Immunocytochemical localization of somatostatin-containing neurons in the rat hypothalamus

Summary The rat hypothalamus was studied at the light microscopic level with the use of single and double immunocytochemical staining methods. It was shown that the rat supraoptic and paraventricular hypothalamic nuclei, and their accessory neurosecretory nuclei, do not contain magnocellular somatostatin neurons. The distribution of the hypothalamic parvocellular somatostatin cells is described. The parvocellular component of the rat hypothalamic paraventricular nucleus is, at least partly, composed of somatostatin cells: they form a fairly well circumscribed periventricular cell mass. The rat suprachiasmatic nuclei contain separate somatostatin neurons and vasopressin neurons. Scattered somatostatin cells are present in the entire arcuate nucleus. In addition to the periventricular somatostatin cells located in the preopticanterior hypothalamic area and in the arcuate nucleus, the rat hypothalamus also contains numerous scattered somatostatin cells located distant from the third ventricle.This investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

Gene expression and chemical diversity in hypothalamic neurosecretory neurons

Hypothalamic neurosecretory neurons transcribe, translate, store, and secrete a large number of chemical messengers. The neurons contain hypothalamic signal substances that regulate the secretion of anterior pituitary hormones as well as the neurohypophysial peptides vasopressin and oxytocin. In addition to the classical hypophysiotropic hormones, a large number of neuropeptides and classical transmitters of amine and amino acid nature are present in the same cells. This is particularly evident in the magnocellular neurons of the supraoptic and paraventricular nuclei, and in parvocellular neurons of the arcuate and paraventricular nuclei. The changes in gene expression induced by experimental manipulations and the colocalization chemical messengers in hypothalamic neurosecretory neurons and its possible significance is summarized in this review.     

Changes in metabolic activity of neurons in the anterior hypothalamus nuclei during hyperthermia, fever, and hypothermia

Differently directed changes in metabolic activity of anterior hypothalamic nuclei's neurons in rats during hyperthermia, fever, and hypothermia were revealed with histochemical methods. During hyperthermia, the activity of energy metabolism enzymes increased as well as RNA content in the neurons of supraoptic, paraventricular and median preoptic anterior hypothalamic nuclei. This is shown by an increase in the metabolic activity of neurons of these nuclei. Metabolic activity in neurons of median preoptic nuclei decreased and was not changed considerably in neurons of supraoptic and paraventricular nuclei during endotoxin-induced fever. The development of hypothermia was characterised by a decrease in metabolic activity of neurons of supraoptic, paraventricular and medium preoptic nuclei. It is supposed that differently directed metabolic activity changes in neurons of anterior hypothalamic nuclei during hyperthermia are connected with the mechanisms of body temperature regulation (median preoptic nuclei) and neurosecretory processes (supraoptic and paraventricular nuclei).     

大鼠下丘脑内一氧化氮合酶阳性神经元的分布

用ＮＡＤＰＨ－ｄ组织化学方法观察了大白鼠下丘脑内一氧化氮合酶（ＮＤＳ）阳性神经元的分布及形态特征。结果显示：在视上核、室旁核的大细胞部、环状核、穹窿周核、下丘脑外侧区、下丘脑腹内侧核、下丘脑背内侧核、乳头体区大部分核团均可见一氧化氮合酶阳性神经元聚集成团。在视前内侧区、视前外侧区、下丘脑前区、下丘脑背侧区、下丘脑后区、室周核、室旁核小细胞部及穹窿内可见散在的一氧化氮合酶阳性神经元。室周核内可见呈阳性反应的接触脑脊液神经元的胞体及突起。一氧化氮合酶阳性神经元大多可见突起,有的突起上可见１～２级分支,并可见膨体。下丘脑大部分区域内可见阳性神经纤维。弓状核内可见许多弧形纤维连于第三脑室室管膜和正中隆起。     

Role of Catecholamines in Regulation of the Functional State of Vasopressinergic Cells of the Rat Hypothalamus

