Gonadotropin-releasing hormone (GnRH) neurons and pathways in the rat brain

Summary Gonadotropin-releasing hormone (GnRH) neurons and their pathways in the rat brain were localized by immunocytochemistry in 6-to 18-day-old female animals, by use of thick frozen or vibratome sections, and silver-gold intensification of the diaminobenzidine reaction product. GnRH-immunoreactive perikarya were observed in the following regions: olfactory bulb and tubercle, vertical and horizontal limbs of the diagonal band of Broca, medial septum, medial preoptic and suprachiasmatic areas, anterior and lateral hypothalamus, and different regions of the hippocampus (indusium griseum, Ammon's horn). In addition to the known GnRH-pathways (preoptico-terminal, preoptico-infundibular, periventricular), we also observed GnRH-immunopositive processes in several major tracts and areas of the brain, including the medial and cortical amygdaloid complex, stria terminalis, stria medullaris thalami, fasciculus retroflexus, medial forebrain bundle, indusium griseum, stria longitudinalis medialis and lateralis, hippocampus, periaqueductal gray of the mesencephalon, and extracerebral regions, such as the lamina cribrosa, nervus terminalis and its associated ganglia. By use of the silver-gold intensification method we present Golgi-like images of GnRH perikarya and their pathways. The possible distribution of efferents from each GnRH cell group is discussed.     

GnRH-prohormone-containing neurons in the primate brain: immunostaining for the GnRH-associated peptide

The structure of the prohormone for mammalian gonadotropin releasing hormone (proGnRH) includes the GnRH decapeptide followed by a 56 amino acid GnRH-associated peptide (GAP). In this study, we compared immunostaining of brain neurons and fibers for GAP and GnRH in fetal rhesus monkeys and juvenile baboons. We used antisera against different portions of human and rat GAP (proGnRH 14-24, proGnRH 40-53, and proGnRH 52-66) or against GnRH and the PAP technique. Liquid phase absorption with GAP or GnRH confirmed the specificity of these antisera. Major accumulations of GAP immunoreactive (GAP+) perikarya occurred in the medial septal and preoptic areas and the nucleus of the diagonal band of Broca (44.6% in rhesus, 49.6% in baboon), supraoptic region including the area dorsal to the optic tract (21.9% in rhesus, 23.0% in baboon), and the medial basal hypothalamus (15.7% in rhesus, 16.4% in baboon), especially at the infundibular lip. Occasional cell bodies were scattered throughout the hypothalamic and forebrain regions studied. GAP+ fibers were widely distributed, but formed well-defined pathways such as the periventricular and ventral hypothalamic tract. In addition, GAP+ nerve terminals with various densities occurred in the lamina terminalis, the zona externa of the infundibulum, and behind the infundibular stalk. Fetal rhesus macaques had more GAP+ cell bodies, denser fiber networks, and more distinct pathways than juvenile baboons. However, fiber and terminal immunostaining was somewhat less intense for GAP than GnRH in comparable regions. These results indicate that proGnRH (GAP) is present in the same population of neurons as GnRH in the primate brain. They also suggest that post-translational products of proGnRH are present in perikarya, axons and terminals, and that GnRH and GAP and/or further cleavage products are consecreted into hypophysial portal blood in the primate.     

Aromatase in axonal processes of early postnatal hypothalamic and limbic areas including the cingulate cortex

