hypothalamic paraventricular nucleus of the rat

To determine whether GABA-ergic axons are anatomically situated to directly influence TRH neurons in the PVN, double-labeling light- and electronmicroscopic immunocytochemistry was performed using antisera against glutamic acid decarboxylase (GAD) and prothyrotropin-releasing hormone (proTRH). In the anterior, periventricular and medial parvocellular subdivisions of the PVN, GAD-immunoreactive (IR) axon varicosities were closely apposed to all proTRH containing cell bodies and proximal dendrites. Ultrastucturally, GAD-IR nerve terminals established symmetric type synapses with both perikarya and dendrites of proTRH-IR neurons, indicating the inhibitory nature of the contacts. Since a subpopulation of neuropeptide Y (NPY) neurons in the hypothalamic arcuate nucleus co-synthesize GABA, and NPY-containing neurons of arcuate nucleus origin densely innervate TRH neurons in the PVN, we performed triple labeling immunocytochemistry to elucidate the origin of the GAD-IR innervation of hypophysiotropic TRH neurons. While axons co-containing GAD and NPY were observed throughout the PVN, only approximately 10% of GAD-IR terminals in contact with TRH neurons were found to contain NPY-immunoreactivity. We conclude that GABA-ergic neurons are in position to act directly on hypophysiotropic TRH neurons and while this innervation arises partly from neurons in the arcuate nucleus that co-synthesize NPY, the majority of the GABA-ergic input arises from other neuronal groups.     

Corticotropin-releasing hormone-synthesizing neurons of the human hypothalamus receive neuropeptide Y-immunoreactive innervation from neurons residing primarily outside the infundibular nucleus

Immunohistochemical single- and double-labeling studies were performed on the hypothalami of postmortem human brains to elucidate the distribution of corticotropin-releasing hormone (CRH)-immunoreactive (IR) neuronal elements and their interaction with the neuropeptide Y (NPY)-ergic neuronal system. The great majority of CRH-IR perikarya were found in the paraventricular nucleus (PVN), whereas a considerable number of CRH-IR neurons were also observed in the periventricular and infundibular nuclei. The dorsomedial nucleus and the perifornical region contained only scattered CRH-IR neurons. Dense CRH-IR fiber networks were found throughout the hypothalamus. However, the medial preoptic, the dorsolateral part of the supraoptic, the suprachiasmatic, the ventromedial, and the different mammillary nuclei showed a relative paucity of fibers. The terminal fields of NPY-IR axons overlapped the distribution of CRH-IR neurons in the hypothalamus. NPY-IR axon varicosities were juxtaposed to both dendrites and perikarya of the majority of CRH-IR neurons residing in the paraventricular, periventricular, and infundibular nuclei. These neurons were frequently contacted by multiple NPY axons that either formed baskets around their perikarya or completely ensheathed the emanating CRH dendrites. Because NPY and agouti-related protein (AGRP) are co-contained in neurons of the human infundibular nucleus, we used AGRP as a marker of NPY fibers originating exclusively from the infundibular nucleus. Only a small proportion of CRH neurons in the PVN was contacted by AGRP-IR axon varicosities, suggesting that NPY-IR innervation of CRH neurons in the PVN derive mainly from regions outside the infundibular nucleus. The present morphological findings support the view that NPY regulates the CRH system of the human hypothalamus and therefore at least some of the effects of NPY on metabolic, autonomic, and endocrine functions may be mediated through CRH.     

Vasoactive intestinal peptide in the hypothalamohypophysial system of the Mongolian gerbil

By use of the indirect peroxidase-antiperoxidase immunohistochemical technique the location of perikarya and fibers exhibiting vasoactive intestinal peptide (VIP)-like immunoreactivity was investigated in the hypothalamus and the posterior pituitary of the Mongolian gerbil (Meriones unguiculatus), because of the involvement of VIP in several neuroendocrine functions. In the hypothalamus, a large number of VIP-immunoreactive perikarya were seen in the ventromedial part of the suprachiasmatic nucleus. Few VIP-positive perikarya were present in the periventricular, paraventricular, and supraoptic nuclei and in the medial preoptic area close to the third ventricle. The perikarya in the paraventricular nucleus projected fibers in the direction of the median eminence. In the median eminence VIP-immunoreactive fibers were present especially in the external layer, concentrated in the perivascular spaces surrounding the portal vessels. Scattered VIP-immunoreactive fibers were also located in the internal layer of the median eminence as well as in the posterior pituitary lobe. In the latter, large VIP-positive Herring-like bodies were observed. With receptor autoradiography a large number of grains were demonstrated in the anterior pituitary lobe in contrast to the neural lobe. Many VIP-positive fibers and some perikarya were observed within the ependyma covering the rostroventral part of the third ventricle. Finally, fibers exhibiting VIP immunoreactivity were also seen in the organum vasculosum laminae terminalis (OVLT). These results support the concept that VIP is released into the portal vascular system and plays a role in the regulation of the activity of the anterior pituitary. In addition, VIP might be secreted into the cerebrospinal fluid of the third ventricle.     

