Immunocytochemical localization of enkephalins in the brain of the African lungfish,Protopterus annectens,provides evidence for differential distribution of Met-enkephalin and Leu-enkephalin

The distribution of various opioid peptides derived from proenkephalin A and B was studied in the brain of the African lungfish Protopterus annectens by using a series of antibodies directed against mammalian opioid peptides. The results show that both Met-enkephalin- and Leu-enkephalin-immunoreactive peptides are present in the lungfish brain. In contrast, enkephalin forms similar to Met-enkephalin-Arg-Phe, or Met-enkephalin-Arg-Gly-Leu, as well as mammalian α-neoendorphin, dynorphin A (1–8), dynorphin A (1–13), or dynorphin A (1–17) were not detected. In all major subdivisions of the brain, the overwhelming majority of Met-enkephalin- and Leu-enkephalin-immunoreactive cells were distinct. In particular, cell bodies reacting only with Leu-enkephalin antibodies were detected in the medial subpallium of the telencephalon, the griseum centrale, the reticular formation, the nucleus of the solitary tract, and the visceral sensory area of the rhombencephalon. Cell bodies reacting only with Met-enkephalin antibodies were found in the lateral subpallium of the telencephalon, the caudal hypothalamus, and the tegmentum of the mesencephalon. The preoptic periventricular nucleus of the hypothalamus exhibited a high density of Met-enkephalin-immunoreactive neurons and only a few Leu-enkephalin-immunoreactive neurons. The distribution of Met-enkephalin- and Leu-enkephalin-immunoreactive cell bodies and fibers in the lungfish brain showed similarities to the distribution of proenkephalin A-derived peptides described previously in the brain of land vertebrates. The presence of Met-enkephalin- and Leu-enkephalin-like peptides in distinct regions, together with the absence of dynorphin-related peptides, suggests that, in the lungfish, Met-enkephalin and Leu-enkephalin may originate from distinct precursors. J. Comp. Neurol. 396:275–287, 1998. © 1998 Wiley-Liss, Inc.     

Distribution of calcitonin gene-related peptide-like immunoreactivity in the brain of the small-spotted dogfish,scyliorhinus canicula L

The distribution of neuropeptides has been useful in comparing neuronal aggregates of elasmobranchs with those in other vertebrates. The distribution of calcitonin gene-related peptide (CGRP)-like immunoreactivity in the brain of the dogfish was examined with an antiserum to rat α-CGRP. Western blot analysis confirms that our antiserum recognizes a single peptide in the dogfish brain very similar to mammalian CGRP. CGRP-like immunoreactivity was located in discrete neuronal groups. CGRP-like-immunoreactive (CGRP-ir) neurons were found in the motor nuclei III, IV, V, VI, VII, IX, and X of the brainstem motor column and in the octavolateral efferent neurons. In the isthmal region, two groups of CGRP-ir neurons appeared in the parabrachial region and reticular substance. Three other CGRP-ir cell groups were observed in the mesencephalon: in the ventral tegmental area, in the substantia nigra, and one widely scattered but numerous population in superficial layers of the optic tectum. In the diencephalon, CGRP-ir cells were observed in the magnocellular preoptic nucleus and the organon vasculosum hypothalami. A population of CGRP-ir cells was also observed in the entopeduncular nucleus in the impar telencephalon. CGRP-ir fibers of central origin were widely distributed in the brain, but the most conspicuous areas were found in the ventral telencephalon, the hypothalamus, the mesencephalic lateral reticular area, and the dorsolateral isthmal region. The neurointermediate lobe of the hypophysis was also richly innervated by CGRP-ir fibers. CGRP-ir sensory fibers of cranial nerves IX and X and of dorsal spinal roots formed very conspicuous terminal fields in the lobus vagi and Cajal's nucleus commissuralis and in the dorsal region of the substantia gelatinosa, respectively. Comparison of the distribution of fibers and perikarya in dogfish and other vertebrates suggests that this CGRP-ir system has been well conserved during evolution. © 1995 Wiley-Liss, Inc.     

