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.     

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.     

Neurotensin-like immunoreactivity in the diencephalon of the adult male cat

Using an indirect immunoperoxidase technique, the location of neurotensin-like fibers and cell bodies was studied in the diencephalon of the cat. The findings showed that the hypothalamus is richer in neurotensin-like-immunoreactive structures than the thalamus, and that neurotensin-like-immunoreactive structures are more widely distributed in the hypothalamus than in the thalamus. A high density of immunoreactive fibers was observed in the hypothalamic regions, area hypothalamica dorsalis, hypothalamus posterior, nucleus (n.) filiformis and n. arcuatus, whereas a moderate density was found in the n. parafascicularis, n. paraventricularis anterior, hypothalamus lateralis, median eminence and n. paraventricularis hypothalami. Other diencephalic regions such as n. lateralis posterior, n. lateralis dorsalis, n. medialis dorsalis, n. habenularis lateralis, n. centrum medianum, n. rhomboidens, n. reuniens, hypothalamus anterior, n. supra chiasmaticus, hypothalamus ventromedialis, n. supraopticus and hypothalamus dorsomedialis had the lowest density of immunoreactive fibers. In addition, the densest clusters of neurotensin-like perikarya were found in the n. arcuatus, n. centralis medialis and hypothalamus posterior, whereas the n. medialis dorsalis, n. paraventricularis anterior, n. reuniens, hypothalamus lateralis and hypothalamus ventromedialis had the lowest density. In the n. lateralis dorsalis, n. supraopticus, area hypothalamica dorsalis and n. supra chiasmaticus the density of immunoreactive perikarya was moderate.     

Galanin-like immunoreactivity in the chicken brain

The anatomical distribution of neurons containing galanin has been studied in the central nervous system of the chicken by means of immunocytochemistry using antisera against rat galanin. Major populations of immunostained perikarya were detected in several brain areas. The majority of galanin-immunoreactive cell bodies was present in the hypothalamus and in the caudal brainstem. Extensive groups of labeled perikarya were found in the paraventricular, periventricular, dorsomedial and tuberal hypothalamic nuclei, and in the nucleus of the solitary tract in the medulla oblongata. In the telencephalon, immunoreactive perikarya were observed in the preoptic area, in the lateral septal nucleus and in the hippocampus. The mesencephalon contained only a few galanin-positive perikarya located in the interpeduncular nucleus. Immunoreactive nerve fibers of varying density were detected in all subdivisions of the brain. Dense accumulations of galanin-positive fibers were seen in the preoptic area, periventricular region of the diencephalon, the ventral hypothalamus, the median eminence, the central gray of the brainstem, and the dorsomedial caudal medulla. The distributional pattern of galanin-immunoreactive neurons suggests a possible involvement of a galanin-like peptide in several neuroregulatory mechanisms.     

Immunofluorescence of somatostatin-producing sites in the hypothalamus of the tadpole,Alytes obstetricans Laur.

Summary Three sites of somatostatin-synthesizing perikarya, or a related antigen, were determined by immunofluorescence in the hypothalamus of the tadpole, Alytes obstetricans (Amphibia, Anura). Two sites of neurosecretory perikarya were localized in the preoptic nuclei of the anterior hypothalamus; the axons extended either to the anterior diencephalon or to the median eminence and the pituitary. The third site was found in the posterior hypothalamus. These neurosecretory cells showed a strong immunofluorescent reaction; their axons all terminated at the level of the median eminence. Somatostatin cells were only found in intact or hypophysectomized tadpoles given somatotropin (STH). The strong reaction observed in hypophysectomized tadpoles was possibly due to the loss of the terminal portion of the neurosecretory pathway (median eminence and pituitary) by which the agent is transported to the site of discharge.     

Distribution of NT-IR perikarya in the brain of the guinea pig with special reference to cardiovascular centers in the medulla oblongata

The occurrence and distribution of neurotensin-immunoreactive (NT-IR) perikarya was studied in the central nervous system of the guinea pig using a newly raised antibody (KN 1). Numerous NT-IR perikarya were found in the nuclei amygdaloidei, nuclei septi interventriculare, hypothalamus, nucleus parafascicularis thalami, substantia grisea centralis mesencephali, ventral medulla oblongata, nucleus solitarius and spinal cord. The distribution of NT-IR perikarya was similar to that previously described in the rat and monkey. In the gyrus cinguli, hippocampus and nucleus olfactorius, though, no NT-IR neurons were detected in this investigation. Additional immunoreactive perikarya, however, were observed in areas of the ventral medulla oblongata, namely in the nucleus paragigantocellularis, nucleus retrofacialis and nucleus raphe obscurus. The relevance of the NT-IR perikarya within the ventral medulla oblongata is discussed with respect to other neuropeptides, which are found in this area, and to cardiovascular regulation.     

