Sexually dimorphic immunoreactivity of galanin and colocalization with arginine vasotocin in the chicken brain (Gallus gallus domesticus)

The bed nucleus of the stria terminalis medialis (BSTM) of adult chickens (Gallus gallus domesticus) was previously shown to synthesize arginine vasotocin (AVT) in males only and coincides spatially and temporally with steroid activity regulating male reproductive behavior. Galanin has been shown to be a potent modulator of the behavioral and neuroendocrine responses in the mammalian BSTM and in other sexually dimorphic brain regions. In the present study of adult chickens the morphological relationship of AVT and galanin was examined by immunohistochemical analysis of two limbic structures, the BSTM and the lateral septum (SL). The analysis also included the hypothalamic nuclei supraopticus (SON) and paraventricularis (PVN). In males galanin and AVT were both synthesized in the BSTM, while in females neither galanin nor AVT was present. Furthermore, in the males galanin and AVT were colocalized in the majority of neurons within BSTM and in fibers of the SL. In both sexes galanin neurons in the PVN were scattered between the distinct clusters of AVT neurons and there was no colocalization of galanin and AVT in single PVN neurons. Furthermore, AVT immunoreactivity was significantly higher in the SON than in the PVN in both sexes. In the SON, galanin was colocalized with AVT in significantly more neurons in hens than in males (P 相似文献   

Forced swimming stimulates the expression of vasopressin and oxytocin in magnocellular neurons of the rat hypothalamic paraventricular nucleus.

Previous studies have shown that a 10-min forced swimming session triggers the release of both vasopressin and oxytocin into the extracellular fluid of the hypothalamic paraventricular (PVN) and supraoptic nuclei (SON) in rats. At the same time oxytocin, but not vasopressin, was released from the axon terminals into the blood. Here we combined forced swimming with in situ hybridization to investigate whether (i) the stressor-induced release of vasopressin and oxytocin within the PVN originates from parvo- or magnocellular neurons of the nucleus, and (ii) central release with or without concomitant peripheral secretion is followed by changes in the synthesis of vasopressin and/or oxytocin. Adult male Wistar rats were killed 2, 4 or 8 h after a 10-min forced swimming session and their brains processed for in situ hybridization using 35S-labelled oligonucleotide probes. As measured on photo-emulsion-coated slides, cellular vasopressin mRNA concentration increased in magnocellular PVN neurons 2 and 4 h after swimming (P < 0.05). Similarly, oxytocin mRNA concentration was significantly increased in magnocellular neurons of the PVN at 2 and 8 h (P < 0.05). We failed to observe significant effects on vasopressin and oxytocin mRNA levels in the parvocellular PVN and in the SON. Taken together with results from previous studies, our data suggest that magnocellular neurons are the predominant source of vasopressin and oxytocin released within PVN in response to forced swimming. Furthermore, in the case of vasopressin, central release in the absence of peripheral secretion is followed by increased mRNA levels, implying a refill of depleted somato-dendritic vasopressin stores. Within the SON, however, mRNA levels are poor indicators of the secretory activity of magnocellular neurons during stress.     

Effect of indomethacin on the c-fos expression in AVP and TH neurons in rat brain induced by lipopolysaccharide

The aim of the present study was to investigate the effect of indomethacin on the Fos expression in arginine vasopressin (AVP)-containing neurons in the hypothalamus and tyrosine hydroxylase (TH)-containing neurons in the locus coeruleus (LC) using dual-labeled immunohistochemistry. In the hypothalamus, intraperitoneal (i.p) injection of different doses [2.5 microg/100 g, 125 microg/100 g body weight (b.w.)] of lipopolysaccharide (LPS) induced a significant Fos expression in AVP neurons in the supraoptic nucleus (SON), the magnocellular division (mPVN) and the parvocellular division (pPVN) of the paraventricular nucleus (PVN). Pretreatment with the cyclooxygenase inhibitor indomethacin (0.8 mg/100 g b.w.) significantly blocked the Fos expression in these AVP neurons induced by a low dose of LPS (2.5 microg/100 g) but had no effect on the Fos expression induced by a high dose of LPS (125 microg/100 g). Similarly, in the brain stem, a large number of TH-positive neurons in the LC expressed Fos after administration of either dose of LPS. Indomethacin prevented the Fos expression induced only by a low dose of LPS, but not by a high dose of LPS. These results suggest that the activation of AVP neurons in PVN and SON and TH neurons in LC response to immune challenge might be mediated-at least partially-by prostaglandins.     

