Effects of leptin on fasting-induced inhibition of neuronal nitric oxide synthase mRNA in the paraventricular and supraoptic nuclei of rats

Fasting induced a reduction in neuronal nitric oxide synthase (nNOS) mRNA in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of rats. The effect of intracerebroventricular (i.c.v.) administration of leptin on the nNOS mRNA level in the PVN and SON of fasting rats was studied by in situ hybridization histochemistry. Leptin (10 microg/kg b.wt) or vehicle was administered i.c.v. at 1700 h on two successive days fasting for 2 days. Fasting for 2 days with i.c.v. administration of vehicle induced a significant reduction of nNOS mRNA in the PVN and SON. Central administration of leptin prevented the fasting-induced reduction of nNOS mRNA in the PVN and SON. Administration of leptin also prevented the fasting-induced reductions of thyrotropin-releasing hormone (TRH) and corticotropin-releasing hormone (CRH) mRNAs in the parvocellular division of the PVN. These results suggest that leptin is associated with fasting-induced reduction of nNOS mRNA in the PVN and SON as well as TRH and CRH mRNAs in the PVN.     

Increased nitric oxide synthase activity and expression in the hypothalamus of hindlimb unloaded rats

Upon return from spaceflight or resumption of normal posture after bed rest, individuals often exhibit cardiovascular deconditioning. Although the mechanisms responsible for cardiovascular deconditioning have yet to be fully elucidated, alterations within the central nervous system have been postulated to be involved. The paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus are important brain regions in control of sympathetic outflow and body fluid homeostasis. Nitric oxide (NO) modulates the activity of PVN and SON neurons, and alterations in NO transmission within these brain regions may contribute to symptoms of cardiovascular deconditioning. The purpose of the present study was to examine nitric oxide synthase (NOS) activity and expression in the PVN and SON of control and hindlimb unloaded (HU) rats, an animal model of cardiovascular deconditioning. The number of neurons exhibiting NOS activity as assessed by NADPH-diaphorase staining was significantly greater in the PVN but not SON of HU rats. Western blot analysis revealed that neuronal NOS (nNOS) but not endothelial NOS (eNOS) protein expression was higher in the PVN of HU rats. In the SON, there was a strong trend for an increase in nNOS (p=0.052) and a significant increase in eNOS expression in HU rats. Our results suggest that increased nNOS in the PVN contributes to autonomic and humoral alterations following cardiovascular deconditioning. In contrast, the functional significance of increases in nNOS and eNOS protein in the SON may be related to alterations in vasopressin release observed previously in HU rats.     

Role of arginine vasopressin and corticotropin-releasing factor in mediating alcohol-induced adrenocorticotropin and vasopressin secretion in male rats bearing lesions of the paraventricular nuclei

In male rats, lesions of the paraventricular nucleus (PVN) of the hypothalamus attenuate, but do not abolish, adrenocorticotropin (ACTH) secretion in response to acute alcohol injection. As the PVN is the major source of corticotropin-releasing factor (CRF) in the median eminence, this observation suggests that extra-PVN brain regions, and/or ACTH secretagogues other than CRF (e.g. arginine vasopressin (AVP)), mediate ACTH stimulation by alcohol. This hypothesis was tested by examining the effect of AVP immunoneutralization in PVN-lesioned (PVNx) rats. Removal of endogenous AVP diminished alcohol-evoked ACTH secretion in both sham-operated and PVNx animals, indicating that AVP from outside the PVN partially mediates the hypothalamic-pituitary-adrenal (HPA) axis response to alcohol. This led us to determine whether alcohol might also regulate AVP steady-state gene expression in the supraoptic nucleus (SON) and PVN, and/or CRF mRNA in the PVN and the central nucleus of the amygdala (AMY). In the magnocellular portion of the PVN, sham-operated animals showed significantly increased PVN levels of both CRF and AVP mRNAs 3 h after alcohol. In the SON, alcohol administration tended to decrease AVP gene expression in PVNx rats, while the drug increased AVP mRNA levels in the SON of sham-operated rats. AMY levels of CRF mRNA were unaffected by these manipulations. Finally, since the regulation of alcohol-induced AVP mRNA levels in the SON appeared to depend on the presence of the PVN, we measured peripheral levels of AVP in both sham-operated and PVNx animals after injection of vehicle or alcohol. Although AVP decreased in all groups, alcohol depressed AVP secretion to a greater extent in PVNx animals, suggesting that AVP systems are more sensitive to inhibition in the absence of the PVN. Our results demonstrate that although AVP of PVN origin may participate in regulating the stimulatory effect to AVP on ACTH secretion, AVP from areas other than the PVN also plays a role. Additionally, regulation of both AVP gene expression in the SON and secretion in the systemic circulation are altered in rats bearing lesions of the PVN.     