It has been shown that sexual dimorphic morphology of certain hypothalamic and limbic areas underlie gender-specific sexual behavior and neuroendocrine mechanisms. The key role played by locally formed estrogen in these developmental events has been revealed during a critical perinatal period. In this study, we aimed to document the presence of estrogen-synthetase (aromatase)-immunoreactive elements in the involved limbic system and hypothalamus of the developing rat brain. On postnatal day 5, animals of both sexes were perfusion-fixed, and sections from the forebrain and hypothalamus were immunolabelled for aromatase using an antiserum that was generated against a 20 amino acid sequence of placental aromatase. Aromatase-immunoreactivity was present in neuronal perikarya and axonal processes in the following limbic structures: the central and medial nuclei of the amygdala, stria terminalis, bed nucleus of the stria terminalis (BNST), lateral septum, medial septum, diagonal band of Broca, lateral habenula and all areas of the limbic (cingulate) cortex. In the hypothalamus, the most robust labelling was observed in the medial preoptic area, periventricular regions, ventromedial and arcuate nuclei. The most striking feature of the immunostaining with this antiserum was its intracellular distribution. In contrast to the heavy perikaryal labelling that can be observed with most of the currently available aromatase antisera, in the present experiments, immunoperoxidase was predominantly localized to axons and axon terminals. All the regions with fiber staining corresponded to the projection fields of neuron populations that have previously been found to express perikaryal aromatase. Our results confirm the presence of aromatase-immunoreactivity in developing limbic and hypothalamic areas. The massive expression of aromatase in axonal processes raises the possibility that estrogen formed locally by aromatase may not only regulate the growth, pathfinding and target recognition of its host neuronal processes, but may also exert paracrine actions on structures in close proximity, including the target cells.     

The organization of prolactin-like-immunoreactive neurons in the rat central nervous system

Summary The localization and distribution of prolactinlike-immunoreactive perikarya and nerve fibers in the rat central nervous system have been studied by a preembedding immunoperoxidase method using well-characterized specific immunsera to rat prolactin. Although the localization of labeled neuronal structures in a number of brain areas correlates with the data of previous immunocytochemical studies, we found prolactin-immunoreactive neurons in various regions not previously reported. In untreated animals, the highest concentrations of prolactinfibers were observed: (i) in the external layers of the median eminence where they exhibited close contact with blood vessels, and (ii) in the bed nucleus of the stria terminalis and in the central nucleus of the amygdala where they closely surrounded unlabeled perikarya. Dense networks of finely varicose prolactin fibers were also observed in the organum vasculosum of the lamina terminalis, in the subfornical organ, and in the dorsolateral regions of the medulla oblongata and the spinal cord. Lastly, a number of large, varicose, intensely immunoreactive fibers were found in the olfactory bulb, the cingulum, and the periventricular regions of the hypothalamus and central gray, whereas isolated fibers could be detected in the caudate nucleus and in the cerebral cortex. In animals treated with colchicine, prolactin-immunoreactive perikarya were essentially located within the periventricular and perifornical regions of the hypothalamus, and within the bed nucleus of the stria terminalis. Although corticotropin (ACTH 17-39)-immunoreactive fibers could be detected in several regions found to contain prolactin fibers, the distribution and organization of both fiber types clearly differed in numerous brain regions, and the regions containing the corresponding perikarya did not overlap. The ultrastructural organization of the prolactin-immunoreactive fibers revealed by electronmicroscopic immunocytochernistry in various brain regions, allowed the characterization of two main types of prolactinergic neurons including: (i) endocrine neurons, whose axons terminated in close vicinity to portal blood vessels in the external median eminence, and (ii) neurons projecting to extrahypothalamic regions, whose axons formed typical synaptic connections with unidentified neuronal units.     