Adrenomedullin 2/intermedin-like immunoreactivity in the hypothalamus and brainstem of rats

Adrenomedullin 2 (AM2) (identical to intermedin)-like immunoreactivity (LI) was examined in the rat brain by immunohistochemistry after intracerebroventricular administration of colchicine (100 microg/rat) and chronic salt loading (2% saline to drink) for 5 days. In both vehicle-treated and euhydrated rats, AM2-LI neurons were observed in the hypothalamus and brainstem, including in the organum vasculosum of the lamina terminalis, the median preoptic nucleus, the supraoptic nucleus (SON), the paraventricular nucleus (PVN), the ventromedial hypothalamic nucleus, the arcuate nucleus, the locus coeruleus, the nucleus of the tractus solitarius and the nucleus ambiguus. In colchicine-treated and salt loaded rats, AM2-LI neurons were visualized more strongly in the SON and the magnocellular part of the PVN than in those in each control. Some AM2-LI neurons appeared in the parvocellular part of the PVN in the colchicine-treated but not salt loaded rats. AM2-LI in the other areas of the hypothalamus and brainstem did not change after colchicine-treatment and chronic salt loading. These results suggest that AM2/intermedin in the hypothalamus and brainstem may play roles on neuroendocrine and autonomic functions, such as water/salt balance, in rats.     

Cardiovascular responses to subseptic doses of endotoxin contribute to differential neuronal activation in rat brain

The contribution of cardiovascular activity in the early central responses to systemic inflammation was assessed in rats following intravenous administration of subseptic doses of lipopolysaccharide (LPS). LPS at 12.5 microg/kg increased heart rate (HR) but did not alter mean arterial pressure (MAP), and induced interleukin-1 beta (IL-1 beta) gene expression at 1 h in circumventricular organs (CVOs), choroid plexus, meninges, blood vessels, and pituitary gland. IL-1 beta mRNA levels were attenuated at 2 h in most regions studied. LPS at 50 microg/kg caused a biphasic change in MAP, increased HR, increased levels of arginine vasopressin heteronuclear RNA in the hypothalamic paraventricular nucleus (PVN), and induced IL-1 beta gene expression in the nucleus of the solitary tract (NTS) at 1 h. LPS (both doses) induced Fos-like immunoreactivity (FLI) in the area postrema, organum vasculosum of the lamina terminalis, NTS, preoptic area, supraoptic nucleus, and PVN at 1 h. In the PVN, neurons with FLI were found primarily in the dorsal and dorsal medial parvocellular divisions after 12.5 microg/kg of LPS whereas neurons with FLI were found throughout the PVN after 50 microg/kg of LPS. After 2 h, FLI was widespread throughout the brain. Plasma ACTH levels were elevated at 1 and 2 h in response to both doses of LPS, and levels of CRF mRNA were increased after 2 h in the parvocellular PVN. Our results reveal that central responses to increasing doses of LPS show different patterns which are related to activation of distinct immune and viscerosensory pathways, and that cardiovascular responses contribute to early neuronal activation as LPS concentrations are increased.     

Colocalization of urocortin and neuronal nitric oxide synthase in the hypothalamus and Edinger-Westphal nucleus of the rat

Different lines of studies suggest that both the corticotropin-releasing hormone-related peptide Urocortin I (Ucn) and the neuromodulator nitric oxide (NO) are involved in the regulation of the complex mechanisms controlling feeding and anxiety-related behaviors. The aim of the present study was to investigate the possible interaction between Ucn and NO in the hypothalamic paraventricular nucleus (PVN), an area known to be involved in the modulation of these particular behaviors. Therefore, we mapped local mRNA and peptide/protein presence of both Ucn and the NO producing neuronal NO synthase (nNOS). This investigation was extended to include the hypothalamic supraoptic nucleus (SON) and the Edinger-Westphal nucleus area (EW), the latter being one of the major cellular Ucn-expressing sites. Furthermore, we compared the two predominantly used laboratory rat strains, Wistar and Sprague-Dawley. Ucn mRNA and immunoreactivity were detected in the SON and in the EW. A significant difference between Wistar and Sprague-Dawley rats was found in mRNA levels in the EW. nNOS was detected in all brain areas analyzed, showing a significantly lower immunoreactivity in the PVN and EW of Sprague-Dawley versus Wistar rats. Contrary to some previous reports, no Ucn mRNA and only a very low immunoreactivity were detectable in the PVN of either rat strain. Interestingly, double-labeling immunofluorescence revealed that in the SON approximately 75% of all cells immunoreactive for Ucn were colocalized with nNOS, whereas in the EW only approximately 2% of the Ucn neurons were found to contain nNOS. These findings suggest an interaction between Ucn and NO signaling within the SON, rather than the PVN, that may modulate the regulation of feeding, reproduction, and anxiety-related behaviors.     