Nociceptive stimulation increases NO synthase mRNA and vasopressin heteronuclearRNA in the rat paraventricular nucleus

Nociceptive stimulation causes neuroendocrine responses such as arginine vasopressin (AVP) release and activation of the hypothalamo-pituitary-adrenal (HPA) axis. We examined the effects of nociceptive stimulation on the expression levels of neuronal nitric oxide synthase (nNOS) mRNA, heteronuclear (hn)RNA for AVP and AVP mRNA in the rat paraventricular nucleus (PVN) and supraoptic nucleus (SON), using in situ hybridization histochemistry. For nociceptive stimulation, formalin (5%) or saline was injected subcutaneously (s.c.) into the bilateral hind paws of rats. The expression of the nNOS gene in the PVN was significantly increased 2 and 6 h after s.c. injection of formalin in comparison with that in untreated and saline injected rats. The expression of the nNOS gene in the SON did not change in the untreated, saline- and formalin-injected rats. The AVP hnRNA in the PVN and SON was also significantly increased 15, 30 min and 2 h after s.c. injection of formalin, though AVP mRNA did not change at any time points that we studied. Plasma concentration of AVP was significantly increased 15 min after s.c. injection of formalin. These results suggest that NO in the PVN may be involved in nociceptive stimulation-induced neuroendocrine responses.     

迷走神经切断对胃肠道给予大鼠伤寒杆菌诱发的下丘脑室旁核和视上核Fos表达的影响

为研究迷走神经在自然感染状态下向脑传递免疫信息的作用。应用免疫组织化学方法,观察了切断隔下迷走神经对大鼠消化道内给予鼠伤寒杆菌刺激诱发的下丘脑室旁核和视上核的Fos表达变化的影响。结果发现,接受细菌刺激的动物与仅给予生理盐水的动物相比,回肠和肠系膜淋巴结有明显炎症存在,室旁结果发现,接受细菌刺激的动物与仅给予生理盐水的动物相比,回肠和肠系膜淋巴结有明显炎症存在,室旁核外侧部和视上核背侧部的Fos阳性细胞数增加;膈下迷走神经切断后,手术 细菌组与假手术 细菌组相比,室旁核的外侧部和视上核背部Fos表达减少。因此迷走神经途径在自然感染性免疫应答过程中,特别是在其早期阶段可能是传递腹腔免疫信息的重要途径之一。     

Depletion of oestrogen receptor-beta expression in magnocellular arginine vasopressin neurones by hypovolaemia and dehydration

Oestrogen receptor (ER)-beta expression correlates inversely with osmotic control of arginine vasopressin (AVP) release such that cellular dehydration induced by 72 h of 2% saline consumption depletes ER-beta in the magnocellular AVP neurones in the supraoptic (SON) and paraventricular nuclei (PVN). The current studies were performed to determine whether other pathways that stimulate AVP release, such as hypovolaemia, also regulate ER-beta expression in these nuclei, and to evaluate the time course of the change in ER-beta expression during water deprivation and subsequent rehydration. ER-beta expression was evaluated immunocytochemically. In rats made hypovolaemic with a subcutaneous injection of 40% polyethylene glycol (PEG), a significant depletion of ER-beta in both SON and magnocellular PVN (P 相似文献   

Effects of cholecystokinin (CCK)-8 on hypothalamic oxytocin-secreting neurons in rats lacking CCK-A receptor