In in vivo and in vitro experiments there have been shown different mechanisms of regulation of hypothalamic vasopressinergic neurons, including regulation due to changes of activity level of brain catecholaminergic and NPY-ergic neurons innervating hypothalamic vasopressinergic cells. We demonstrated in in vitro experiments that dopamine and noradrenaline had no effects on vasopressin expression, but inhibited its release from cell perikarya in supraoptic and paraventricular nuclei of hypothalamus. Besides, activity of vasopressinergic neurons might probably be regulated via activation of synthesis of these neurotransmitters in vasopressinergic cells themselves in the supraoptic and paraventricular nuclei. To activate synthesis of various neurotransmitters, in our case, catecholamines and NPY, in vasopressinergic neurons, different stimuli adequate to trigger or activate synthesis of these substances are required. Synthesis of catecholamines in vasopressinergic cells of supraoptic and paraventricular nuclei was revealed after immobilization stress and adrenalectomy. NPY is synthesized in neurons of hypothalamic neurosecretory centers in norm, and its synthesis increases at disturbances of NPY-ergic innervation of vasopressinergic cells.     

Immunocytochemical localization of neuropeptides in the hypothalamus of the Japanese long-fingered bat,Miniopterus schreibersii fuliginosus

Summary To elucidate the role of hypothalamic neuropeptides in regulation of reproductive phenomena of seasonally breeding feral mammals, we used Japanese long-fingered bats, Miniopterus schreibersii fuliginosus, for immunocytochemical study of distribution of the following neuropeptides in the hypothalamus: arginin vasopressin, oxytocin, luteinizing hormone-releasing hormone, somatostatin, corticotropin-releasing factor, and growth hormone-releasing factor. The size, shape and location of supraoptic, paraventricular, suprachiasmatic, and arcuate nuclei of the bat were determined. Arginin vasopressin-and oxytocin-immunoreactive magnocellular neurons were found in the supraoptic and paraventricular nuclei, where they exhibited separate distribution into two distinct groups. Parvocellular arginin vasopressin neurons occurred only in the suprachiasmatic nucleus. The hibernating bats exhibited slightly increased numbers of vasopressin and oxytocin neurons in the supraoptic and paraventricular nuclei. The pregnant bat displayed further increased numbers of vasopressin and oxytocin neurons in both nuclei. Somatostatin-immunoreactive neurons in the paraventricular nucleus were also immunopositive to anti-oxytocin serum, while those in the ventromedial and arcuate nuclei reacted solely to anti-somatostatin serum. They projected to the anterior median eminence and infundibular stalk. Luteinizing hormone-releasing hormone-immunoreactive perikarya were scattered throughout the basal hypothalamus, being particularly abundant in the arcuate nucleus. They were larger in size in hibernating bats than those in normal (non-pregnant) and pregnant females. They projected fibers mainly to the internal layer of the median eminence and infundibular stalk. A few luteinizing hormone-releasing hormone-reactive fibers were also observed in the organum vasculosum laminae terminalis, lateral habenular nuclei, pineal stalk, retroflexus fasciculus, and olfactory tubercle. Corticotropin releasing factor-immunoreactive perikarya were distributed in the paraventricular nucleus and medial preoptic area and projected into the external layer of the anterior median eminence, while growth hormone-releasing factor-immunoreactive perikarya occurred only in the arcuate nucleus and projected into the posterior part of the median eminence.     

Neurosecretory and microstructural changes in the hypothalamus of rats following administration of lithium chloride and 3H-thymidin

Adult male rats were intraperitoneally administered aqueous solution of lithium chloride (LiCl). Studies, including neurosecretory and microstructural changes within particular neurocytes in supraoptic (NSO) and paraventricular nuclei (NPV) were performed on hypothalamic sections. In the experimental rats the administered LiCl increased the level of GOMORI-positive neurosecretory material both in supraoptic and paraventricular nuclei. Great amounts of the neurosecretory material were markedly conspicuous in the above areas after 20 days of LiCl administration. Investigations carried out on cellular nuclei of particular neurocytes showed a significant enlargement of the nuclei, and statistical calculations revealed that, in comparison with the basic control, the difference was essentially significant (p less than 0.001). 3H-thymidin administration to the rats which had previously been on LiCl for 20 days demonstrated also that within supraoptic nuclei the incorporation of the isotope in cellular nuclei took a faster course than in control animals.     