Organization and synaptic interconnections of GABAergic and cholinergic elements in the rat amygdaloid nuclei: single- and double-immunolabeling studies

The aim of this study was to describe the localization of cholinergic and GABAergic neurons and terminals in the amygdaloid nuclei of the rat. Double immunolabeling was performed to study cholinergic-GABAergic synaptic interconnections. Cholinergic elements were labeled by using a monoclonal antibody to choline acetyltransferase (ChAT), the acetylcholine synthesizing enzyme. Antibodies against glutamate decarboxylase (GAD), the GABA- synthesizing enzyme, were employed to identify GABAergic perikarya and terminals. The tissue sites of the antibody bindings were detected by using either Sternberger's peroxidase-antiperoxidase (PAP) method or a biotinylated secondary antibody and avidinated ferritin. These two contrasting immunolabels allowed us to study GABAergic-cholinergic interconnections at the electron microscopic level. Our study revealed a characteristic distribution of GABAergic and cholinergic elements in the various amygdaloid nuclei: 1) Large, ChAT-immunopositive cells with heavily labeled dendrites were observed in the anterior amygdaloid area and in the lateral and medial zones of the central nucleus. These cells seem to constitute the intraamygdaloid extension of the magnocellular basal nucleus. Their dendrites invaded other amygdaloid nuclei, in particular the intercalated nuclei, the lateral olfactory tract nucleus, and the central zone of the central nucleus. These ChAT-immunoreactive dendrites formed synaptic contacts with GAD-positive terminals. GABAergic terminals probably thus exert an inhibitory amygdaloid influence onto cholinergic neurons of the magnocellular basal nucleus. 2) Two amygdaloid nuclei-the basal dorsal nucleus and the lateral olfactory tract nucleus-contained a dense network of ChAT-immunoreactive fibers and terminals, but they also contained numerous GAD-positive perikarya. Double-immunolabeling experiments revealed cholinergic terminals forming synaptic contacts on GAD-immunopositive cell bodies, dendritic shafts, and spines. 3) The central and medial nucleus seem to be the main target of GABAergic fibers to the amygdala. Both nuclei contained a dense plexus of GAD-immunoreactive terminals that may arise, at least in part, from the GABAergic neurons in the basal dorsal nucleus. Inhibition of the centromedial "excitatory" region through intraamygdaloid GABAergic connections may reduce excitatory amygdaloid influence onto hypothalamus and brainstem.     

Peptide- and transmitter-containing neurons in the mediobasal hypothalamus and their relation to GABAergic systems: possible roles in control of prolactin and growth hormone secretion

Indirect immunofluorescence histochemistry was used to study the relation among GABAergic, catecholaminergic, cholinergic, and peptidergic neurons in the rat mediobasal hypothalamus. By employing a direct double-labelling procedure using sheep antiserum against glutamic acid decarboxylase (GAD), mouse monoclonal and rabbit antibodies to neurotensin (NT) and rabbit antisera to tyrosine hydroxylase (TH), choline acetyltransferase (ChAT), galanin (GAL), growth hormone-releasing factor (GRF), or somatostatin (SOM), it was demonstrated that GAD-positive fibers and terminals in the external part of the median eminence co-contained immunoreactivity for TH, NT, GAL or GRF, but not for SOM. In the internal part of the median eminence-infundibular stalk, GAD-positive/NT-, GAL-, and GRF-negative and GAD-positive/TH-positive fiber plexa were shown. When a recently developed direct triple-labelling procedure with biotin-conjugated mouse secondary antibodies in conjunction with diethylaminocoumarin (DAMC)-conjugated avidin was employed, presence of GAD/GAL/NT- as well as GAD/GRF/NT-containing varicosities could be demonstrated close to hypophysial portal vessels. In colchicine-pretreated animals, GAD was shown to coexist with TH, NT, or GAL in cell bodies in both the dorsomedial and ventrolateral domains of the arcuate nucleus, but with GRF only in the ventrolateral division. ChAT-positive neurons in the ventrolateral region were also TH-positive. In the ventrolateral arcuate nucleus, triple-labelling followed by elution-restaining showed GAD/NT/GAL/TH-immunoreactivities in the same cells. Similarly, double-labelling with two following elution-restaining steps showed several NT/GAL/GRF/TH-containing cell bodies in this part of the arcuate nucleus. GAD-positive cells in the anterior hypothalamic periventricular area and fibers in the pituitary neurointermediate lobe were also TH-positive. The results demonstrate complex patterns of storage of chemical messengers in neurons of the arcuate nucleus-median eminence complex. Possible neuroendocrine interactions of these systems in the control of prolactin and growth hormone secretion are discussed.     