LHRH-systems in the brain of the golden hamster

Summary Vibra tome sections of male hamster brains were treated immunohistochemically with LHRH antiserum, and the anatomical distribution of LHRH immunoreactive cells and nerve fibers was assessed. LHRH-cell bodies are found in the ventral hypothalamus that includes its preoptic, anterior and central parts, in the septum, the olfactory tubercle, the main and accessory olfactory bulb, and the prepiriform cortex. In addition, extracerebral LHRH-neurons and ganglia exist in LHRH-positive nerves at the ventromedial surface of the olfactory tubercle and bulb as well as in olfactory nerves. Dense networks of LHRH-immunoreactive fibers are found in all regions where LHRH-cell bodies exist. Intraseptal connections reach the organum vasculosum of the lamina terminalis, the subfornical organ, and the lateral ventricle. Dorsolateral projections from the septum can be traced via the fimbria hippocampi and alveus to the ventral hippocampus, via the stria terminalis to the amygdala and piriform cortex. Ventrolateral projections extend from the level of the olfactory tubercle and preoptic-anterior hypothalamic area via the ventral amygdalofugal pathway to the prepiriform and piriform cortex as well as the amygdala. Dorsal supracallosal projections via the stria longitudinalis are seen in the induseum griseum and the cingulate cortex. Caudal efferents reach the habenula, interpeduncular nucleus, midbrain raphe, and central gray of the rostral fourth ventricle via the stria medullaris and fasciculus retroflexus and by a ventral projection via the periventricular and subventricular hypothalamus. A major portion of this ventrocaudal projection gives rise to a dense network in the median eminence. Anatomical relationships of LHRH-fibers to certain regions of the inner ventricular and outer brain surface are noted.Postdoctoral fellow of the Deutsche ForschungsgemeinschaftSupported by US PHS grant NS09914 and NRCHD grant HD03110     

Localization of luteinizing hormone-releasing hormone in the forebrain of the pig

The distribution of luteinizing hormone-releasing hormone (LHRH)-immunostained perikarya and processes was examined in the forebrains of six sexually mature female pigs by use of indirect biotin-avidin horseradish peroxidase immunocytochemistry. Two primary antisera (Drs. Y.F. Chen and V.D. Ramirez CRR11B73 and Miles-Yeda UZ-4) yielded positive staining. Adjacent sections treated either primary antiserum preabsorbed with LHRH or with normal rabbit serum substituted for primary antiserum lacked positive staining. The greatest proportion of LHRH-immunostained perikarya were found in the medial preoptic area adjacent to the organum vasculosum of the lamina terminalis. The LHRH-immunostained perikarya were also scattered rostrally in the diagonal band of Broca, and within the lateral hypothalamic area, paraventricular nucleus, periventricular zone, suprachiasmatic nucleus, and medial basal hypothalamus. LHRH-immunostained processes, which extended from the medial preoptic area, coursed either along the ventral surface to the median eminence or medially and ventrally along the third ventricular wall ventrally to the median eminence and caudally to the level of the mammillary bodies. Extrahypothalamic processes were located adjacent to the lateral ventricular floor and the third ventricle from the lateral septal area (stria terminalis) to the level of the habenular nucleus. LHRH-immunostained neurons were unipolar, bipolar, and multipolar. Close associations between individual LHRH-immunostained neurons were observed.     

Processing of the luteinizing hormone-releasing hormone precursor in the preoptic area and hypothalamus of the rat

The LHRH precursor is known to contain the decapeptide and a 56 amino acid peptide termed gonadotropin-releasing hormone-associated peptide (GAP). The purpose of our study was to characterize the proLHRH and its processed products from the cell body and fiber region and from the nerve terminal region of LHRH neurons. The median eminence (ME) and a tissue block containing the preoptic area and hypothalamus (POH) were dissected separately. Tissues were homogenized and peptides were separated according to mol wt. Three different LHRH antisera bound to one immunoreactive (IR) substance which eluted at approximately 1200 mol wt. Subsequently, this material coeluted with synthetic LHRH on a reversed-phase column as a single peak. There was approximately 1.6-fold more LHRH-like IR in the ME than in the POH. The four different GAP antisera recognized multiple mol wt forms of GAP-like IR at approximately 16,000 to 14,000, 8,200, 6,500, 3,500, and 2,800 mol wt. There were more of the high mol wt materials and less of the 6500 and lower mol wt materials in the POH than in the ME. The most abundant species in both regions was the 6500 mol wt form. This IR substance coeluted with synthetic rat GAP1-56 on a reversed-phase column as a single peak. These experiments demonstrate 1) that multiple IR forms of the LHRH prohormone exist in the POH of the rat and 2) that nerve terminals of the LHRH neurons contain LHRH, GAP1-56, and some lower mol wt GAP-like substances. These results provide the first information concerning the processing scheme for the LHRH prohormone in the rat brain.     

