Glucagon-like immunoreactivity in hypothalamic neurons of the rat.

Antisera specific for three different regions of pancreatic proglucagon were used to examine the distribution of such immunoreactivity in rat hypothalamus. Neurons in the supraoptic and paraventricular nuclei were immunoreactive with an antiserum against glucagon, but not with antisera directed towards the aminoterminal region of proglucagon (glicentin) or the glucagon-like peptide I sequence in the carboxyl-terminal region of proglucagon. These findings confirm a previous report of glucagon-like immunoreactivity in the supraoptic and paraventricular nuclei, but indicate that, while this material is immunochemically related to glucagon, it is not derived from a proglucagon-like precursor.

Hyperphagia in obesity is associated with a central peptidergic dysregulation in rats.

Hyperphagia and obesity are often associated, and the origins of the biochemical modifications leading to these syndromes might be in the hypothalamus. Indeed, food intake is regulated by numerous neuropeptides in various hypothalamic nuclei, including the paraventricular (PVN), arcuate (ARC), ventromedian (VMN) and suprachiasmatic (SCH) nuclei. Among these peptides, neuropeptide Y (NPY) is the most potent inducer of food intake whereas neurotensin (NT) decreases food intake. We measured these two peptides in microdissected hypothalamic nuclei in obese Zucker rats that ate 30% more food than their lean counterparts. Neuropeptide Y and neurotensin levels varied in opposite directions: In the hyperphagic obese Zucker rats, the NPY concentrations were significantly greater than those in the lean normophagic rats in the ARC (+30%), PVN (+60%) and SCH (+94%) nuclei, whereas the NT levels were significantly lower in the ARC (-40%), PVN (-31%) VMN (-66%) and SCH (-47%) nuclei. Both these variations tend to increase food intake. Feeding periodicity might also be modified because large variations of the two peptides have been measured in the supra-chiasmatic nucleus, which is considered the most important regulator of feeding rhythm. The results reinforce the hypothesis that hyperphagia in obesity is associated with a biochemical modification in the central nervous system because the peripheral status of NT and NPY was not modified in the obese rats. Because levels of other hypothalamic peptides, such as opioid peptides and somatostatin, are also slightly modified, it can be concluded that hyperphagia in obesity is associated with a central peptidergic dysregulation. Research on drugs reacting specifically with the receptor of these peptides might have interesting implications for the treatment of hyperphagia and, therefore, of obesity.

Coexistence of CRF peptide and oxytocin mRNA in the paraventricular nucleus.

Several studies have reported coexistences of peptides in parvocellular neurons of the paraventricular nucleus (PVN). However, the coexistence of peptides in the magnocellular PVN is less clear. Controversy exists in particular about the coexistence of corticotropin-releasing factor (CRF) and oxytocin (OX). Although these peptides are present in distinct areas of the PVN, some overlap may exist. This study investigated a potential coexistence of OX and CRF in magno- and parvocellular PVN. The data demonstrate with clarity that neurons containing both the mRNA for OX and the peptide CRF are present in subpopulations of magnocellular and parvocellular neurons of the PVN.

Atrial natriuretic factor in central nervous system regulatory mechanisms: effect of experimental alterations in water and salt homeostasis and blood pressure.

Atrial natriuretic factor (ANF) is widely distributed in the preoptic area and the hypothalamus, it is present there both in cell bodies and nerve terminals. Effect of experimental alterations in the salt and water balance was examined on preoptic-hypothalamic ANF levels measured in ten microdissected nuclei. Immunohistochemical analysis was also performed to confirm radioimmunological results. Following interventions were performed in adult male rats: adrenalectomy (5 days), daily 0.9% NaCl, aldosterone (5 micrograms/100 g) and dexamethasone (2 micrograms/ml drinking water) treatments in both intact and adrenalectomized groups, and in rats with diabetes insipidus (Brattleboro rats) and DOC-salt hypertension. Although no appreciable alterations were observed in the intensity of ANF-like immunoreactivity in sections of the preoptic-hypothalamic region, ANF levels altered markedly in the periventricular structures (organum vasculosum laminae terminalis, preoptic and periventricular nuclei). Little or no changes were measured in ANF levels of other hypothalamic nuclei (except the perifornical nucleus). Adrenalectomy depleted ANF levels which were restored by NaCl drinking. Aldosterone elevated ANF concentrations both in intact and adrenalectomized animals while dexamethasone treatment was without any significant effect on ANF levels in the periventricular preoptic nucleus. Diabetes insipidus or DOC-salt hypertension had little or no effect on ANF levels in this brain area. Unchanged ANF concentrations were also measured in the vasopressin-containing supraoptic nucleus following adrenalectomy or in diabetes insipidus rats.