Indomethacin Attenuates Oxytocin and Hypothalamic-Pituitary-Adrenal Axis Responses to Systemic Interleukin-1β

Systemic administration of the cytokine IL-1β produces a significant release of ACTH into the plasma and activation of hypothalamic oxytocin (OT) and corticotropin releasing factor (CRF) cells. However, the mechanism(s) by which systemic IL-1β induces these responses is not clear. In the present study, we have investigated the proposal that catecholamine cells of the ventrolateral medulla (VLM) and nucleus of the solitary tract (NTS) can relay circulating IL-1 signals via a prostaglandin-dependent mechanism to effect the HPA axis responses in the rat. Intra-arterial administration of IL-1β (1 pg/kg) to otherwise untreated animals produced a prominent release of ACTH into the plasma, substantial c-fos expression in paraventricular medial parvocellular (mPVN) corticotropin releasing factor (CRF) cells, supraoptic (SON) and paraventricular nucleus (PVN) OT cells, area postrema cells, NTS and VLM catecholamine cells and cells of the central amygdala. Pretreatment with the prostaglandin synthesis inhibitor, indomethacin (10 mg/kg body weight ia) 15 min before IL-1β administration (1 pg/kg ia) significantly reduced plasma ACTH release and c-fos expression in PVN and SON OT cells and MPVN CRF cells. In addition, the area postrema, A1 and C1 catecholamine cell groups of the VLM and A2 and C2 catecholamine cell groups of the NTS, all exhibited concomitant reductions in c-fos expression. Conversely indomethacin administration did not alter the IL1β-induced expression of c-fos in the central amygdala. These data suggest that central pathways involved in the IL-1β-induced activation of the HPA axis and OT cells are, at least in part, dependent upon prostaglandin synthesis. It is proposed that neurons in the area postrema, NTS and VLM might mediate this IL-1β-induced activation of hypothalamic CRF and OT cells and release of ACTH into the plasma.     

Corticotropin-releasing factor type-1 receptor mRNA is not induced in mouse hypothalamus by either stress or osmotic stimulation

In rats, acute stress substantially increases corticotropin-releasing factor (CRF) type 1 receptor (CRFR-1) mRNA expression in the paraventricular nucleus (PVN) and osmotic stimulation induces both CRF and CRFR-1 mRNA in magnocellular PVN and supraoptic nucleus (SON). However, these phenomena have not been analysed in other species. We compared CRF and CRFR-1 expression in rat and mouse hypothalamus. Male C57BL/6 mice and Wistar rats were exposed to acute restraint stress for 3 h, or to hypertonic saline ingestion for 7 days. Restraint stress increased CRF and c-fos mRNA expression in both rat and mouse PVN. CRFR-1 mRNA was barely detectable in controls, whereas restraint stress substantially increased CRFR-1 mRNA in rat PVN, but not in mouse. Hypertonic saline ingestion induced CRF mRNA in magnocellular PVN and SON of the rat, but did not alter CRF mRNA levels in mouse hypothalamus. CRFR-1 mRNA was also induced in magnocellular PVN and SON of the rat in response to osmotic stimulation, but not in mouse. Immunohistochemistry demonstrated that CRFR-1-like immunoreactivity (ir) was distributed within parvocellular and magnocellular PVN of mouse and rat. CRFR-1-ir in rat PVN was increased by acute stress and osmotic stimulation. By contrast, these treatments did not alter CRFR-1-ir in mouse PVN. Combined immunohistochemistry and in situ hybridization revealed that CRFR-1-ir was most frequently colocalized to CRF in mouse PVN, whereas only a small percentage of oxytocin and vasopressin-producing cells coexpressed CRFR-1-ir. These results indicate that (i) by contrast to rats, neither acute stress nor osmotic stimulation induces CRFR-1 mRNA expression in the mouse PVN; (ii) osmotic stimulation does not alter CRF mRNA expression in parvocellular and magnocellular neurones of mouse PVN; and (iii) acute stress increases c-fos and CRF mRNA to a similar degree in mouse and rat PVN. Thus, differences may exist between mouse and rat in the regulation of CRF and CRFR-1 gene expression in hypothalamus following stress and osmotic stimulation.     