Anatomical localization of corticotropin-releasing activity in the human brain

Corticotropin-releasing activity (CRa) and arginine-vasopressin (AVP) content were measured in seven human hypothalami. The hypothalami were obtained from routine autopsy of patients suffering from no obvious neuroendocrinological abnormality. Twelve distinct hypothalamic areas were dissected in the frozen state and extracted in aqueous solution. CRa was measured by a bioassay measuring the aCTH released by rat pituitary cells in vitro, and vasopressin by direct radioimmunoassay. CRa was detectable in almost every area with the highest values in the supraoptic, paraventricular and infundibular (arcuate) areas. Vasopressin concentrations were maximum in the supraoptic nucleus, followed by the paraventricular and infundibular nuclei. We conclude that: hypothalami obtained from routine autopsy at a general hospital can be used for consistent CRa and vasopressin assay. Vasopressin and CRa are similarly distributed in man and in the rat. In both species, high CRa, which is not explained by AVP, is found in the paraventricular nucleus. The infundibular (arcuate) nucleus seems to display non AVP-dependent CRa much greater in the human than in the rat.     

Glucagon-related peptides in the rat hypothalamus

Summary Immunohistochemically, nerve fibers and terminals reacting with anti-N-terminal-specific but not with anti-C-terminal-specific glucagon antiserum were observed in the following rat hypothalamic regions: paraventricular nucleus, supraoptic nucleus, anterior hypothalamus, arcuate nucleus, ventromedial hypothalamic nucleus and median eminence. Few fibers and terminals were demonstrated in the lateral hypothalamic area and dorsomedial hypothalamic nucleus. Radioimmunoassay data indicated that the concentration of gut glucagon-like immunoreactivity was higher in the ventromedial nucleus than in the lateral hypothalamic area. In food-deprived conditions, this concentration increased in both these parts. This was also verified in immunostained preparations in which a marked enhancement of gut glucagon-like immunoreactivity-containing fibers and terminals was observed in many hypothalamic regions. Several immunoreactive cell bodies were found in the ventromedial and arcuate nuclei of starved rats. Both biochemical and morphological data suggest that glucagon-related peptides may act as neurotransmitters or neuromodulators in the hypothalamus and may be involved in the central regulatory mechanism related to feeding behavior and energy metabolism.     

APM/CD13 and FOS in the hypothalamus of monosodium glutamate obese and food deprived rats

Protein (western blotting) and gene (PCR) expressions, catalytic activity of puromycin-insensitive membrane-bound neutral aminopeptidase (APM/CD13) and in situ regional distribution of CD13 and FOS immunoreactivity (ir) were evaluated in the hypothalamus of monosodium glutamate obese (MSG) and/or food deprived (FD) rats in order to investigate their possible interplay with metabolic functions. Variations in protein and gene expressions of CD13 relative to controls coincided in the hypothalamus of MSG and MSG-FD (decreased 2- to 17-fold). Compared with controls, the reduction of hypothalamic CD13 content reflected a negative balance in its regional distribution in the supraoptic, paraventricular, periventricular and arcuate nuclei. CD13-ir increased in the supraoptic nucleus in MSG (2.5-fold) and decreased in the paraventricular nucleus (2-fold) together with FOS-ir (1.5-fold) in FD. In MSG-FD, FOS-ir decreased (7-fold) in the paraventricular nucleus, while CD13-ir decreased in the periventricular (5.6-fold) and the arcuate (3.7-fold) nuclei. It was noteworthy that all these changes of CD13 were not related to catalytic activity of APM. Data suggested that hypothalamic CD13 plays a role in the regulation of energy metabolism not by means of APM enzyme activity.     