Peripheral administration of cholecystokinin (CCK)-8 selectively activates oxytocin (OXT)-secreting neurons in the supraoptic (SON) and the paraventricular nuclei (PVN) with the elevation of plasma OXT level in rats. We examined the effects of intravenous (iv) administration of CCK-8 on the neuronal activity of hypothalamic OXT-secreting neurons and plasma OXT level in Otsuka Long-Evans Tokushima Fatty (OLETF) rats that have a congenital defect in the expression of the CCK-A receptor gene. In situ hybridization histochemistry (ISH) for c-fos mRNA revealed that the expression of the c-fos gene was not induced in the SON, the PVN, the nucleus of the tractus solitarius (NTS) and the area postrema (AP) 30 min after iv administration of CCK-8 (20 and 40 microg/kg) in OLETF rats. In Long-Evans Tokushima Otsuka (LETO) rats (controls), c-fos mRNA was detected abundantly in those nuclei 30 min after iv administration of CCK-8 (20 microg/kg). Immunohistochemistry for c-fos protein (Fos) showed that the distributions of Fos-like immunoreactivity (LI) were identical to the results obtained from ISH. Dual immunostaining for OXT and Fos revealed that Fos-LI was mainly observed in OXT-secreting neurons in the SON and the PVN of LETO rats 90 min after iv administration of CCK-8 (20 microg/kg). Radioimmunoassay for OXT and arginine vasopressin (AVP) showed that iv administration of CCK-8 did not cause significant change in the plasma OXT and AVP levels in OLETF rats, while iv administration of CCK-8 caused a significant elevation of plasma OXT level without changing the plasma AVP level in LETO rats. These results suggest that peripheral administration of CCK-8 may selectively activate the hypothalamic OXT-secreting neurons and brainstem neurons through CCK-A receptor in rats.     

Distinct distribution and time-course changes in neuronal nitric oxide synthase and inducible NOS in the paraventricular nucleus following lipopolysaccharide injection

Nitric oxide (NO) is known to be involved in the modulation of neuroendocrine function. To clarify the role of different isoforms of NO synthase (NOS) in the neuroendocrine response to immune challenge, the expressions of neuronal NOS (nNOS) and inducible NOS (iNOS) genes in the hypothalamus following lipopolysaccharide (LPS) injection were examined using in situ hybridization. NOS activity was also determined by NADPH-diaphorase (NADPH-d) histochemistry. LPS (25 mg/kg) or sterile saline was injected intraperitoneally to male Wistar rats and the rats sacrificed 30 min, or 1, 2, 3, 5, 12 or 24 h after injection. nNOS mRNA expression in the paraventricular nucleus (PVN) was significantly increased 2 h after LPS injection. iNOS mRNA, which was not detected until 2 h after LPS injection, was significantly increased in the PVN 3 h after LPS injection. Both RNA expressions had returned to basal levels by 12 h after LPS injection. The number of NADPH-d positive cells was significantly increased 5 h after LPS injection. iNOS expression was more robust in parvocellular PVN, while nNOS was distributed mainly in the magnocellular PVN. Double in situ hybridization histochemistry revealed that some of the iNOS- (48.4%) or nNOS-positive cells (34. 3%) in the parvocellular PVN expressed CRF mRNA. The results demonstrate that LPS-induced sepsis causes significant increases in nNOS and iNOS gene expression with different time-courses and distributions, and that iNOS mRNA was more frequently co-localized with CRF-producing parvocellular neurons in the PVN. Thus, NO produced by iNOS and nNOS may play an important role in the neuroendocrine response to an immune challenge. Distinct differences in the distribution and time-course changes of iNOS and nNOS suggest different roles for the hypothalamic-pituitary-adrenal axis and/or neurohypophyseal system.     

Adenovirus-mediated gene delivery to cells of the magnocellular hypothalamo-neurohypophyseal system

The objective of the present study was to define the optimum conditions for using replication-defective adenovirus (Ad) to transfer the gene for the green fluorescent protein (GFP) to the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei and cells of the neurohypophysis (NH). As indicated by characterizing cell survival over 15 days in culture and in electrophysiological whole cell patch-clamp studies, viral concentrations up to 2 x 10(7) pfu/coverslip did not affect viability of transfected PVN and NH cultured cells from preweanling rats. At 2 x 10(7) pfu, GFP gene expression was higher (40% of GFP-positive cells) and more sustained (up to 15 days). Using a stereotaxic approach in adult rats, we were able to directly transduce the PVN, SON, and NH and visualize gene expression in coronal brain slices and in the pituitary 4 days after injection of Ad. In animals receiving NH injections of Ad, the virus was retrogradely transported to PVN and SON neurons as indicated by the appearance of GFP-positive neurons in cultures of dissociated cells from those brain nuclei and by polymerase chain reaction and Western blot analyses of PVN and SON tissues. Adenoviral concentrations of up to 8 x 10(6) pfu injected into the NH did not affect cell viability and did not cause inflammatory responses. Adenoviral injection into the pituitary enabled the selective delivery of genes to the soma of magnocellular neurons. The experimental approaches described here provide potentially useful strategies for the treatment of disordered expression of the hormones vasopressin or oxytocin.     