Distribution of neuropeptide FF-like immunoreactivity in the brain of Dermophis mexicanus (Amphibia; Gymnophiona): comparison with FMRFamide immunoreactivity

Neuropeptide FF (NPFF) is an FMRFamide-related peptide widely distributed in the mammalian brain. NPFF immunohistochemistry labeled cell bodies in a few locations and dense fiber networks throughout the brain. Recently, the distribution of NPFF immunoreactive (NPFF-ir) cells and fibers in the brain of anuran and urodele amphibians was studied and, as in mammals, significant species differences were noted. To further assess general and derived features of the NPFF-containing neuron system in amphibians, we have investigated the distribution of NPFF-ir cell bodies and fibers in the brain of the gymnophionan Dermophis mexicanus by means of an antiserum against bovine NPFF. This distribution was compared to that of FMRFamide immunoreactivity. Major traits shared with anurans and urodeles were the abundant fiber labeling in the ventral telencephalon, hypothalamus, isthmus, ventrolateral medulla and dorsal spinal cord. In addition, in the three amphibian orders the majority of the NPFF-ir cells were located in the preoptic-hypothalamic region. However, distinct particular features were present in the gymnophionan such as the lack of NPFF-ir cells in the telencephalon, brainstem and spinal cord and the absence of NPFF-ir fibers in the hypophysis and the olfactory bulbs. This pattern was distinct from that observed for FMRFamide distribution. Striking differences were noted in the pallium, caudal hypothalamus and midbrain tegmentum where FMRFamide-containing cells were localized. The present results in Dermophis support the idea that data from gymnophionans must be included when stating the amphibian condition of a given system because important variations are obvious when gymnophionans are compared with anurans and urodeles.     

Organization of the histaminergic system in the brain of the turtle Chinemys reevesii

To accumulate phylogenetic information on the central histaminergic system, we investigated the histaminergic system in the brain of the Reeves turtle, Chinemys reevesii, using the indirect immunofluorescent method with antiserum against histamine. Histaminergic neuronal cell bodies were found exclusively in the posterior part of the ventral hypothalamus. Histaminergic varicose fibers innervated almost all parts of the turtle brain, but tended to be concentrated in several areas. Very dense innervation was observed in the medial part of the telencephalon, ventrolateral part of the hypothalamus, nucleus habenularis lateralis, and ventromedial part of the tegmentum. Medium density of innervation was seen in the olfactory bulb, nucleus medialis amygdalae, and tectum. Only a few fibers were detected in the lateral part of the telencephalon, dorsal part of the hypothalamus, thalamus, rhombencephalon, and spinal cord. The main ascending fibers were observed in the lateral part of the hypothalamus, sending dense fiber bundles to the cortices dorsomedialis and medialis and nucleus habenularis lateralis. Descending fibers appeared to run in the ventral tegmental area, passing through the dorsal and ventral parts of the midline of the brain stem to the spinal cord. These findings indicate that the general morphological features of the histaminergic system in the turtle brain are similar to those in the mammalian and frog brains.     

Prodynorphin peptide distribution in the forebrain of the Syrian hamster and rat: a comparative study with antisera against dynorphin A, dynorphin B, and the C-terminus of the prodynorphin precursor molecule

The neuroanatomical distribution of the prodynorphin precursor molecule in the forebrain of the male Syrian hamster (Mesocricetus auratus) has been studied with a novel antiserum directed against the C-terminus of the leumorphin [dynorphin B (1-29)] peptide product. C-peptide staining in sections from colchicine-treated hamsters is compared to staining in sections from untreated animals. In addition, the pattern of C-peptide immunostaining in hamster brain is compared to that in the rat brain. Finally, the C-peptide immunolabeling patterns in hamsters and rats are compared to those obtained with antisera to dynorphin A (1-17) and dynorphin B (1-13). Areas of heaviest prodynorphin immunoreactivity in the hamster include the hippocampal formation, lateral septum, bed nucleus of the stria terminalis, medial preoptic area, medial and central amygdaloid nuclei, ventral pallidum, substantia nigra, and numerous hypothalamic nuclei. Although this C-peptide staining pattern is similar to dynorphin staining reported previously in the rat, several species differences are apparent. Whereas moderate dentate gyrus granule cell staining and no CA4 cell staining have been reported in the rat hippocampal formation, intense immunostaining in the dentate gyrus and CA4 cell labeling are observed in the hamster. In addition, the medial preoptic area, bed nucleus of the stria terminalis, and medial nucleus of the amygdala stain lightly for prodynorphin-containing fibers and cells in the rat, compared to heavy cell and fiber staining in the hamster in all three of these regions. In the rat there is no differential staining between tissues processed with the C-peptide, dynorphin A, and dynorphin B antisera, but numerous areas of the hamster brain show striking differences. In most hamster brain areas containing prodynorphin peptides, the C-peptide antiserum immunolabels more cells and fibers than the dynorphin B antiserum, which in turn labels more cells and fibers than dynorphin A antiserum. However, exceptions to this hierarchy of staining intensity are found in the lateral hypothalamus, substantia nigra, arcuate nucleus, and habenula. The differences in staining patterns between rat and hamster are greatest when C-peptide antiserum is used; apparent species differences are present, though less pronounced, in dynorphin B- and dynorphin A-immunostained material.     