Distribution of LHRH in the rat and mouse brain with special reference to the tanycytes

Summary The distribution of luteinizing hormone-releasing hormone (LHRH) was studied in the rat and mouse brain by means of light and electron microscopic immunohistochemistry using the peroxidase-antiperoxidase method. An immunoreactive product to LHRH antiserum was found near the blood vessels of the vascular organ of the lamina terminalis. In the arcuate nucleus-median eminence region, an immunoreactive material occurred bilaterally in the hypothalamic tissue around the tuberoinfundibular sulci. Electron microscopy revealed that immunoreactive fibers observed light microscopically contain numerous granules 100–130 nm in diameter. No immunoreactive product was located in the tanycytes of the median eminence, the perikarya of hypothalamic neurons, and the parenchyma of several circumventricular organs (subfornical organ, subcommissural organ, pineal organ, area postrema).Supported by grants from the Ministry of Education of Japan and the Ford Foundation     

Localization of neurokinin B in the central nervous system of the rat.

The distribution of neurokinin B (NKB) was determined by immunocytochemistry with antisera directed toward its amino terminus. Immunoreactive perikarya were detected in the main and accessory olfactory bulbs, cortical regions, the olfactory tubercle, the bed nucleus of the stria terminalis, the diagonal band of Broca, the nucleus accumbens, the septum, the neostriatum, several hypothalamic nuclei, the superior colliculus, the central gray, the substantia nigra, the medullary reticular formation, and the external cuneate nucleus. The distribution of NKB-containing perikarya revealed by immunocytochemistry was similar to the distribution of protachykinin B-containing cells previously visualized by in situ hybridization. Immunoreactive nerve fibers and terminals were detected in all major subdivisions of the brain. The levels of NKB measured by radioimmunoassay were highest in the hypothalamus. The distribution of NKB in the rat brain was similar to the distribution of substance P; however, there were several regions where the two distributions were clearly different.     

Immunocytochemical localization of corticotropin-releasing factor (CRF) in the rat brain

The immunocytochemical localization of neurons containing the 41 amino acid peptide corticotropin-releasing factor (CRF) in the rat brain is described. The detection of CRF-like immunoreactivity in neurons was facilitated by colchicine pretreatment of the rats and by silver intensification of the diaminobenzidine end-product. The presence of immunoreactive CRF in perikarya, neuronal processes, and terminals in all major subdivisions of the rat brain is demonstrated. Aggregates of CRF-immunoreactive perikarya are found in the paraventricular, supraoptic, medial and periventricular preoptic, and premammillary nuclei of the hypothalamus, the bed nuclei of the stria terminalis and of the anterior commissure, the medial septal nucleus, the nucleus accumbens, the central amygdaloid nucleus, the olfactory bulb, the locus ceruleus, the parabrachial nucleus, the superior and inferior colliculus, and the medial vestibular nucleus. A few scattered perikarya with CRF-like immunoreactivity are present along the paraventriculo-infundibular pathway, in the anterior hypothalamus, the cerebral cortex, the hippocampus, and the periaqueductal gray of the mesencephalon and pons. Processes with CRF-like immunoreactivity are present in all of the above areas as well as in the cerebellum. The densest accumulation of CRF-immunoreactive terminals is seen in the external zone of the median eminence, with some immunoreactive CRF also present in the internal zone. The widespread but selective distribution of neurons containing CRF-like immunoreactivity supports the neuroendocrine role of this peptide and suggests that CRF, similarly to other neuropeptides, may also function as a neuromodulator throughout the brain.     