Functional interrelationships of the sympathetic nervous system and the hypothalamo-hypophyseo-adrenal complex]

Adrenal and hypothalamic structure has been studied in mice at functional inhibition of the sympathetic region of the vegetative nerve system by means of antibodies against the nerve growth factor. Routine histological, histochemical, morphometric, electron microscopic and radioautographic methods have been applied. In the experiment the hypothalamic region is in the state of a continuous functional stress which is evident from enlargement of neurons and their nuclei in the macrocellular neurosecretory nuclei of the hypothalamus. The method of radioautography proves increasing activity in synthetic processes taking place in supraoptic, paraventricular and ventromedial nuclei of the hypothalamus and in the medial habenular nucleus of the epithalamus. In 30-day-old mice certain structural changes have been revealed in the adrenals demonstrating an increasing activity in their cortex: blood vessels are dilated, cholesterine, lipid and ascorbic acid granules are not evenly distributed, enzymatic activity in cytoplasm of adrenocorticocytes is increased. Ultramicroscopic destructive and regenerative alterations have no definite zonal specificity, nevertheless they are more pronounced in the fascicular zone. Radioautographic and morphometric investigations demonstrate an increasing functional activity in glomerular and fascicular adrenal zones. Taking into account morphologic demonstration of functional stress in hypophysial adenocytes in the same animals (Molostov O. K., 1974), it is possible to conclude that lesions in the sympathetic system center result in adaptive reaction of the hypothalamo-hypophysial system which, in its turn, produces reactions in the adrenals and in the vascular complex. This interconnection is accompanied by the reaction depending on breaking off direct sympathetic effects in the adrenals and vessels.     

Distribution of Mahogany/Attractin mRNA in the rat central nervous system

The Mahogany/Attractin gene (Atrn) has been proposed as a downstream mediator of Agouti signaling because yellow hair color and obesity in lethal yellow (A(y)) mice are suppressed by the mahogany (Atrn(mg)) mutation. The present study examined the distribution of Atrn mRNA in the brain and spinal cord by in situ hybridization. Atrn mRNA was found widely distributed throughout the central nervous system, with high levels in regions of the olfactory system, some limbic structures, regions of the brainstem, cerebellum and spinal cord. In the hypothalamus, Atrn mRNA was found in specific nuclei including the suprachiasmatic nucleus, the supraoptic nucleus, the medial preoptic nucleus, the paraventricular hypothalamic nucleus, the ventromedial hypothalamic nucleus, and the arcuate nucleus. These results suggest a broad spectrum of physiological functions for the Atrn gene product.     

Functional changes in neurosecretory cells of the anterior hypothalamic nuclei after stimulation of the midbrain reticular formation

The functional activity of the neurosecretory cells in the supraoptic and paraventricular nuclei was studied in male Wistar albino rats at various intervals after electric stimulation of the midbrain reticular formation. These studies showed that such stimulation elicits higher functional activity of the neurosecretory cells in the anterior hypothalamic nuclei, characterized by increased secretory synthesis by these cells and rapid transport of the neurosecretion. These changes were most pronounced 1 h after stimulation of the reticular formation. The changes observed were unidirectional in both neurosecretory centers, but their manifestation was different: in the supraoptic nucleus the reaction was more intense but short lived, in the paraventricular it was less intense but lasted longer.A. A. Bogomolets Institute of Physiology, Academy of Sciences, Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 3, No. 4, pp. 394–400, July–August, 1971.     

Different distributions of nitric oxide synthase-containing neurons in the mouse and rat hypothalamus.

The distribution of nitric oxide synthase-containing neurons was studied in the rat and mouse hypothalamus by immunohistochemistry and NADPH-diaphorase histochemistry. Immunostaining and NADPH-diaphorase staining of hypothalamic neurons were comparable in all hypothalamic nuclei of either species except in the arcuate nucleus that stained positive for nitric oxide synthase immunoreactivity but negative for NADPH-diaphorase reactivity. The presence of nitric oxide synthase-immunopositive neurons in the arcuate nucleus was confirmed by nitric oxide synthase immunofluorescence viewed under the confocal microscope at 1 microm thickness. Cross-species comparison showed that, in general, the number and intensity of nitric oxide synthase-containing neurons were much higher in the rat than in the mouse hypothalamus. Differences in the distribution of nitric oxide synthase-containing neurons between these two rodents were found in most hypothalamic nuclei. In particular, two dense clusters of nitric oxide synthase-containing neurons were found in the paraventricular and supraoptic nuclei of the rat hypothalamus in contrast to their scarcity in the same nuclei of the mouse hypothalamus.     