Distribution of c-fos mRNA in the brain following intracerebroventricular injection of nitric oxide (NO)-releasing compounds: possible role of NO in central cardiovascular regulation

We injected nitric oxide (NO)-releasing compounds and NO synthase (NOS) inhibitors into the brains of conscious, freely moving rats and measured the effects on mean arterial blood pressure (MAP) and heart rate, as well as on the expression of c-fos mRNA, neuronal NOS (nNOS) mRNA and NADPH-diaphorase, an indicator of NOS activity. When administered i.c.v., the NO donor, NOC-18, caused a significant fall in MAP and heart rate, whereas the NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), induced a significant rise in MAP. The same dose of NOC-18 or L-NAME when administered i.v. did not affect MAP and heart rate. Centrally administered NOC-18 induced c-fos mRNA expression in several regions of the brain involved in the baroreceptor response, including the nucleus of the solitary tract, the area postrema and the rostral ventrolateral medulla, as well as areas involved in the integration of autonomic, neuroendocrine and behavioural responses, including the medial preoptic area, the organum vasculosum lamina terminalis, the bed nucleus of stria terminalis, the paraventricular nucleus (PVN), the supraoptic nucleus (SON), the central nucleus of amygdala (CeA) and the locus coeruleus. Most of the areas that expressed c-fos also contained nNOS mRNA and/or NADPH-d-positive neurones and fibres. i.c.v. injection of L-NAME induced c-fos mRNA expression in PVN, SON, locus coeruleus and NTS, suggesting a tonic inhibition of neuronal activity by NO or stimulation of neuronal activity by endogenous NO. i.v. injection of NOC-18 or L-NAME did not induce any significant c-fos mRNA expression in rat brain. These results demonstrate that NO acts directly in the brain to reduce the systemic blood pressure, and that the endogenous NO pathway may play a role in cardiovascular and autonomic regulation by modulating neuronal activities in discrete regions of the brain.     

Area postrema ablation attenuates activation of neurones in the paraventricular nucleus in response to systemic adrenomedullin

Adrenomedullin (ADM) is a potent vasodilator in the periphery which also acts centrally to increase blood pressure and inhibit drinking, feeding and salt appetite. This study was designed to study the effects of circulating ADM on neuronal activation in autonomic centres in the rat brain and to examine whether neuronal nitric oxide (NO) may participate in these processes. We identified activated neurones 1 h after intravenous (i.v.) injections of ADM (2 nmol/kg) using immunohistochemistry for Fos. The nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemical reaction was used to localize putative NO-producing neurones and double labelling for Fos and NADPH-d was used to identify activated NO producing neurones. To separate baroreceptor-induced neuronal activation in autonomic centres by ADM from other effects which it may have, i.v. infusions of sodium nitroprusside (NP) were used to mimic the hypotensive effects of ADM in control rats. Significantly greater numbers of activated neurones were found in the paraventricular nucleus of the hypothalamus (PVN) and especially in the dorsolateral medial parvocellular division, the nucleus of the solitary tract, and the area postrema (AP) of ADM-treated rats compared to control rats. In addition, the number of activated NO-producing neurones in the PVN was significantly higher in ADM-treated rats compared to rats treated with NP. To determine whether AP is one of the possible routes through which systemic ADM enters the brain to exert its central effects, the APs of rats were ablated by aspiration. One hour after i.v. injections of ADM, significantly fewer PVN neurones were activated in AP ablation rats compared to AP sham ablation rats. Similarly, the number of activated NO-producing neurones in the PVN was significantly lower in AP ablation rats compared to AP sham ablation rats. In conclusion, our results suggest that systemic ADM gains access to the brain through the AP to regulate neuronal activity in autonomic centres and that neuronal NO might be involved in central autonomic and/or neuroendocrine regulation by ADM.     