Localization of 3H-prazosin binding sites in the supraoptic and paraventricular nuclei of the human hypothalamus.

Using 3H-prazosin, we have examined the distribution of alpha 1-adrenergic receptors in the supraoptic and paraventricular nuclei of the human hypothalamus. Our studies show that binding sites for 3H-prazosin in human hypothalamus possess pharmacological characteristics similar to those of rat brain. Autoradiographic studies revealed discrete localization of 3H-prazosin to the paraventricular and supraoptic nuclei.

Endothelin: a novel peptide in the posterior pituitary system.

Endothelin (ET), originally characterized as a 21-residue vasoconstrictor peptide from endothelial cells, is present in the porcine spinal cord and may act as a neuropeptide. Endothelin-like immunoreactivity has now been demonstrated by immunohistochemistry in the paraventricular and supraoptic nuclear neurons and their terminals in the posterior pituitary of the pig and the rat. The presence of ET in the porcine hypothalamus was confirmed by reversed-phase high-pressure liquid chromatography and radioimmunoassay. Moreover, in situ hybridization demonstrated ET messenger RNA in porcine paraventricular nuclear neurons. Endothelin-like immunoreactive products in the posterior pituitary of the rat were depleted by water deprivation, suggesting a release of ET under physiological conditions. These findings indicate that ET is synthesized in the posterior pituitary system and may be involved in neurosecretory functions.

Exogenous triiodothyronine lowers thyrotropin-releasing hormone concentrations in the specific hypothalamic nucleus (paraventricular) involved in thyrotropin regulation and also in posterior nucleus.

Recent evidence indicates that thyroid hormones can regulate thyrotropin secretion in vivo in part by inhibiting thyrotropin-releasing hormone (TRH) secretion itself. Therefore, to explore whether triiodothyronine (T3) interacts with the specific hypothalamic area involved in thyrotropin (TSH) secretory regulation, the paraventricular nucleus (PVN), Palkovitz micropunches from eight nuclear regions were obtained from 1,000-microns frozen coronal brain slices for immunoassay determinations of TRH. Rats were treated either with parenteral L-T3 for 6 days to induce experimental thyrotoxicosis, or 0.15 M saline. The induction of thyrotoxicosis was confirmed by demonstrating that mean plasma TSH concentrations fell from 108 to less than 10 microU/ml (p less than 0.01). TRH concentrations in the PVN were reduced concomitantly after L-T3 from 1.9 to 1.1 ng/mg protein (p less than 0.05). No reductions in TRH concentrations during T3 treatment occurred in other nuclear groupings except in the posterior hypothalamic nucleus. Total TRH content in the median eminence declined also in T3-treated animals from 1.77 to 1.29 ng, representing a 32% reduction (p less than 0.01). No significant change was seen in the median eminence content of the TRH structurally related dipeptide, cyclo(His-Pro). The data herein indicate that experimental thyrotoxicosis in the rat is associated with a selective reduction in TRH concentrations in the PVN, documenting T3 effects upon hypothalamic TRH metabolism per se.

Vasopressin gene expression in the normal and Brattleboro rat: a histological analysis in semi-thin sections with biotinylated oligonucleotide probes.

We analyzed expression of the vasopressin (AVP) gene in semi-thin sections in normal and Brattleboro rats by using in situ hybridization and immunohistochemistry. AVP mRNA was detected as follows: vibratome sections of rat hypothalamus were hybridized with a biotinylated oligonucleotide probe, embedded in Araldite, and cut into semi-thin sections which were reacted with streptavidin-alkaline phosphatase and the appropriate substrate. Adjacent serial sections were treated by immunohistochemistry to detect AVP or oxytocin immunoreactivity. In normal rat, AVP mRNA can be detected in magnocellular neurons of the supraoptic and paraventricular nuclei and in parvocellular neurons of the suprachiasmatic nucleus. AVP mRNA was present throughout the cytoplasm of the cell bodies, their processes, and in punctate structures in the vicinity of the AVP cell bodies. Most neurons containing AVP mRNA also contain AVP immunoreactivity, but the staining intensity was not consistently correlated for each reaction. A few neurons contained AVP mRNA without detectable AVP immunoreactivity. In the Brattleboro rat, staining intensity of the reaction was lower than in normal rat and the AVP mRNA was restricted mostly to the periphery of the cytoplasm. In this strain, the neurons containing the AVP mRNA did not contain AVP or oxytocin immunoreactivity. These results demonstrate that neuropeptide mRNA can be detected in semi-thin sections with a biotinylated oligonucleotide probe, and that AVP gene deletion provokes modification of the intracellular localization of the AVP mRNA.