Distribution of aromatase-immunoreactive cells in the mouse forebrain

Summary The distribution of aromatase-immunoreactive cells was studied by immunocytochemistry in the mouse forebrain using a purified polyclonal antibody raised against human placental aromatase. Labeled perikarya were found in the dorso-lateral parts of the medial and tuberal hypothalamus. Positive cells filled an area extending between the subincertal nucleus in the dorsal part, the ventromedial hypothalamic nucleus in the ventral part, and the internal capsule and the magnocellular nucleus of the lateral hypothalamus in the lateral part. The same distribution was seen in the two strains of mice that were studied (Jackson and Swiss), and the number of immunoreactive perikarya did not seem to be affected by castration or testosterone treatment. No immunoreactivity could be detected in the medial regions of the preoptic area and hypothalamus; these were expected to contain the enzyme based on assays of aromatase activity performed in rats and on indirect autoradiographic evidence in mice. Our data raise questions concerning the distribution of aromatase in the brain and the mode of action of the centrally produced estrogens.     

The somatostatin system of the brain and hibernation in the European hamster (Cricetus cricetus)

The depression of physiological processes characteristic of mammalian hibernation is precisely regulated by the central nervous system, especially by the neuropeptidergic apparatus of the hypothalamus. Because of inhibitory influences on neuronal circuits within the brain and suppressive effects on the metabolism via the endocrine axis, somatostatin has been implicated in the regulation of hibernation. The somatostatin system of the brain was investigated with immunocytochemistry, in situ hybridization, and radioimmunoassays in euthermic summer, euthermic winter, and hibernating European hamsters (Cricetus cricetus). Numerous somatostatin-immunoreactive perikarya were observed in the periventricular hypothalamic nucleus. The striatum, amygdala, and cortex contained only scattered immunoreactive perikarya. These entities also contained immunoreactive fiber profiles, although the highest density of immunoreactive fibers was found in the median eminence. Immunocytochemistry and radioimmunoassays showed that the number of somatostatin-immunoreactive perikarya and fibers and the content of somatostatin in the hypothalamus and the median eminence was conspicuously lower in euthermic winter animals than in euthermic summer animals. This decrease was more pronounced in hibernating specimens. In situ hybridization also demonstrated a decrease in the expression and synthesis rate of somatostatin in euthermic winter animals; again, this was even more dramatic in hibernating hamsters. These changes were less pronounced or non-significant in the extrahypothalamic somatostatin-immunoreactive perikarya and fiber systems, as shown by immunocytochemistry and radioimmunoassay, respectively.     

Gonadotropin-releasing hormone neurons and pathways in the brain of the female mink (Mustela vison)

Summary The distribution of gonadotropin-releasing hormone-immunoreactive neurons and processes was mapped in the female mink brain using coronal, horizontal and sagittal sections. Perikarya were found along a ventral continuum including the olfactory tubercle, the diagonal band of Broca, the lateral septum, the preoptic and anterior hypothalamic area and the mediobasal hypothalamus; 80% of the perikarya were counted in the mediobasal hypothalamus. Fibres were mainly observed in the organum vasculosum of the lamina terminalis and the median eminence. A few processes terminated in the ependymal cells lining the third and lateral ventricles. The total number of immunoreactive perikarya was the highest in the brains of females sacrificed in July; it then significantly decreased until December. This variation is discussed in relation to the annual breeding cycle.     

Development of melanin-concentrating hormone-immunoreactive elements in the brain of gilthead seabream (Sparus auratus)