Regulation of rat APJ receptor messenger ribonucleic acid expression in magnocellular neurones of the paraventricular and supraopric nuclei by osmotic stimuli

The novel apelin receptor (APJ receptor, APJR) has a restricted expression in the central nervous system suggestive of an involvement in the regulation of body fluid homeostasis. The endogenous ligand for APJR, apelin, is also highly concentrated in regions that are involved in the control of drinking behaviour. While the physiological roles of APJR and apelin are not fully known, apelin has been shown to stimulate drinking behaviour in rats and to have a regulatory effect on vasopressin release from magnocellular neurones of the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei. To determine the role of APJR in the regulation of water balance, this study examined the effects of osmotic stimulation on the expression of APJR mRNA in the magnocellular PVN (mPVN) and SON of salt-loaded and water-deprived rats. Intake of 2% NaCl and water deprivation for 48 h induced expression of APJR mRNA in the mPVN and SON. Using dual-label in situ hybridization histochemistry, we also investigated whether APJR is colocalized within vasopressin neurones in control, salt-loaded and water-deprived rats. APJR mRNA was found to colocalize with a small population of vasopressin-containing magnocellular neurones in control and water-deprived rats. Salt-loading resulted in an increased colocalization of APJR and vasopressin mRNAs in the SON. These data verify a role for APJ receptors in body fluid regulation and suggest a role for apelin in the regulation of vasopressin-containing neurones via a local autocrine/paracrine action of the peptide.     

Water deprivation upregulates the three calmodulin genes in exclusively the supraoptic nucleus of the rat brain

Calmodulin (CaM), the ubiquitous intracellular calcium-binding protein, is coded by three bona fide CaM genes (CaM I, CaM II and CaM III) in mammals. They code for the same protein and are transcribed at particularly high levels in the brain, where CaM plays an essential role in basic neuronal functions. In this study, the expression of the three CaM genes in response to osmotic stimuli by water deprivation was investigated in the rat brain, with particular interest as concerns the hypothalamic magnocellular nuclei. CaM mRNA levels were determined by quantitative in situ hybridization autoradiography with gene-specific [35S]cRNA probes. In response to osmotic challenge, it was found that upregulation of the three CaM genes participates in the activation of the hypothalamo-hypophyseal system in the supraoptic nucleus (SON) (126% to 169%), but not in the magnocellular part of the paraventricular hypothalamic nucleus (PVN) (-10%). CaM mRNA levels decreased by 10%-15% in the suprachiasmatic nucleus (SCh) and many other extrahypothalamic brain areas. The opposite responses of the CaM gene expression in the SON and the magnocellular part of the PVN suggest a functional difference between them. Moreover, the significantly different magnitudes of the changes in the CaM mRNA levels in the SON nucleus (138%, 126% and 169% for CaM I, CaM II and CaM III, respectively) exemplify the precise differential control of the CaM gene expression in the brain.     

APJ receptor mRNA expression in the rat hypothalamic paraventricular nucleus: regulation by stress and glucocorticoids

The apelin receptor (APJ receptor, APJR) has recently come to prominence following the isolation and identification of its endogenous ligand, apelin, from bovine stomach tissue extracts. Investigation of APJR mRNA expression has revealed a hypothalamic distribution similar to that of vasopressin suggesting that the apelin-APJR system may be involved in the regulation of the hypothalamic-adrenal-pituitary (HPA) stress axis. To investigate whether APJR is involved in the regulation of hypothalamic function during stress, APJR mRNA expression levels were measured by in situ hybridization in the hypothalamus of rats subjected to acute and repeated restraint stress. Acute stress caused an increase in APJR mRNA expression in the hypothalamic parvocellular paraventricular nucleus (pPVN) while repeated restraint stress induced a sustained up-regulation of pPVN APJR mRNA expression in intact rats. Removal of endogenous glucocorticoids by adrenalectomy also resulted in an increased expression of APJR mRNA in the PVN, suggesting a negative regulation of APJR mRNA expression by glucocorticoids. The role of glucocorticoids in mediating these stress-induced changes was investigated by analysing the effects of acute and repeated restraint stress on APJR mRNA levels in adrenalectomized rats. In these rats, APJR mRNA expression levels did not change above the already elevated levels of adrenalectomized-control rats. These data suggest that acute and repeated stress exert a stimulatory influence on APJR mRNA expression at the hypothalamic level that may be dependent on basal levels of circulating glucocorticoids, and further suggest a role for APJR in the regulation of hypothalamic function.     