Distribution of immunoreactive growth hormone releasing factor (1–44)NH2 in the tuberoinfundibular system of the rhesus monkey

Using an antiserum which reacts with the carboxyl terminus of GRF(1-44)NH2, the distribution of immunoreactive growth hormone releasing factor (GRF) in the rhesus monkey hypothalamus was delineated by peroxidase immunocytochemistry. Immunoreactive material was present in dense terminal fields in the median eminence closely associated with portal capillaries but in a location distinct from that noted for immunoreactive thyrotropin-releasing hormone (TRH) or somatostatin. GRF-immunoreactive cell bodies were identified in the arcuate nucleus and ventromedial nucleus. These studies provide evidence for the presence of GRF(1-44)NH2 in the primate brain and demonstrate that in the hypothalamus it is localized exclusively in cells and fibers corresponding to the tuberoinfundibular system.     

Distribution of opioid peptides in the preoptic region: immunohistochemical evidence for a steroid-sensitive enkephalin sexual dimorphism

The distribution of cells and fibers that contain opioid peptides within the preoptic region of the rat was examined immunohistochemically. Cells and/or fibers that contain peptides derived from each of the three major opioid peptide families were differentially stained by using antisera that recognize unique derivatives of each precursor molecule and do not cross-react with members of the other opioid peptide families. A beta-endorphin (beta E) antiserum was used to stain fibers that contain peptides derived from the proopiomelanocortin molecule, and dynorphin-containing cells were identified by using an antiserum directed toward dynorphin B (Dyn B) that does not show detectable cross-reactivity with enkephalin-related peptides. An antiserum raised against peptide E (PE), which does not appear to cross-react significantly with dynorphin peptides, was used to localize enkephalin cells and fibers. Each family of opioid peptides showed a unique distribution in the preoptic region. beta E-immunoreactive fibers were primarily localized to the preoptic part of the periventricular nucleus, with moderate densities of fibers contained in the anteroventral periventricular nucleus (AVPv) and medial preoptic nucleus (MPN). Dyn B-immunoreactive fibers showed a somewhat more uniform distribution throughout the region, and only a few Dyn B-stained cells bodies were found within the medial preoptic area. In contrast, the preoptic region contained hundreds of PE-immunoreactive cells, which were particularly numerous within the AVPv, MPN, and anterodorsal preoptic nucleus. The AVPv and MPN also contained discretely localized plexuses of PE-stained fibers. Although the overall distributions of opioid peptide-containing fibers within the preoptic region were quite similar in male and female rats, differential distributions of fibers were found in certain nuclei such as the AVPv and MPN, and they were correlated with previously identified cytoarchitectonic sexual dimorphisms. Such differential distributions were particularly distinct for enkephalin-containing fibers. Although the AVPv is larger in female rats, it contained more PE-immunoreactive cell bodies in male rats, and we have shown here that this sexual dimorphism appears to be at least partially dependent on perinatal levels of gonadal steroids. In contrast, no difference in the number of PE-stained cells was found within the anterodorsal preoptic nucleus of male and female animals, indicating that sexual differences are not a general characteristic of enkephalinergic cells in the preoptic region of the rat.(ABSTRACT TRUNCATED AT 400 WORDS)     

Several distinct receptor binding enkephalins in olivocochlear fibers and terminals in the organ of corti