Neuronal expression of fos protein in the forebrain of diabetic rats

We sought to identify the areas that have altered neuronal activity within the hypothalamus of diabetic rats by mapping neuronal expression of c-fos protein (Fos) and Fos-related antigens. After a standard PAP immunocytochemical protocol, Fos-like immunoreactivity was observed in the paraventricular nucleus (PVN), supraoptic nucleus (SON), median preoptic area (MnPO), anterior hypothalamus (AH) and posterior hypothalamus (PH) of control (vehicle; n=6) and diabetic rats (Sprague-Dawley rats injected with STZ 65 mg/kg/ip 4 weeks prior to the experiment; n=6). Blood glucose levels were significantly elevated in the diabetic group (370+/-8 mg/dl) compared to control group (104+/-3 mg/dl). Diabetic rats had a significantly higher number of Fos-positive cells in PVN (2.5x), SON (7x) and MnPO (2x) compared to the control rats. However, diabetic rats had significantly fewer Fos-positive cells in the AH (0.3x) and no difference was observed in the PH between the diabetic and control rats. Despite the elevated number of Fos-positive cells in the diabetic rats, dehydration (water withdrawal for 24 h) or hypertonic challenge (1.5 ml of 0.1 M NaCl i.p. injection) produced a further increase in the number of Fos-positive cells in the PVN, SON and MnPO. Dehydration did not alter the number of Fos-positive cells in the AH or PH, but hypertonic challenge produced a significant increase in the Fos-positive cells in both the AH and PH of diabetic rats. This study demonstrates that: (1) there is increased basal neuronal activity in the PVN, SON and MnPO, a decrease in neuronal activity in the AH and no change in neuronal activity in the PH as indicated by Fos staining in diabetic rats; and (2) dehydration or hypertonic challenge produces a further increase in the number of Fos-positive cells in the PVN, SON, and MnPO which is comparable to control rats. These data support the conclusion that vasopressin producing neurons in the PVN and SON and autonomic areas within the lamina terminalis and hypothalamus are activated during diabetes and may contribute to the elevated levels of vasopressin and autonomic dysfunction during diabetes.     

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.     

Activation of hypothalamic neuronal nitric oxide synthase in lithium-induced diabetes insipidus rats

The expression of the neuronal nitric oxide synthase (nNOS) gene in the paraventricular (PVN) and supraoptic nuclei (SON) in rats with lithium (Li)-induced polyuria was examined by using in situ hybridization histochemistry. The state of the thyroid axis in these rats was also examined by in situ hybridization histochemistry for thyrotropin-releasing hormone (TRH) and thyroid-stimulating hormone (TSH) mRNAs and radioimmunoassay for circulating thyroid hormones. Adult male Wistar rats consuming a diet that contained LiCl (60 mmol/kg) for 4 weeks developed remarkable polyuria. The urine in the Li-treated rats was hypotonic and had a large volume and low ionic concentration. The nNOS mRNA in the PVN and SON was significantly increased in the Li-treated rats in comparison with that in control. The increased levels of the nNOS mRNA in the PVN and SON were confirmed by NADPH-diaphorase histochemical staining. There were no differences of TRH mRNA in the PVN, TSH mRNA in the anterior pituitary and plasma concentrations of free T3 and free T4 between Li-treated rats and control rats. These results suggest that Li-induced diabetes insipidus may activate nNOS in the PVN and SON without change of the thyroid axis.     

Effects of alpha-melanocyte-stimulating hormone on magnocellular oxytocin neurones and their activation at intromission in male rats

The peptides alpha-melanocyte-stimulating hormone (alpha-MSH) and oxytocin have very similar effects on several behaviours, including male sexual behaviour. Both induce penile erection and enhance copulatory behaviour when given centrally, suggesting that their central actions are not independent. Here, we used intromission as a physiological stimulus to investigate whether some central effects of alpha-MSH during male sexual behaviour are mediated by oxytocin neurones. We used the expression of the immediate-early gene product Fos to investigate oxytocin neurone activation at intromission and after intracerebroventricular (i.c.v.) administration of alpha-MSH (1 microg/5 microl) and studied the effects of i.c.v. administration of a MC4 receptor antagonist on Fos expression and on the latency of male rats to exhibit sexual behaviour in the presence of a receptive female. In rats that showed intromission, Fos was expressed in magnocellular oxytocin neurones in both the paraventricular nucleus (PVN) and the supraoptic nucleus (SON), but there was no significant activation of parvocellular oxytocin neurones of the PVN. Similarly, alpha-MSH increased Fos expression in magnocellular oxytocin neurones but had little or no effect in parvocellular oxytocin neurones. In male rats that achieved intromission, central injection of a MC4 receptor antagonist significantly attenuated the increase in Fos expression in magnocellular oxytocin neurones in both the PVN and the SON and increased mount and intromission latencies compared to vehicle-injected controls. Together, the results indicate that magnocellular oxytocin neurones are involved in the central regulation of male sexual behaviour, and that some of the central effects of alpha-MSH are likely to be mediated by magnocellular oxytocin neurones.     