Dietary amino acid imbalance and neurochemical changes in three hypothalamic areas.

The impact of feeding imbalanced amino acid diets on monoamine, metabolite and amino acid concentrations was measured in the ventromedial hypothalamus (VMH), lateral hypothalamus (LH) and paraventricular nucleus (PVN). After rats were fed either an isoleucine imbalanced diet, a threonine imbalanced diet, or the appropriate basal or corrected control diets, regional differences were found in neurochemical concentrations. Contrary to our expectations, the limiting amino acid was unchanged in the imbalanced groups, tending to be decreased only in the isoleucine imbalanced-diet group in the PVN. This is the first report that the limiting amino acid was not reduced uniformly in the brain after imbalanced amino acid feeding. In the VMH, norepinephrine (NE) was increased by 22% and 63% in the threonine and isoleucine imbalanced-diet groups, respectively. Since the concentration of NE was affected even before the decrease in feeding, both in the VMH, and, as previously reported, in the prepyriform cortex, the NE system may be involved in very early responses to imbalanced amino acid diets.

Thyroid hormone regulation of neurons staining for a pro-TRH-derived cryptic peptide sequence in the rat hypothalamic paraventricular nucleus.

The influence of thyroid hormones on neurons staining for a pro-TRH-derived cryptic peptide sequence in the rat hypothalamic paraventricular nucleus was assessed by using quantitative immunocytochemistry. The antiserum used for immunocytochemical studies recognized a cryptic fragment of pro-TRH located in the Phe 178-Ser 186 region of the precursor. Hypothyroid female rats obtained following 1 month administration of 6-n-propyl-2-thiouracil (PTU) as well as euthyroid animals were treated with a low (3 micrograms/kg/day) or high (67 micrograms/kg/day) dose of L-thyroxine (T4) for 4-10 days before sacrifice. All the animals received an intraventricular injection of colchicine 2 days prior to perfusion with 4% paraformaldehyde. PTU treatment induced an increase (45% over control) in the number of immunostained neurons. The administration of a low dose of T4 to hypothyroid animals slightly increased the number of staining for a pro-TRH-derived cryptic sequence neurons, while being uneffective in euthyroid rats. In hypothyroid animals, a 4-day treatment with the high dose of T4 did not produce any changes whereas the 7-day treatment reduced the number of positive cells to 42% of control. In euthyroid rats treated with the high dose of T4 for 4 and 7 days, the number of stained neurons was reduced to 60 and 37% of control, respectively. These results indicate that thyroid hormones can modify the amounts of staining for a pro-TRH-derived cryptic sequence in hypothalamic paraventricular neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Synaptic regulation of paraventricular arginine vasopressin-containing neurons by neuropeptide Y-containing monoaminergic neurons in rats. Electron-microscopic triple labeling.

Synaptic regulation of arginine vasopressin (AVP)-containing neurons by neuropeptide Y (NPY)-containing monoaminergic neurons was demonstrated in the paraventricular nucleus of the rat hypothalamus. NPY and AVP were immunolabeled in the pre- and the post-embedding procedures, respectively, and monoaminergic fibers were marked by incorporating 5-hydroxydopamine (5-OHDA), a false neurotransmitter. The immunoreaction for NPY was expressed by diaminobenzidine (DAB) chromogen, and that for AVP by gold particles. The DAB chromogen was localized on the surface of the membrane structures, such as vesicles or mitochondria, and on the core of large cored vesicles. Gold particles were located on the core of the secretory granules within the AVP cell bodies and processes. The incorporated 5-OHDA was found as dense cores within small or large vesicular structures. From these data, three types of nerve terminals were discernible: NPY-containing monoaminergic, NPY-containing non-aminergic, and monoaminergic fibers. The AVP cell bodies appeared to have synaptic junctions formed by these nerve terminals as well as by the unlabeled nerve terminals which have small clear vesicles and large cored vesicles. These different types of nerve terminals were frequently observed in a closely apposed position on the same AVP cell bodies. The functional relationships of these three types of neuronal terminals are discussed.

Galanin and vasopressin coexist in the rat hypothalamo-neurohypophyseal system.