The Effect of Acute and Chronic Restraint on the Central Expression of Prepro-Neuropeptide Y mRNA in Normotensive and Hypertensive Rats

Neuropeptide Y (NPY), one of the most abundant neuropeptides found in the central nervous system (CNS), has been implicated in the regulation of many autonomic functions, including cardiovascular control and the central stress response. The present study represents a detailed investigation of the effects of acute and chronic restraint stress on the expression of the mRNA encoding the NPY precursor, prepro-NPY, in the CNS of normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) using in situ hybridization histochemistry. Basal (unstressed) levels of prepro-NPY mRNA expression were found to be significantly increased in the hypothalamic arcuate nucleus of SHR compared to WKY rats, with similar levels of prepro-NPY mRNA expression found in the remaining central nuclei. Following exposure to both acute and chronic restraint, significant changes in prepro-NPY mRNA expression were found in a variety of central regions in both strains, including the arcuate nucleus and hippocampus (both strains), medial amygdala and cortex (WKY only), and dentate gyrus, nucleus of the solitary tract and ventrolateral medulla (SHR only). A comparison of the temporal response to restraint revealed that significant differences between strains existed in regions such as the arcuate nucleus, hippocampus and dentate gyrus, providing further evidence that hypertensive rats apparently have an impaired neural stress response. The present study demonstrates that exposure to restraint results in significant changes in prepro-NPY mRNA expression in specific nuclei of both WKY and SHR that are components of not only the central circuitry regulating the stress response, but also the neural network modulating autonomic function.     

Food or Water Deprivation Modulate Nitric Oxide Synthase (NOS) Activity and Gene Expression in Rat Hypothalamic Neurones: Correlation with Neurosecretory Activity?

Nitric oxide (NO) is produced by the enzyme NO synthase (NOS) and may be involved in the regulation of nutrient and endocrine homeostasis via actions on neurones of the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei. The effects of water deprivation or food deprivation for 4 days on the abundance of messenger RNA encoding NOS in these nuclei in rats were examined using in situ hybridization. Water deprivation markedly increased the abundance of NOS mRNA in both the SON and PVN (225±11% of control, P<0.05 and 261±34% of control, P<0.01 respectively). NOS mRNA abundance also appeared to be increased in magnocellular accessory nuclei. Food deprivation decreased NOS mRNA abundance in the SON and PVN (42±6% and 52±7% of control respectively, both P<0.05), while withdrawal of both food and water produced no significant net changes in the abundance of NOS mRNA. Treatment-induced alterations in NOS mRNA abundance were reflected by changes in NOS activity, as assessed by NADPH-diaphorase histochemistry, and NADPH-diaphorase staining was observed in neurones both positive and negative for oxytocin-like immunoreactivity. These findings suggest that NOS mRNA abundance, NOS enzymatic activity and presumably NO production are modulated in an activity-dependent manner in hypothalamic (magnocellular and parvocellular) neurones by alterations in fluid and nutrient homeostasis, and support data from other studies suggesting a role for NO in the central regulation of water and food intake in the rat.     