Immunohistochemical evidence for synaptic connections between neuropeptide Y-containing axons and periventricular somatostatin neurons in the anterior hypothalamus in rats

By employing a pre-embedding double immunolabeling technique, we examined light and electron microscopically synaptic associations between neuropeptide Y (NPY)-containing axons and somatostatin (SRIH)-containing neurons in the anterior periventricular area (APV) of the rat hypothalamus. For light microscopy, the immunoreactions for NPY and SRIH were visualized with silver-gold and diaminobenzidine (DAB), respectively, and the reverse labeling was used for electron microscopy. Light microscopy disclosed many brown SRIH perikarya surrounded by several black beads of NPY fibers in the APV. In electron microscopy, immunoreactive SRIH neurons revealed silver-gold particles scattered throughout the cytoplasm and accumulated in the Golgi area and the secretory granules. SRIH perikarya and dendritic processes indicated synaptic associations with DAB-labeled NPY fiber terminals and immunonegative fibers. NPY presynaptic terminals possessed numerous small clear vesicles and a few dense core visicles; vesicular membranes and cores were labeled with DAB chromogen. Both the pre- and postsynaptic membranes were thickened equally to be a symmetric synapse. These findings suggest that NPY neurons are involved in the regulation of growth hormone secretion from the pituitary by affecting periventricular SRIH neurons.     

Organization of vasoactive intestinal peptide-like immunoreactive system in the brain, olfactory organ and retina of the zebrafish, Danio rerio, during development

The localization of vasoactive intestinal peptide (VIP)-like immunoreactivity was investigated in the brain, olfactory system and retina of the zebrafish, Danio rerio, during development and in juvenile specimens, by using the indirect immunofluorescence and the peroxidase-antiperoxidase methods. In 24 h post fertilization (hpf) embryos, VIP-like immunoreactive cells were present in the olfactory pit, the retina, and several regions of the brain, including the dorsal telencephalon, the diencephalon, the tegmentum of the mesencephalon, the caudal rhombencephalon and the anterior pituitary. In 48 hpf embryos, additional VIP-like immunoreactive cell bodies were found in the ventral telencephalon, whereas in the diencephalon VIP-like immunopositive cells were more concentrated within the ventro-caudal hypothalamus. During the 7 day larval period, a dense plexus of VIP-like immunoreactive fibers first appeared in the olfactory bulbs. In 15-day-old larvae, two new groups of positive cells were observed in the periventricular preoptic nucleus and in the dorsal rhombencephalon. In 1 month/2 months old animals, VIP-like immunoreactive elements were confined to the olfactory organ, the olfactory bulbs, the periventricular preoptic nucleus and the pituitary, pars distalis. At 3 months stage, a large number of cells was observed in the periventricular preoptic nucleus. Western immunoblot analysis confirmed that VIP-like peptides, with molecular weight similar to that of synthetic VIP, are present early during the development of zebrafish. These results show that VIP-like immunoreactive structures appear early during ontogeny both in the olfactory pit, retina and brain. Transient expression of positive cells was found in the retina, telencephalon, diencephalon and brainstem. The location of VIP-like immunoreactivity indicates that, during development, VIP could be involved in several neuromodulatory functions, including the processing of visual and olfactory informations, as well as growth or survival promotion activities. The presence of VIP-like immunopositive cells in the pituitary, pars distalis, suggest that, during development, VIP may influence the secretion of pituitary hormones.     