The development of the hypothalamic melanin-concentrating hormone (MCH) system of the teleost Sparus auratus has been studied by immunocytochemistry using an anti-salmon MCH serum. Immunoreactive perikarya and fibers are found in embryos, larvae, and juvenile specimens. In juveniles, most labeled neurons are present in the nucleus lateralis tuberis; some are dispersed in the nucleus recessus lateralis and nucleus periventricularis posterior. From the nucleus lateralis tuberis, MCH neurons project a conspicuous tract of fibers to the ventral hypothalamus; this penetrates the pituitary stalk and reaches the neurohypophysis. Most fibers end close to the cells of the pars intermedia, and some reach the adenohypophysial rostral pars distalis. Immunoreactive fibers can also be seen in extrahypophysial localizations, such as the preoptic region and the nucleus sacci vasculosi. In embryos, MCH-immunoreactive neurons first appear at 36 h post-fertilization in the ventrolateral margin of the developing hypothalamus. In larvae, at 4 days post-hatching, perikarya can be observed in the ventrolateral border of the hypothalamus and in the mid-hypothalamus, near the ventricle. At 26 days post-hatching, MCH perikarya are restricted to the nucleus lateralis tuberis. The neurohypophysis possesses MCH-immunoreactive fibers from the second day post-hatching. The results indicate that MCH plays a role in larval development with respect to skin melanophores and cells that secrete melanocyte-stimulating hormone. Received: 4 April 1995 / Accepted: 17 July 1995     

Immunocytochemical studies of vasotocin and mesotocin in the hypothalamo-hypophysial system of the chicken

Summary The hypothalamo-hypophysial system of the adult chicken has been studied with a monoclonal antibody that cross-reacts with arginine vasotocin and mesotocin. We have used this antibody on thick (100 m) sections in conjunction with a peroxidase-conjugated rabbit antimouse antibody that permits the visualization not only of entire perikarya, but also of long portions of their axons and dendrites. Our results confirm older concepts based on classical methods, but the more sensitive immunocytochemical method reveals that the system is more extensive than previously recognized. Immunostained neurons in the chicken are widely scattered in the hypothalamus. In the rostral preoptic region, there are three immunostained neuronal cell groups: 1) a prominent closely packed group that extends along the ventromedial surface, 2) a diffusely distributed lateral group, and 3) an external group that surrounds the lateral aspect of the septomesencephalic tract. Caudally in the preoptic area and in the anterior hypothalamus, the same groups are present; but there are also conspicuous periventricular perikarya. Many of them have processes that project to the lumen of the third ventricle, as well as parallel axons that arch lateroventrally in the hypothalamus. In the midhypothalamic area, the periventricular perikarya and processes are particularly numerous at the level of the palliai commissure. The dorsal periventricular group located at the level of the dorsomedial anterior nucleus of the thalamus are the most caudal perikarya. They extend laterally in a wing-like formation. The immunostained axons from all of these perikarya form a compact hypothalamo-hypophysial tract as they run from the midhypothalamus to the median eminence and converge beneath the third ventricle. Axons branching from this tract innervate the zone externa of the anterior median eminence; another group of axons running in the fibrous layer of the zona interna proceeds to the neural lobe.     

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.     

The location of LH-RH neurons in the rat hypothalamus and their pathways to the median eminence

Summary The location of the perikarya of LH-RH neurons in the rat hypothalamus and their pathways to the median eminence were studied by immunohistochemistry and radioimmunoassay after placing stereotaxic electrolytic lesions in several parts of the hypothalamus. The principal location of the cell somata was found to be in the ventral part of the medial preoptic area; their pathways were classified into a main baso-lateral pathway and an accessory descending pathway branching off from the former. The main pathway was found to cross in the vicinity of the corresponding neuronal perikarya. The central median eminence and the dorsal and ventral walls of the tubero-infundibular sulcus of the caudal part of the median eminence are innervated mainly by the baso-lateral pathway. On the other hand, the rostral and most caudal portions of the median eminence are innervated principally by the descending pathway and have a subsidiary dual innervation. The projection of LH-RH neurons to the OVLT is believed to originate from perikarya adjacent to this circumventricular organ.This work was supported in part by a grant (No. 248093, 321426) from the Ministry of Education, Science and Culture, Japan     

Immunocytochemical localization of CRF in the ovine hypothalamus

A population of neuronal cell bodies and their fiber pathways have been elucidated within the ovine hypothalamus. The immunoreactive neurons were located in the anterior and dorsal hypothalamus interspersed throughout the paraventricular nucleus. These perikarya were only observed when an antiserum that was generated against the C-terminal of CRF was employed. A dense fiber projection traversed the medial-basal hypothalamus and ended within the palisade-contact zone of the median eminence and neural stem. Fibers were revealed by antisera generated against both the N-terminal and the C-terminal of CRF. Antisera pre-absorbed with synthetic CRF failed to yield immunoreactivity.     