Using indirect immunofluorescence methods and antisera raised against galanin (GAL) and vasopressin (VP), we have demonstrated both peptides coexisting in the very same cell bodies in the supraoptic and magnocellular paraventricular nuclei and the magnocellular accessory cells of the lateral hypothalamic area. Furthermore, dehydration and salt loading, which is known to cause release and depletion of VP and oxytocin from the neurohypophysis, also caused a marked reduction of GAL-like immunoreactivity in the posterior lobe of the pituitary but had no effect on hypothalamic GAL immunoreactivity. Systemically administered GAL caused a brief small increase in blood pressure with no effect on heart rate. A thousandfold molar concentration of GAL, compared of VP, was required to induce comparable effects on blood pressure. GAL itself had no modulatory effect on VP-induced pressor response. Systemically administered GAL resulted in mild diuresis whereas VP caused complete and sustained inhibition of diuresis. GAL had no effect on VP-induced anti-diuresis effects. The significance of the coexistence and corelease of GAL and VP remains to be elucidated.

Differential responses in vasopressin and oxytocin gene expression in distinct hypothalamic nuclei after hypothalamoneurohypophyseal disconnection and vasopressin substitution.

A possible feedback of circulating vasopressin (VP) on the expression of its gene in the hypothalamoneurohypophyseal system was studied in rats with depleted stores of neurohypophyseal hormones. The neural lobe of the pituitary gland was disconnected by anterolateral deafferentation of the basal hypothalamus using a Halasz knife cut. One group of lesioned animals was substituted continuously with VP using mini-osmopumps. After 1 week, the lesion had caused a complete disappearance of neurohypophyseal hormones from the neurointermediate pituitary and induced a 2-fold increase in water intake and an increase in plasma osmolality of approximately 7 m0sm/kg. VP mRNA levels of the supraoptic nucleus (SON), paraventricular nucleus (PVN) and suprachiasmatic nucleus (SCN) were measured in punched tissue by Northern blot analysis and dot-blot analysis. VP mRNA levels of the SON and PVN were reduced by 50% due to the deafferentiation, which might have been caused by degeneration. VP substitution of the lesioned animals normalized water intake but not plasma osmolality. In the VP-substituted animals, VP mRNA levels of the SON were 2-fold reduced compared to the nonsubstituted lesioned animals. The VP mRNA levels of the PVN were not affected by VP substitution. The VP mRNA levels of the SCN increased 2-fold after the lesion but were not influenced by VP substitution. Concomitant determination of oxytocin (OT) mRNA showed that OT mRNA levels in the PVN and SON were not affected by the deafferentiation and did not respond to VP substitution.(ABSTRACT TRUNCATED AT 250 WORDS)

Corticotropin-releasing hormone in the human hypothalamus. Free-floating immunostaining method.

We have clearly demonstrated corticotropin-releasing hormone (CRH) immunoreactive cell bodies and nerve fibers in the human hypothalamus by immunocytochemistry using free-floating sections instead of paraffin-embedded sections. Human hypothalami were obtained at autopsy, fixed and cryostat-sectioned at 40 microns. Free-floating sections were immunostained with antibody to CRH using the Vector ABC system. Most of CRH immunoreactive nerve fibers from the paraventricular nucleus pass under the fornix, while some CRH immunoreactive nerve fibers pass beyond the fornix and some through the fornix. Then the CRH immunoreactive nerve fibers run downward, medially to the supraoptic nucleus and toward the pituitary stalk. This method of immunocytochemistry is a very sensitive and suitable means for immunocytochemical studies of neuropeptides in the human brain.

Distribution and coexistence of corticotropin-releasing factor-, neurotensin-, enkephalin-, cholecystokinin-, galanin- and vasoactive intestinal polypeptide/peptide histidine isoleucine-like peptides in the parvocellular part of the paraventricular nucleus.

The distribution of several peptides, corticotropin-releasing factor (CRF), neurotensin (NT), enkephalin (ENK), vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI), cholecystokinin (CCK), thyrotropin-releasing hormone (TRH) and galanin (GAL) was studied in detail with immunohistochemistry in the various subdivisions of the parvocellular part of the rat hypothalamic paraventricular nucleus (PVN). Using a double-staining method and elution-restaining technique, the coexistence of CRF- and NT-like immunoreactivities (LI) with other neuropeptides was analyzed. Our results indicate that coexistence of two or more peptides in the PVN is common, and revealed that about 30% of the CRF neurons contain NT-LI and about 20% ENK-LI, whereas other peptides only occur in small fractions of the CRF cells. Thus, it seems possible to define three major subpopulations of CRF neurons, one containing NT-LI, another one containing ENK-LI and a third one apparently lacking these peptides. Conversely, about 60% of both NT- and ENK-immunoreactive neurons lacked CRF-LI. A large proportion of the small population of VIP/PHI neurons contained NT-LI. TRH neurons represented a neuron population completely distinct from the CRF neurons. Also, it did not seem to contain any of the other peptides studied with the rare exception of ENK-LI. Neuropeptides present in the PVN and presumably in nerve fibers of the external layer of the median eminence may participate in the control of the anterior pituitary hormone secretion. Whereas the role of CRF and TRH is well established, the physiological role of the other peptides studied here is still unclear.