Biochemical studies centering on the use of reverse-phase high-performance liquid chromatography (HPLC) and radioimmunoassays (RIA) demonstrate the presence in the guinea pig organ of Corti of at least 3 enkephalin-related peptides, two of which are identified as Met- and Leu-enkephalin, respectively. Enkephalins were identified and quantitated by HPLC-RIA in the isolated second turn of the organ of Corti, but were not found in stria vascularis or auditory nerve dissected from the cochlea. Three enkephalin-immunoreactive HPLC fractions inhibited the binding of labeled naloxone to rat brain membranes. All enkephalins identified by the combined HPLC-RIA procedure had an apparent molecular weight similar to that of Met- and Leu-enkephalin peptide standards. Immunocytochemistry, performed with the best-characterized Met-enkephalin antiserum used in the RIAs, localized the enkephalin-like immunoreactivity to lateral efferent fibers and terminals under inner hair cells of the organ of Corti. Other antisera raised against Met-enkephalin, not used for RIA, visualized enkephalin-like immunoreactivity in medial efferent fibers under outer hair cells as well. This enkephalin-like immunoreactivity may reflect the presence in the medial efferent system of other structurally similar peptides in addition to those detected biochemisally. Efferent fiber lesion, by evulsion of the vestibular nerve close to the vestibulocochlear anastomosis in which the olivocochlear fibers run, eliminated enkephalin-like immunoreactivity and the enkephalin-related peptides identified by HPLC-RIA.     

Comparative neuroanatomy of the histaminergic system in the brain of the frog Xenopus laevis

The distribution of the histaminergic neuronal system in the brain of the clawed frog Xenopus laevis was mapped with an antiserum against carbodiimide-fixed histamine and compared to that in mammals. The histamine-immunoreactive cell bodies were located in a small area of the posterolateral hypothalamus, close to the dorsal infundibular nucleus, which contains catecholaminergic and serotonergic neurons. This area may be homologous to the tuberomammillary nucleus in mammals. A thick process extended from each cell between the ependymal cell layer and terminated in the ventricle lumen. The number of histaminergic cell bodies in adult Xenopus brain was relatively low, as compared with the mammalian brain. Preliminary analysis of adjacent sections stained with antisera against GABA or serotonin indicated that the histamine cells were not immunoreactive for these. The pathways and distribution of histaminergic fibers in Xenopus brain showed many similarities to mammals. The densest fiber networks were present in the medial basal forebrain, particularly in the medial amygdala and septum. A distinct cluster of fibers was concentrated around the cell bodies of nucleus accumbens. In most pallial areas, the density was moderate to low. In the primordial piriform cortex and the striatum, very few fibers were seen. In diencephalon, highest fiber densities were found in the anterior and ventral thalamus and posterior and lateral hypothalamus. In hindbrain, the density was highest in the medullary central gray, as in some mammals. The results suggest that the general pattern of the histaminergic system in vertebrate brain is conserved from amphibians to mammals.     

Characterization of proenkephalin B-derived opioid peptides in the human hypothalamo-neurohypophyseal axis

Proenkephalin B-derived opioid peptides, such as dynorphin1-17, dynorphin1-8, dynorphin B, alpha-neo-endorphin and beta-neo-endorphin in the human hypothalamo-neurohypophyseal tract were quantitated and characterized by the combined use of various radioimmunoassays, gel filtration, high performance liquid chromatography and enzymatic cleavage. Chromatographic analysis of immuno-reactive peptide levels determined that, in each case, these were comprised almost exclusively of the authentic peptides both in the neurohypophysis and hypothalamus. Concentrations of authentic proenkephalin B-peptides were 100-5000-fold lower in the human as compared to the rat neurohypophysis. However, in the paraventricular nucleus (PVN), supraoptic nucleus (SON) and certain other nuclei of the human hypothalamus concentrations of authentic peptides were found to be in the same range as those in the rat hypothalamus. The ratio of proenkephalin B-peptides in PVN and SON to those of the neurohypophysis in the rat was ca. 1:50. Conversely, in man these ratios were shown to be 80:1 for dynorphin B, 6:1 for alpha-neo-endorphin and 1:1 for all other peptides evaluated. Examination of postmortem degradation of peptides indicated that these lower levels in the neurohypophysis are not due to a higher rate of postmortem breakdown. Since levels of both vasopressin and beta-endorphin were very high, these deficits in proenkephalin B-peptides were selective and do not represent a generalized property of the human pituitary. Experiments involving enzymatic cleavage demonstrated the occurrence of higher molecular weight forms containing the Leu-enkephalin sequence which were not recognized by the antisera employed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of Opioid Binding Sites in the Neural and Intermediate Lobe of the Rat Pituitary Gland by Quantitative Receptor Autoradiography