The medial basal hypothalamus and luteinizing hormone release in the rat: Where are the LH-RH neurons responsible for tonic gonadotropin secretion?

Careful review of the literature demonstrates conflicting results concerning the ability of the deafferented medial basal hypothalamus to support gonadotropin release in the rat and thus one may question the existence of LH-RH neurons in the medial basal hypothalamus. The direct search for the LH-RH perikarya in the rat hypothalamus has not settled the question of whether these releasing hormone neurons are located in the medial basal hypothalamus. Most investigators do agree that following complete hypothalamic deafferentation there is a reduction of the immunoassayable LH-RH in the medial basal hypothalamus; however, these results do not necessarily prove that LH-RH originates outside the hypothalamus. It is argued that the completely deafferented medial basal hypothalamus may be so altered by the deafferentation procedure that it may be inadequate as a means to study neuroendocrine function.     

Distribution of neuropeptide Y-like immunoreactivity in the brain and hypophysis of the cloudy dogfish,Scyliorhinus torazame

Summary Using a specific antiserum raised against synthetic neuropeptide Y, we examined the localization of immunoreactivity in the brain and hypophysis of the cloudy dogfish, Scyliorhinus torazame, by the peroxidase-antiperoxidase method. Immunoreactive perikarya were demonstrated in the ganglion of the nervus terminalis, the dorsocaudal portions of the pallium dorsale, the basal telencephalon, and the nucleus lateralis tuberis and the nucleus lobi lateralis in the hypothalamus. Labeled perikarya were also found in the tegmentum mesencephali, the corpus cerebelli, and the medulla oblongata. Some of the immunoreactive neurons in the hypothalamus were of the CSF-contacting type. The bulk of the labeled fibers in the nervus terminalis ran toward the basal telencephalon, showing radial projections and ramifications. Large numbers of these fibers coursed into the nucleus septi caudoventralis and the nucleus interstitialis commissurae anterioris, where they became varicose and occasionally formed fine networks or invested immunonegative perikarya. In the diencephalon, immunoreactive fibers were observed throughout the hypothalamus, e.g., in the pars neurointermedia of the hypophysis, the subependymal layer of the lobus inferior hypothalami, and in the neuropil of the posterior (mammillary) recess organ. Labeled fibers were scattered throughout the rest of the brain stem and were also seen in the granular layer of the cerebellum. These results suggest that, in the dogfish brain, neuropeptide Y or a related substance is involved in a variety of physiological processes in the brain, including the neuroendocrine control of the hypophysis.     

Immunocytochemical evidence for anti-LHRH and anti-ACTH activity in the "F" antiserum.

An antiserum which has previously been thought to be specific for LHRH-like immunoreactive material was shown to contain two populations of antibodies, one demonstrating anti-LHRH activity and the other anti-ACTH(1-24) activity. In rat and mouse, ACTH(1-24)-like immunoreactive substance is present in perikarya within the basal hypothalamus and in fibers in arcuate, periventricular and dorsomedial nuclei. LHRH-like immunoreactivity is present in fibers within the median eminence and arcuate nucleus, in a few fibers running along the ventral border of the hypothalamus, and in a small number of cell bodies within the medial basal hypothalamus.     

Cyto-immunological study of the somatostatin hypothalamic peptidergic system in the fox (Vulpes vulpes)]

Immunocytological investigations on the hypothalamus of the red fox with rabbit anti-SRIF showed large or small perikarya in the supra-optical and paraventricular nuclei. In the median eminence SRIF-positive nerve fibres and their endings have a characteristic distribution in areas different from those of LH-RH containing nerve fibres.     

Ultrastructural localization of the receptor for leptin in the rat hypothalamus

Ultrastructural localization of the leptin receptor in the rat hypothalamus was studied by immunocytochemistry. The antiserum against the leptin receptor which was used specifically recognized the carboxy terminal of the cytoplasmic domain. Intense leptin receptor immunoreactivity was detected in the arcuate, paraventricular, and ventromedial nuclei of the hypothalamus and in the lateral hypothalamic area. At the ultrastructural level, leptin receptor-like immunoreactivity appeared to be concentrated predominantly in perikarya and dendrites of these areas and strong immunolabeling for the leptin receptor was detected in the plasma membrane, rough endoplasmic reticulum, Golgi apparatus, and cytoplasmic matrix. This study provides the first detailed fine structure of leptin receptor-immunoreactive neurons in the rat hypothalamus. It may help to provide better understanding of the functions of leptin in the rat hypothalamus.