LHRH in the human hypothalamus. Immunohistochemical mapping in normal newborn infants or in sudden death infants

The topographical distribution of neurons containing LHRH has been investigated in newborn hypothalamus using the peroxidase anti-peroxidase technique. In control subjects, LHRH immunoreactive (LHRH-IR) perikarya have been mainly observed essentially in the infundibular nucleus. The preoptic region displayed a moderate density of LHRH-IR cell bodies. High LHRH innervation was observed in the anterior hypothalamus in the lamina terminalis and in the mediobasal hypothalamus in the median eminence, and in the peri- and paraventricular regions. In sudden death infant syndrome, a comparable mapping was observed, except a low density in the mediobasal peri- and paraventricular areas.     

Immunoreactive pancreatic polypeptide (PP) occurs in the central and peripheral nervous system: Preliminary immunocytochemical observations

Summary Pancreatic polypeptide (PP) is a candidate hormone of unknown physiological significance. It is produced by a population of endocrine cells in the pancreas. In the present study a PP-like peptide was found to occur in the mammalian and avian central and peripheral nervous systems. Immunoreactive nerve fibres and nerve cell bodies were widely distributed in the brain. Dense accumulations of nerve fibres occurred in the following areas: nucleus accumbens, interstitial nucleus of the stria terminalis, para- and periventricular hypothalamic nuclei, and medial preoptic area. In addition, nerve fibres were regularly seen in cortical areas. Immunoreactive perikarya were observed in the following regions: cortex, nucleus accumbens, neostriatum and septum. In the gut, immunoreactive nerve fibers were distributed in the myenteric plexus, in smooth muscle, around blood vessels, and in the core of the villi. Immunoreactive perikarya occurred in the submucosal and myenteric plexus, suggesting that PP immunoreactive nerves are intrinsic to the gut.In the species examined, the neuronal PP-like peptide could be demonstrated with an antiserum raised against avian PP, but not with those raised against bovine or human PP. Thus, neuronal PP is distinct from the PP that occurs in pancreatic endocrine cells.     

Biosynthesis of LHRH: Inferences from immunocytochemical studies

Inferences regarding biosynthesis of LHRH in rats are made from immunocytochemical studies using LHRH antisera with varied and specific binding requirements. Immunoreactive perikarya were observed with antisera that could bind putative large molecular weight precursors of LHRH. No cells were detected with an antiserum that requires free decapeptide terminals and could not bind extended precursors. No such differential immunoreactivity was apparent in neuronal processes and neurovascular terminals. Features of intracellular processing of LHRH which can be inferred from these immunocytochemical data are: (1) the decapeptide is initially synthesized within neuronal cell bodies as a larger molecular weight peptide, extended at both the N- and C-terminals; (2) processing occurs as the newly synthesized material is transported along neuronal processes; and (3) intermediate molecular forms are converted to the active decapeptide primarily in distal portions of neuronal fibers, including the neurovascular terminal. Immunocytochemical observations in other mammalian species (humans, monkeys, ferrets and bats) allow us to further suggest that the dynamics of maturation of this hormone may differ among mammals.     

Immunocytochemical demonstration of GAP-like immunoreactive neuronal elements in the human hypothalamus and pituitary

GnRH-associated peptide (GAP)-like immunonreactive elements located in the human hypothalamus were investigated by PAP immunocytochemistry using specific antiserum against [pro-GnRH (14-69) OH]. Immunoreactive neuronal perikarya were distributed in the MPOA, PVN and infundibular nucleus, with the largest numbers of GAP-like immunoreactive perikarya found in the infundibular nucleus. We also detected the coexistence of GAP-like and GnRH-like immunoreactivities in the same neuronal perikarya in the MPOA by using a double immunolabelling procedure. In addition to the above regions immunoreactive neuronal perikarya were present in the region dorsal to the medial mammillary nucleus. GAP-like immunoreactive fibers were distributed in same areas that immunoreactive perikarya were observed. Many immunoreactive terminals were found adjacent to capillaries in the infundibulum. Immunoreactive dots, presumably terminals, were observed in the posterior pituitary and these were particularly evident along the margin adjacent to the anterior pituitary. The distribution pattern and density of GAP-like immunoreactive neuronal elements are compared with those of other mammalian species. We also compared GAP-like immunoreactive elements with that of GnRH as has been previously observed in the human hypothalamus.     