Peptide GEGLSS-Like Immunoreactivity in the Rat Central Nervous System

A rabbit antiserum was raised against the N-terminal fragment peptide, GEGLSS (Gly-Glu-Gly-Leu-Ser-Ser) of bovine neuropeptide AF (NPAF, A18Famide). NPAF is an octadecapeptide isolated from the bovine brain together with neuropeptide FF (NPFF). GEGLSS-like immunoreactivity was localized with immunofluorescence technique in colchicine-treated rats in neuronal cell bodies of the supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei. A few neurons were also observed in the retrochiasmatic part of the SON. GEGLSS-like immunoreactivity was also localized to nerve terminals of the posterior pituitary. No GEGLSS-ir neuronal cell bodies were observed in the medial hypothalamus, in an area that contains NPFF-ir neurons. GEGLSS immunoreactivity was also seen in the fibers and terminals of nucleus of the solitary tract. We injected a retrograde tracer, fluorogold, to the posterior pituitary gland and visualized GEGLSS-ir neuronal cell bodies double-labeled with the tracer in SON, PVN, and SOR. The pituitary stalk transsection totally abolished the GEGLSS-ir structures from the posterior pituitary. Our results suggest that GEGLSS immunoreactivity in the rat brain has a more limited distribution than NPFF immunoreactivity. GEGLSS immunoreactivity was partially colocalized with arginine-vasopressin and oxytocin in neuronal cell bodies in the SON and PVN. Considering the fact that the known rat NPFF-NPAF precursor does not contain GEGLSS structure, the detected GEGLSS immunoreactivity may be derived from a previously unknown precursor.     

Vasopressin mRNA regulation in individual hypothalamic nuclei: a northern and in situ hybridization analysis

The present study examines the relative levels of vasopressin (AVP) mRNA within the paraventricular (PVN), supraoptic (SON), and suprachiasmatic (SCN) nuclei of the rat hypothalamus, and details the rates at which these levels change over the course of a 6 d salt-loading regimen. The quantitation of vasopressin mRNA was achieved by using three different procedures: (1) cell-free translation in rabbit reticulocyte lysate or (2) Northern analysis of poly(A)RNAs isolated from micro-punch dissected SON, PVN, and SCN, and (3) in situ hybridization histochemistry. The former involved the quantitative immunoprecipitation of the neurophysin precursors containing arginine8-vasopressin (AVP) or oxytocin, and the latter two techniques employed a radiolabeled synthetic oligodeoxynucleotide complementary to the 3' region of the AVP mRNA. Both the cell-free studies and the Northern gel analyses detected a sevenfold increase of AVP mRNA in the SON, a fivefold increase in the PVN, and no significant change in the SCN following 6 d of salt-loading. After the initiation of salt-drinking, these increases were shown to occur between 24 and 48 hr in the SON and between 48 and 72 hr in the PVN. The in situ hybridization studies revealed the anatomically correct hybridization of either 32P- or 3H-labeled AVP oligonucleotide to magnocellular perikarya within both the SON and PVN. Autoradiographic grains could be shown to be confined to the cytoplasm of these cells, and could be co-localized with immunoreactivity directed against the carboxy terminus of the AVP percursor. Comparison of x-ray level autoradiograms of control and 6 day salt-loaded SON revealed up to a sevenfold increase in specific signal in the salt-loaded sections. It is concluded that the response of AVP mRNA to osmotic stimuli in the three hypothalamic nuclei is heterogeneous, and that this heterogeneity can be explained by separating AVP neurons into two systems: one responsible for eliciting the antidiuretic actions of AVP via plasma AVP levels, and the other involved in CNS activities not directly involved with antidiuresis.     

Chondroitin sulfate proteoglycan phosphacan/RPTPbeta in the hypothalamic magnocellular nuclei

The hypothalamo-neurohypophysial system synthesizes and releases arginine vasopressin (AVP) and oxytocin (OXT) with physiological stimulation. In the present study, we investigated localization of a chondroitin sulfate proteoglycan (CSPG), phosphacan/RPTPbeta, in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of adult rats at both the light and electron microscopic levels. Immunohistochemical analyses demonstrated stronger phosphacan/RPTPbeta immunoreactivity within the SON and PVN compared with adjacent hypothalamic areas. Double labeling experiments showed phosphacan/RPTPbeta immunoreactivity constituting punctate networks to surround the somata and dendrites of AVP- and OXT-secreting magnocellular neurons. Electron microscopic examination further revealed strong phosphacan/RPTPbeta immunoreactivity at extracellular membrane surface of some axons, somata, and dendrites of the SON, but not of synaptic junctions. Interestingly, phosphacan/RPTPbeta immunoreactivity was also observed at extracellular surface membrane between astrocytic processes and neurons rather than between magnocellular neurons. The present results indicate the high expression of the CSPG, phosphacan/RPTPbeta at the extracellular space in the hypothalamic AVP- and OXT-secreting magnocellular neurons.