Previous studies have suggested an involvement of enkephalins in regulation of oxytocin (OXT) and vasopressin (AVP) release, which seems to disagree with the very low affinities of Met- and Leu-enkephalin for the kappa opioid receptor. As opioid receptors in the neural lobe exclusively exist of kappa receptors, we studied the binding characteristics of larger pro-enkephalin derived peptides for opioid binding sites in the neural lobe by means of light microscopic receptor autoradiography. In addition, the pharmacological characteristics of opioid binding sites in the neural lobe were compared with those in other parts of the pituitary. In the neural as well as the intermediate lobe both high and low affinity ³H-bremazocine binding sites were present. Binding to these sites was completely displaceable by both naloxone and nor-binaltorphimine, suggesting that these sites represent kappa opioid receptors. Also with regard to selectivity and affinity characteristics to other ligands, opioid binding sites in the neural and intermediate lobe were quite similar. In the anterior lobe a very low level of bremazocine binding was present, which could not be displaced by nor-binaltorphimine. Displacement studies with pro-enkephalin and pro-dynorphin derived peptides showed that both groups of peptides could bind to opioid binding sites in the neural and intermediate lobe. Especially the relatively large pro-dynorphin and pro-enkephalin derived peptides, such as dynorphin 1–17 and BAM22, appeared to be very potent ligands for these opioid binding sites and were much more potent than smaller fragments, such as dynorphin 1–8, and Met- and Leu-enkephalin. These results contradict the existence of a mismatch in the neural (and intermediate) lobe with regard to the local type of opioid peptides and receptors present.     

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.     

Steady-state levels of pro-dynorphin-related end-products from the brain of the amphibian, Xenopus laevis

Steady-state analyses of prodynorphin-derived opioid peptides were conducted on acid extracts of the brain of the frog. Xenopus laevis. Radioimmunoassays specific for dynorphin A(1-17), dynorphin A(1-8), alpha-neoendorphin and dynorphin B coupled with gel filtration chromatography and reverse phase high performance liquid chromatography were used. The major prodynorphin-related end-product detected was alpha-neoendorphin. Interestingly, Leu-enkephalin was also detected. Since the Xenopus proenkephalin precursor does not contain the Leu-enkephalin sequence, these data suggest that some of the prodynorphin-related end-products had been cleaved to yield Leu-enkephalin.     

Immunocytochemical localization of the mGluR1b metabotropic glutamate receptor in the rat hypothalamus

The mGluR1 metabotropic glutamate receptor is a G-protein-coupled receptor that exists as different C-terminal splice variants. When expressed in mammalian cells, the mGluR1 splice variants exhibit diverse transduction mechanisms and also slightly differ in their apparent agonist affinities. In the present study, we used an affinity-purified antiserum, specifically reactive to the mGluR1b splice variant, in combination with a highly sensitive preembedding immunocytochemical method for light microscopy to investigate the distribution of this receptor in the rat hypothalamus. An intense immunoreactivity for mGluR1b was observed in distinct hypothalamic nuclei. Thus, neuronal cell bodies and dendrites were stained in the preoptic area, suprachiasmatic nucleus, dorsal hypothalamus, lateral hypothalamus, dorsomedial nucleus, tuberomammilary nucleus, and lateral mammilary body. The ventromedial nucleus exhibited neuropil immunostaining but neuronal cell bodies were not labeled. Strong mGluR1b immunoreactivity was observed in magnocellular neurons of the neuroendocrine supraoptic, paraventricular, and arcuate nuclei. Also, neuronal cell bodies were heavily labeled in the retrochiasmatic nucleus, anterior commissural nucleus, and periventricular nucleus. These immunocytochemical observations, together with previous studies, suggest that mGluR1b is coexpressed with other class I mGluRs in some nuclei throughout the hypothalamus. However, mGluR1b is so far the only receptor of this class strongly expressed in the supraoptic, paraventricular, and arcuate nuclei, which might have relevant implications in the physiological control of the neuroendocrine hypothalamic-pituitary system. J. Comp. Neurol. 390:225–233, 1998. © 1998 Wiley-Liss, Inc.     