A comparison of neuropeptide immunocytochemistry in fluid-fixed and freeze-dried brains

Summary Immunocytochemical staining of luteinizing hormone-releasing hormone (LHRH), somatostatin, and neurophysin was compared in rat brains fixed with 1) formalin, 2) Bouin's solution, 3) freeze-dried (FD), or 4) freeze-dried + paraformaldehyde vapor perfused (FDV). The distribution of LHRH fibers was similar in all preparations; however, beads of granular reaction product often appeared finer and more numerous in the median eminence of FD- and FDV brains. Positively stained LHRH perikarya were not observed in any of the preparations. In contrast, somatostatin-immunoreactive perikarya were present in the fluid-fixed and FD brains, although few were observed in FDV brains. Somatostatin-immunoreactive fibers were present in all preparations, but appeared most numerous in the median eminence of FD brains. Staining of neurophysin-containing perikarya and fibers was similar in all preparations. These observations suggest that the FD brain can provide a suitable tissue substrate for immunocytochemistry, demonstrating staining comparable to or surpassing that of more conventional preparations. However, staining of antigens in FD brain was not uniformly successful and may depend on stereochemical characteristics of each antigen as well as properties of the primary antisera used in the staining procedure.     

Localization of immunoreactive lamprey gonadotropin-releasing hormone in the rat brain

A highly specific antiserum against lamprey gonadotropin-releasing hormone (GnRH) was used to localize 1-GnRH in areas of the rat brain associated with reproductive function. Immunoreactive 1-GnRH-like neurons were observed in the ventromedial preoptic area (POA), the region of the diagonal band of Broca and the organum vasculosum lamina terminalis, with fiber projections to the rostral wall of the third ventricle and the organum vasculosum lamina terminalis. Another population of 1-GnRH-like neurons was localized in the dorsomedial and lateral POA, with nerve fibers projecting caudally and ventrally to terminate in the external layer of the median eminence. Other fibers apparently projected caudally and circumventrically to terminate around the cerebral aqueduct in the mid-brain central gray. By using a highly specific antiserum directed against mammalian luteinizing hormone-releasing hormone (m-LHRH), the localization of the LHRH neuronal system was compared to that of the 1-GnRH system. There were no LHRH neurons in the dorsomedial or the lateral region of the POA that contained the 1-GnRH neurons. As expected, there was a large population of LHRH neurons in the ventromedial POA associated with the diagonal band of Broca and organum vasculosum lamina terminalis. In both of these regions, there were many more LHRH neurons than 1-GnRH neurons and the LHRH neurons extended more dorsally and laterally than the 1-GnRH neurons. The LHRH neurons seemed to project to the median eminence in the same areas as those that were innervated by the 1-GnRH neurons. Absorption studies indicated that 1-GnRH cell bodies were eliminated by adding 1 microg of either 1-GnRH-I or 1-GnRH-III, but not m-LHRH to the antiserum before use. Fibers were largely eliminated by the addition of 1 microg 1-GnRH-III to the antiserum. No chicken GnRH-II neurons or nerve fibers could be visualized by immunostaining. Because the antiserum recognized GnRH-I and GnRH-III equally, we have visualized an 1-GnRH system in rat brain. The results are consistent with the presence of either one or both of these peptides within the rat hypothalamus. Because 1-GnRH-I has only weak nonselective gonadotropin-releasing activity, whereas 1-GnRH-III is a highly selective releaser of follicle-stimulating hormone, and because 1-GnRH neurons are located in areas known to control follicle-stimulating hormone release selectively, our results support the hypothesis that 1-GnRH-III, or a closely related peptide, may be mammalian follicle-stimulating hormone-releasing factor.     