Inter- and intra-nuclear differences in galanin expression between the hypothalamic paraventricular and supraoptic nuclei in colchicine-untreated rats

Not much is known of the topography of galanin expression in the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei neurons in colchicine (an axoplasmic inhibitor)-untreated animals. Insight into the biological implication(s) of galanin expression in the PVN and SON will depend, at least in part, on the nature of its distribution in colchicine-untreated animals. In this study therefore, the topographical distribution of galaninergic profiles was examined in the PVN and SON of colchicine-untreated rats. Staining in the parvocellular PVN (PVN(p)) was predominantly as varicose thin galanin fiber processes while the magnocellular PVN (PVN(m)) contained large cell soma and fiber processes. The relative fiber density was higher in the anterior, periventricular and medial PVN(p) than in the dorsal, lateral and posterior subdivisions. Large-sized cells and thick fibers were limited to the posterior PVN(m) while the anterior and medial PVN(m) contained varicose profiles. Light- and intensely-stained galanin-positive cells as well as large- and small-diameter (varicose or non-varicose) fibers were observed in the SON. The large and thin fibers exhibit preferential ventral and dorsal distribution, respectively. Together with the complexity of specific afferent and efferent connections within the PVN and SON, these observations underscore heterogeneous galanin expression and raise potential implications for understanding the biological role of galanin by physiologically challenging stimuli.     

Refeeding-induced expression of neuronal nitric oxide synthase in the rat paraventricular nucleus

We have previously reported that food deprivation decreases the expression of neuronal nitric oxide synthase (nNOS) in the hypothalamic paraventricular nucleus (PVN) of rats, and this reduction is inhibited by blockade of glucocorticoid receptors. In this study, we examined whether the fasting-induced decrease in nNOS gene expression in the PVN is restored by refeeding. The number of nNOS immunopositive cells in the PVN, which was markedly decreased by 48 h of food deprivation, increased significantly after 6 h of refeeding and was fully restored by 24 h after refeeding. The plasma corticosterone level, which was markedly increased by food deprivation, decreased significantly within 30 min after refeeding and returned to the free fed control level by 6 h. Synthetic glucocorticoid dexamethasone blocked the refeeding-induced nNOS expression in the PVN without suppressing food intake. Refeeding with a non-caloric food mash for 5 h failed to restore the fasting-induced decrease in the PVN-nNOS but did, however, successfully restore the plasma corticosterone level. These results suggest that the refeeding-induced nNOS expression in the PVN is a nutrient-directed event and that plasma glucocorticoids may play an inhibitory role in the regulatory pathway. Additionally, glucocorticoid disinhibition alone does not appear to be sufficient to induce nNOS expression in the PVN; nNOS expression in the PVN upon refeeding may require both nutrient supplementation and glucocorticoid disinhibition.     

Physiological studies of stress responses in the hypothalamus of vasopressin-enhanced green fluorescent protein transgenic rat

Arginine vasopressin (AVP) plays an important role in stress-induced activation of the hypothalamic-pituitary adrenal axis. In the present study, AVP-enhanced green fluorescent protein (eGFP) transgenic rats were used to investigate changes in AVP-eGFP expression in the hypothalamic paraventricular nucleus (PVN) and the median eminence (ME) upon exposure to stress conditions. The eGFP fluorescence in the parvocellular division of the PVN (pPVN) was markedly increased 5 days after bilateral adrenalectomy (ADX) and it was colocalised with corticotrophin-releasing hormone-like immunoreactivity in the pPVN. Peripheral administration of dexamethasone completely suppressed the increase of eGFP fluorescence in the pPVN and the external layer of the ME (eME) after bilateral ADX. Significant increases of eGFP fluorescence were observed in the pPVN 6, 12, 24 and 48 h after intraperitoneal (i.p.) administration of lipopolysaccharide (LPS). In the eME, eGFP fluorescence was significantly increased 48 h after i.p. administration of LPS. By contrast, eGFP fluorescence changed neither in the magnocellular division of the PVN, nor the internal layer of the ME after i.p. administration of LPS. Our results indicate that AVP-eGFP transgenic rats are useful animal model to study dynamic changes of AVP expression in the hypothalamus under stressful conditions.