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.     

Immunohistochemical localization of enkephalin in rat brain and spinal cord.

The distribution of immunoreactive enkephalin in rat brain and spinal cord was studied by immunoperoxidase staining using antiserum to leucine-enkephalin ([Leu5]-enkephalin) or methionine-enkephalin ([Met5]-enkephalin). Immunoreactive staining for both enkephalins was similarly observed in nerve fibers, terminals and cell bodies in many regions of the central nervous system. Staining of perikarya was detected in hypophysectomized rats or colchicine pretreated rats. The regions of localization for enkephalin fibers and terminals include in the forebrain: lateral septum, central nucleus of the amygdala, area CA2 of the hippocampus, certain regions of the cortex, corpus striatum, bed nucleus of the stria terminalis, hypothalamus including median eminence, thalamus and subthalamus; in the midbrain: nucleus interpeduncularis, periaqueductal gray and reticular formation; in the hind brain: nucleus parabrachialis, locus ceruleus, nuclei raphes, nucleus cochlearis, nucleus tractus solitarii, nucleus spinalis nervi trigemini, motor nuclei of certain cranial nerves, nucleus commissuralis and formatio reticularis; and in the spinal cord the substantia gelatinosa. In contrast enkephalin cell bodies appear sparsely distributed in the telencephalon, diencephalon, mesencephalon and rhombencephalon. The results of the histochemical staining show that certain structures which positively stain for enkephalin closely correspond to the distribution of opiate receptors in the brain and thus support the concept that the endogenous opiate peptides are involved in the perception of pain and analgesia. The localization of enkephalin in the preoptic-hypothalamic region together with the presence of enkephalin perikarya in the paraventricular and supraoptic nuclei suggest a role of enkephalin in the regulation of neuroendocrine functions.     

An immunocytochemical study of the olfactory projections in the three-spined stickleback, Gasterosteus aculeatus, L

The distribution of olfactory fibers in the brain of the three-spined stickleback was visualized by means of immunohistochemistry. The labeling of the olfactory fibers was produced by serum containing antibodies against somatostatin-14. Olfactory fibers were observed entering the olfactory bulbs, where they terminated in the glomerular layer or collected into fascicles that coursed through the bulbs into the telencephalon without participating in the formation of the glomerules. In the telencephalon the fascicles, which belonged to the medial olfactory tract, formed two fiber systems: ventral descending fibers and dorsal descending fibers. The ventral descending fibers could be followed through the ventral telencephalon to the vicinity of the lateral tuberal nucleus. The dorsal descending fibers coursed via the anterior commissure to the posterior part of the telencephalon. Part of the postcommissural fibers of the dorsal descending system coursed to the posterior zone of the area dorsalis telencephali while others left the telencephalon via the medial forebrain bundle and could be followed to the periventricular hypothalamus. Some axons formed synaptic contacts with unlabeled cell bodies in the nucleus of the terminal nerve which, in this species, is situated immediately behind the bulbs. In addition, an extensive terminal field associated with the dorsal descending fibers was found in the ventromedial aspects of the telencephalon. It is unlikely that the labeling represents immunoreactive somatostatin-14 because: 1) the labeling persisted after the absorption of the antiserum with synthetic somatostatin-14; 2) antiserum against somatostatin-14 from another manufacturer did not have this labeling property; and 3) the production of the absorbable labeling depended on the choice of fixative whereas the production of the unabsorbable labeling did not.     