Localization of avian LHRH-immunoreactive neurons in the hypothalamus of the domestic fowl,Gallus domesticus,and the Japanese quail,Coturnix coturnix

The localization of LHRH-containing perikarya and nerve fibers in the hypothalami of the domestic fowl and Japanese quail was investigated by means of the specific immunoperoxidase ABC method, using antisera against chicken LHRH-I ([Gln8]-LHRH), chicken GnRH-II ([His5-Trp7-Tyr8]-LHRH[2-10]) and mammalian LHRH ([Arg8]-LHRH). Chicken LHRH-I-immunoreactive perikarya were sparsely scattered in the nucleus preopticus periventricularis (POP), nucleus filiformis (FIL) and nucleus septalis medialis (SM), and in bilateral bands extending from these nuclei into the septal area in both species. A few reactive perikarya were also observed in the nucleus accumbens (Ac) and lobus parolfactorius (LPO). Numerous cLHRH-I-immunoreactive fibers were widely scattered in the preoptic, septal and tuberal areas, and were densely concentrated in the external layer of the median eminence and in organum vasculosum of the lamina terminalis (OVLT) in both species. Anti-mammalian LHRH serum cross-reacted weakly with perikarya and fibers immunoreactive to anti-cLHRH-I serum in normal chicken and quail. Anti-cGnRH-II[2-10] serum immunoreacted with magnocellular neurons distributed in the rostral end of the mesencephalon along the midline close to the nervus oculomotorius (N III). These perikarya were apparently different from cLHRH-I immunoreactive neurons. No immunoreactive cells and fibers against anti-cGnRH-II[2-10] were observed in the hypothalamus and median eminence of the chicken or quail. Anti-cGnRH-II[2-10] bound specifically with cGnRH-II.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunohistochemical localisation of natriuretic peptides in the brains and hearts of the spiny dogfishSqualus acanthias and the Atlantic hagfishMyxine glutinosa

Summary The avidin-biotin peroxidase technique was used to determine the distribution of natriuretic peptides in the hearts and brains of the dogfishSqualus acanthias and the Atlantic hagfishMyxine glutinosa. Three antisera were used: one raised against porcine brain natriuretic peptide which cross-reacts with atrial natriuretic and C-type natriuretic peptides (termed natriuretic peptide-like immunoreactivity); the second raised against porcine brain natriuretic peptide which cross-reacts with C-type natriuretic peptide, but not with atrial natriuretic peptide (termed porcine brain natriuretic peptide-like immunoreactivity); and the third raised against rat atrial natriuretic peptide (termed rat atrial natriuretic peptide-like immunoreactivity). Only natriuretic peptide-like immunoreactivity was observed in the heart ofS. acanthias which was most likely due to the antiserum cross-reacting with C-type natriuretic peptide. No immunoreactivity was found in theM. glutinosa heart. In the brain ofS. acanthias, natriuretic peptide-like immunoreactive fibres were located in many areas of the telencephalon, diencephalon, mesencephalon, rhombencephalon, and spinal cord. Extensive immunoreactivity was observed in the hypothalamo-hypophyseal tract and the neurointermediate lobe of the hypophysis. Natriuretic peptide-like immunoreactive perikarya were found in ventromedial regions of the telencephalon and in the nucleus preopticus. Most perikarya had short, thick processes which extended toward the ventricle. Another group of perikarya was observed in the rhombencephalon. Porcine brain natriuretic peptide-like immunoreactive fibres were observed in the telencephalon, diencephalon, mesencephalon, and rhombencephalon, but perikarya were only present in the preoptic area. In theM. glutinosa brain, natriuretic peptide-like immunoreactive fibres were present in all regions. Immunoreactive perikarya were observed in the pallium, primordium hippocampi, pars ventralis thalami, pars dorsalis thalami, nucleus diffusus hypothalami, nucleus profundus, nucleus tuberculi posterioris, and nucleus ventralis tegmenti. Procine brain natriuretic peptide-like immunoreactive perikarya and fibres had a similar, but less abundant distribution than natriuretic peptide-like immunoreactive structures. Although the chemical structures of natriuretic peptides in the brains of dogfish and hagfish are unknown, these observations show that a component of the natriuretic peptide complement is similar to porcine brain natriuretic peptide or porcine C-type natriuretic peptide. The presence of natriuretic peptides in the brain suggest they could be important neuromodulators and/or neurotransmitters. Furthermore, there appears to be divergence in the structural forms of natriuretic peptides in the hearts and brains of dogfish and hagfish.     