Distribution of proenkephalin-derived peptides in the brain of Rana esculenta

The immunocytochemical distribution of authentic proenkephalin-containing perikarya and nerve fibers in the brain of Rana esculenta was determined with antisera directed toward different epitopes of preproenkephalin. The pattern of proenkephalinlike immunoreactivity was similar with antisera directed toward [Met5]-enkephalin, [Met5]-enkephalin-Arg6, [Met5]-enkephalin-Arg6-Phe7, [Leu5]-enkephalin, and metorphamide; however, the intensity of the labelling varied depending on the target antigen. Proenkephalin-containing perikarya were located in all major subdivisions of the brain except the metencephalon. In the telencephalon, immunoreactive perikarya were detected in the dorsal, medial, and lateral pallium; the medial septal nucleus; the dorsal and ventral striatum; and the amygdala. In the diencephalon, immunoreactive perikarya were detected in the preoptic nucleus, in the dorsal and ventral caudal hypothalamus, and in an area that appeared to be homologous to the paraventricular nucleus. In the mesencephalon, numerous immunoreactive perikarya were detected in layer 6 of the optic tectum and a few scattered perikarya were detected in layer 4 of the optic tectum. Immunoreactive perikarya also occurred in the laminar nucleus of the torus semicircularis. In the rhombencephalon, immunoreactive perikarya were detected in the obex region and the nucleus of the solitary tract. Immunoreactive fibers of varying density were observed in all major subdivisions of the brain with the densest accumulations of fibers occurring in the dorsal pallium, the lateral and medial forebrain bundles, the amygdala, the periventricular hypothalamus, the superficial region of the caudolateral brainstem, and in a tract that appeared to be homologous to the tractus solitarius. The extensive system of proenkephalin-containing perikarya and nerve fibers in the brain of an amphibian showed many similarities to the distribution of proenkephalin-containing perikarya and nerve fibers previously described for the amniote brain.     

Neuropeptides and gamma-aminobutyric acid in the vestibular nuclei of the rat: an immunohistochemical analysis. I. Distribution

The distribution of substance P, Leu-enkephalin and gamma-aminobutyric acid (GABA) containing structures in the rat vestibular nuclei were investigated by means of an indirect immunofluorescent method using specific antisera to substance P, Leu-enkephalin and glutamic acid decarboxylase (GAD), respectively. Numerous positive neurons and fibers containing these three substances were found in the medial vestibular nucleus. Most of them were situated in the caudal part of the nucleus and those in the rostral part were concentrated dorsally. In the descending vestibular nucleus, a large number of substance P, Leu-enkephalin and GAD containing neurons were evenly distributed among longitudinally directing fiber bundles. A number of positive fibers with these substances were also observed. The lateral vestibular nucleus contained numerous coarse GAD-immunoreactive fibers surrounding Deiters' neurons, while substance P-immunoreactive and Leu-enkephalin-immunoreactive fibers were rather poorly distributed in this nucleus as well as in the superior vestibular nucleus.     

Distribution of phenylethanolamine-N-methyltransferase-immunoreactive perikarya and fibers in the brain of the lizard Gekko gecko

The distribution of phenylethanolamine N-methyltransferase (PNMT)-immunoreactive (PNMTi) cell bodies and fibers in the brain of the lizard Gekko gecko was studied by antibodies raised in rabbits against purified bovine adrenal PNMT. The PNMTi cell bodies were observed in the ventrolateral rhombencephalic tegmentum at the level of the obex. No immunoreactive perikarya were found in the nucleus of the solitary tract, the medial longitudinal fascicle or the hypothalamus. An extensive network of PNMTi fibers is present throughout the brain, extending rostrally as far as the olfactory peduncle. In the telecenphalon, moderate to dense plexuses of PNMTi fibers were observed in the medial part of the nucleus accumbens, the medial septal nucleus, the nucleus of the diagonal band, the caudoventral septal region and the central amygdaloid nucleus. In the diencephalon, the periventricular and lateral zones of the preoptic and hypothalamic areas, the medial forebrain bundle and the dorsomedial thalamic nucleus contain many PNMTi fibers. Brainstem structures innervated by PNMTi fibers are the ventral tegmental area, the substantia nigra, the periaqueductal gray, the locus coeruleus, the parabrachial region, the nucleus of the solitary tract, the dorsal motor nucleus of the vagus and the ventrolateral region of the caudal brainstem. Although the brain of Gekko appears to lack PNMTi cells in areas comparable to the C2 and C3 cell groups in rats, the distribution of PNMTi fibers is nevertheless strikingly similar in both groups.