Association of dopaminergic fibers with corticotropin releasing hormone (CRH)-synthesizing neurons in the paraventricular nucleus of the rat hypothalamus.

Catecholamines are known to exert a central influence on the hypothalamo-hypophyseal-adrenal neuroendocrine system. The selective dopaminergic innervation of the hypothalamic paraventricular nucleus (PVN) and putative relationships between dopaminergic fibers and corticotropin releasing hormone (CRH)-synthesizing neurons were studied in the male rat by means of immunocytochemistry following the elimination of noradrenergic and adrenergic inputs to the hypothalamus. A 3.0-mm-wide coronal cut was placed unilaterally in the brain at the rostral level of the mesencephalon. All neuronal structures from the cortex to the ventral surface of the brainstem, including the ascending catecholaminergic fiber bundles were transected. This surgical intervention resulted in the accumulation of dopamine-beta-hydroxylase (DBH)-immunoreactivity in axons proximal to the cut, and an almost complete disappearance of DBH activity in those located distal to the lesion. Two weeks following the operation, DBH immunoreactivity was significantly diminished in the PVN located on the side of lesion, while tyrosine hydroxylase (TH)-immunoreactivity was present in a substantial number of fibers in the same nucleus. Both DBH- and TH-immunoreactive axons were preserved in the contralateral PVN. Simultaneous immunocytochemical localization of either DBH- or TH-IR fibers and corticotropin releasing hormone-synthesizing neurons in the hypothalami from brainstem-lesioned, colchicine treated animals revealed that the distribution of catecholaminergic fibers and CRH neurons is homologous within the PVN of the intact side. Only a few scattered DBH-immunoreactive axons were detected among CRH-producing neurons in the PVN on the side of the lesion. In contrast, many tyrosine hydroxylase containing neurons and neuronal processes were observed on the lesioned side and the TH-IR fibers established juxtapositions with CRH-synthesizing neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Potential involvement of galanin in the regulation of fluid homeostasis in the rat.

A physiological role for galanin, a 29-amino acid neuropeptide, has not been established. However, anatomical studies have demonstrated the presence of galanin in brain regions associated with the control of water balance in the rat, most notably in the paraventricular nucleus (PVN) of the hypothalamus and the neurointermediate lobe of the pituitary gland (NIL). In the PVN, galanin coexists with arginine vasopressin (AVP) in magnocellular neurons. The present study demonstrates that homozygous Brattleboro rats, which lack AVP, produce galanin. Galanin concentrations in the median eminence (ME) of the homozygous Brattleboro rat do not differ from the galanin concentrations in the ME of either heterozygous Brattleboro or Sprague-Dawley rats. However, galanin concentrations in the NIL of the homozygous Brattleboro rat were reduced by 75%. Similarly, dehydration induced by salt-loading reduced galanin concentrations in the NIL and produced transient changes in the ME. These data demonstrate that galanin concentrations are influenced by changes in fluid homeostasis and suggest that galanin may be an important component in the regulation of neurohypophyseal function and AVP secretion.

Angiotensin II receptors in paraventricular nucleus, subfornical organ, and pituitary gland of hypophysectomized, adrenalectomized, and vasopressin-deficient rats.

Angiotensin II has been implicated in the regulation of adrenocorticotropin and vasopressin secretion. Angiotensin II may influence the secretion of these hormones either directly at the pituitary gland or by increasing corticotropin-releasing hormone or vasopressin release from cells that are located in the paraventricular hypothalamic nucleus. Pituitary hormone release may also be influenced by circulating angiotensin II through receptors outside the blood-brain barrier in the subfornical organ. We have used alterations in angiotensin II receptors in hypophysectomized, adrenalectomized, and vasopressin-deficient Brattleboro rats as indicators of the activity of angiotensin II in the regulation of adrenocorticotropin and vasopressin secretion. Angiotensin receptor number in the paraventricular nucleus and the subfornical organ, but not in the anterior pituitary gland, was significantly decreased by adrenalectomy, and this effect was reversed by corticoids. Vasopressin deficiency decreased angiotensin receptors in the subfornical organ and increased them in the anterior pituitary gland but did not affect angiotensin II binding in either magnocellular or parvocellular subnucleus of the paraventricular nucleus. Our results suggest that angiotensin II may have a corticoid-dependent role in the regulation of corticotropin-releasing hormone secretion, which could be important in the adaptation to elevated corticosterone secretion in stress.