Immunocytochemical demonstration of GAP-like immunoreactive neuronal elements in the human hypothalamus and pituitary

Summary GnRH-associated peptide (GAP)-like immunoreactive elements located in the human hypothalamus were investigated by PAP immunocytochemistry using specific antiserum against [pro-GnRH (14–69) OH]. Immunoreactive neuronal perikarya were distributed in the MPOA, PVN and infundibular nucleus, with the largest numbers of GAP-like immunoreactive perikarya found in the infundibular nucleus. We also detected the coexistence of GAP-like and GnRH-like immunoreactivities in the same neuronal perikarya in the MPOA by using a double immunolabelling procedure. In addition to the above regions immunoreactive neuronal perikarya were present in the region dorsal to the medial mammillary nucleus. GAP-like immunoreactive fibers were distributed in same areas that immunoreactive perikarya were observed. Many immunoreactive terminals were found adjacent to capillaries in the infundibulum. Immunoreactive dots, presumably terminals, were observed in the posterior pituitary and these were particularly evident along the margin adjacent to the anterior pituitary. The distribution pattern and density of GAP-like immunoreactive neuronal elements are compared with those of other mammalian species. We also compared GAP-like immunoreactive elements with that of GnRH as has been previously observed in the human hypothalamus.     

Localization of proopiomelanocortin (POMC) immunoreactive neurons in the forebrain of the pig

The distribution of proopiomelanocortin (POMC)-immunoreactive neurons was examined in the forebrains of nine sexually mature female pigs by indirect biotin-avidin horseradish peroxidase immunocytochemistry. Primary antiserum against ovine beta-endorphin (Bioflex #BF-EP-3-1) yielded positive staining of neuronal perikarya and processes. Adjacent control sections treated either with primary antiserum preabsorbed with beta-endorphin or substituted with normal rabbit serum lacked specific staining. POMC-immunoreactive cells were located in the anterior and intermediate lobe of the pituitary gland. POMC-immunoreactive perikarya were located in the arcuate nucleus and periarcuate area. The pituitary stalk/median eminence contained sparsely distributed POMC-immunoreactive fibers, which were confined to the zona interna. POMC-immunoreactive fibers were located in the arcuate nucleus and extended rostrally from the arcuate nucleus into the telencephalon coursing adjacent to the wall of the third ventricle as well as through the anterior hypothalamus, suprachiasmatic, supraoptic nuclei and preoptic areas to the nucleus accumbens, diagonal band of Broca, olfactory tubercle, bed nucleus of the stria terminalis and the ventro-lateral aspect of the septum. Caudal projections extended along the wall of the third ventricle to the level of the mammillary bodies and also coursed dorsally, passing through the periventricular, paraventricular, and dorsal medial nuclei of the hypothalamus to the midline thalamic nuclei and habenular nucleus. Lateral projections extended from the arcuate nucleus along the dorsal aspect of the optic tract and terminated in the amygdaloid complex. The distribution of POMC-immunoreactive perikarya and fibers is similar to that of the luteinizing hormone-releasing hormone (LHRH) fiber network. Therefore the opportunities exist, anatomically, for interactions between the POMC and the LHRH systems.