Immunocytochemical localization of tissue kallikrein in brain ventricular epithelium and hypothalamic cell bodies

A specific monoclonal antibody against rat tissue kallikrein was used as the primary antibody for indirect immunoperoxidase staining of rat hypothalamus. Kallikrein was localized in the epithelial cells (ependyma) lining the third ventricle as well as in cell bodies of arcuate, supraoptic, paraventricular, and ventromedial nuclei.     

Lateral septal projections onto tubero-infundibular neurons in the hypothalamus of the guinea pig

Efferent projections of the lateral septal nucleus (LS) to the preoptic area and the hypothalamus were identified in 20 female guinea pigs after iontophoretic injection of the anterograde axonal tracer Fluoro-Ruby. Tubero-infundibular (TI) neurons of the preoptic area and the hypothalamus were retrogradely labeled after intracardiac injection of Granular Blue or Fluoro-Gold. Magnocellular neurons of the supraoptic and paraventricular nuclei were also labeled. The double labeling procedure allowed an estimation of the extent of the direct relationship between LS efferents and TI neurons. Contacts between lateral septal fibers and TI cell bodies were mainly observed at the light-microscopical level in the preoptic area. A group of labeled fibers coursing along the third ventricle established sparse connections with hypothalamic periventricular TI neurons. A few appositions was observed in the infundibular (arcuate) nucleus, suggestive of a monosynaptic regulation of TI neurons by a septo-arcuate tract. Close association with labeled magnocellular neurons was also noted at the edge of the supraoptic and paraventricular nuclei. The sparse but direct connections between LS and TI neurons may be involved in the neuroendocrine functions of the LS.     

Immunohistochemical identification of neurons containing corticotropin-releasing factor in the rat hypothalamus

A specific rabbit anti-CRF serum and the immunoperoxidase technique were used to show that CRF-containing neurons are mainly distributed in the paraventricular and supraoptic nuclei of the rat hypothalamus. In addition, immunoreactive neurons are scattered in other hypothalamic regions. These neurons are 20--30 micrometers in diameter. From the present and previous investigations it may be concluded that the hypothalamic magnocellular nuclei, i.e., paraventricular and supraoptic, and other hypothalamic accessory nuclei, are the producing sites not only for vasopressin and oxytocin, but also for corticotropin-releasing factor.     

Changes in RNA content of the hypothalamus of mice following insulin administration.

Cytoplasmic total RNA in neurons of the supraoptic nucleus, paraventricular nucleus, suprachiasmatic nucleus, and arcuate nucleus was determined cytophotometrically following a single intramuscular dose of 4 IU/kg insulin semilente. A significant decrease was observed after 3, 6, and 12 hours. A return towards normal values was evident after 24 hours and was full after 48 hours only in suprachiasmatic nucleus. Other hypothalamic nuclei did not reach the full normal value in the time interval examined, i.e. 48 hours post-injection.     

Quantitative histoenzymatic analysis of the hypothalamic neurosecretory system in rat ontogeny

In experiments on 6 and 16 days old rats cytophotometric studies have been made of histochemical reactions for succinate, lactate and glutamate dehydrogenases, alpha-glycerophosphate dehydrogenase and glucose-6-phosphate dehydrogenase in the supraoptic nucleus, paraventricular hypothalamic nucleus and the posterior hypophysis. It was found that heterochronous development of the neurones in the supraoptic and paraventricular nuclei, as well as the development of their axons in the posterior hypothalamus depend on the rate of maturation of the enzymic systems in postnatal life. In consolidation of the unique structure of the hypothalamic neurosecretory system, the key role is played by afferent influences, succinic dehydrogenase being involved into their realization. The data obtained indicate the importance of heterochronous development of the enzymic activities in the formation of bifunctional properties of neurosecretory hypothalamic neurones and reveal the primary development of neurotransmittery function as compared to the excretory one.