Proopiomelanocortin peptide immunocytochemistry in rhesus monkey brain

The immunocytochemical distribution of proopiomelanocortin (POMC) peptides (beta-endorphin, ACTH, alpha-MSH, 16K fragment) was studied in the brain of the rhesus monkey (Macaca mulatta). Some animals were administered colchicine intracerebroventricularly prior to sacrifice to enhance the visualization of perikaryal immunoreactivity. Immunoreactive perikarya are localized to hypothalamic infundibular nucleus, giving rise to several distinct projections. Rostral projections extend through midline diencephalic and preoptic areas, and enter the telencephalon. Along this course, immunoreactive fibers are seen in midline hypothalamic and preoptic nuclei, nucleus of the diagonal band, olfactory tubercle, nucleus accumbens, bed nucleus of stria terminalis, septum, and other limbic structures in telencephalon. Caudal to the anterior commissure, some fibers ascend dorsally to enter the midline thalamus, which they innervate. Lateral projections of the infundibular perikarya course through the medial-basal hypothalamus, dorsal to the optic tracts, and enter the amygdala region where they innervate more medially situated amygdaloid nuclei. Caudal projections of the POMC neurons also extend through midline diencephalon, some coursing along a periventricular path to innervate midline hypothalamic and thalamic nuclei. This projection extends into the mesencephalic substantia grisea centralis and may also contribute to the innervation of more dorsally situated nuclei in the pons and medulla, such as the parabrachial nuclei and nucleus tractus solitarius. Other caudal projections originating in the hypothalamus course through the ventral tegmentum of mesencephalon and pons and may contribute to the innervation of midline raphe and other ventrally situated nuclei in the pons and medulla. The distribution of immunoreactive perikarya and fibers in the brain of rhesus monkey is strikingly similar to that found in the rat brain. However, subtle differences appear to exist in the innervation patterns of particular brain regions.     

Nicotine-induced alterations in peripheral tissue concentrations of native and cryptic Met- and Leu-enkephalin

This study examined the peripheral tissue distribution of native and cryptic Met- and Leu-enkephalin, and regulation of tissue enkephalins by nicotine. Met- and Leu-enkephalin concentrations showed widespread variation in tissue concentration and degree of processing. HPLC characterization of homogenate of spleen revealed that both native and cryptic immunoreactive Met-enkephalin are comprised of two peaks, one representing authentic Met-enkephalin pentapeptide and the other its sulfoxide. Subacute repeated administration of nicotine 0.1 mg/kg ip, six times at 30 min intervals, increased native Met- and Leu-enkephalin in adrenal medulla without affecting cryptic Met- and Leu-enkephalin concentrations, consistent with increased processing of larger peptides to Met- and Leu-enkephalin. Subacute nicotine decreased splenic concentrations of native and cryptic Met-enkephalin and native Leu-enkephalin, consistent with increased release of Met- and Leu-enkephalin from spleen and decreased synthesis of proenkephalin A or inadequate processing of larger peptides to enkephalin pentapeptides in spleen to compensate for the increased release during this period. HPLC characterization revealed that nicotine-induced decrease in native Met-enkephalin in spleen resulted from reductions in both pentapeptide and its sulfoxide. Nicotine also increased native Met-enkephalin in jejunum, decreased cryptic Met-enkephalin in heart atrium, increased native Leu-enkephalin in anterior pituitary and decreased cryptic Leu-enkephalin in jejunum. Nicotine may produce some of its effects through alterations in release of enkephalins from peripheral tissues.     

Immunocytochemical demonstration of dynorphin(PH-8P)-like immunoreactive elements in the human hypothalamus

PH-8P (dynorphin[1-8])-like immunoreactive neuronal perikarya, processes, and terminals located within the human hypothalamus were investigated by the avidin-biotin peroxidase complex (ABC) immunocytochemical procedure. Immunopositive neurons were distributed throughout the hypothalamus. The distributional pattern was found to be similar to that in other mammalian species by the use of antisera against dynorphin. A large number of immunoreactive neuronal perikarya were detected in the supraoptic nucleus (SON) and the magnocellular portion of the paraventricular nucleus (PVN). Their processes appeared to project to the posterior pituitary via the internal layer of the median eminence and their distribution seemed to be less dense than in other mammalian species. PH-8P and vasopressin were colocalized in the neuronal perikarya in the human SON unlike the colocalization of these peptides in the rat SON and PVN. There were a few immunoreactive terminals in the external layer of the median eminence; their immunoreactive substances may be released into the portal veins to act on anterior pituitary cells. In addition, PH-8P-like immunoreactive neurons in the human hypothalamus may project to the extrahypothalamic area.