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.     

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.     

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.     

Exaggerated response of arginine vasopressin-enhanced green fluorescent protein fusion gene to salt loading without disturbance of body fluid homeostasis in rats

We examined the effects of chronic salt loading on the hypothalamic expressions of the enhanced green fluorescent protein (eGFP), arginine vasopressin (AVP) and oxytocin (OXT) genes in AVP-eGFP transgenic rats that expressed eGFP in the hypothalamic AVP-containing neurones. In these rats, salt loading for 5 days caused a marked increase of the eGFP fluorescence in the magnocellular divisions of the paraventricular nucleus (PVN), the supraoptic nucleus (SON) and the internal layer of the median eminence. Expression of the eGFP gene was increased seven- to eight-fold in the PVN and SON of salt-loaded rats in comparison with euhydrated rats. By contrast, none of these changes were observed in the suprachiasmatic nucleus. The expression of the AVP and OXT genes was increased 1.5- to two-fold in the PVN and SON of salt-loaded nontransgenic (control) and transgenic rats. There were no differences in the expression levels of the AVP and OXT genes in the PVN and SON between nontransgenic (control) and transgenic animals under normal conditions and after salt loading. In the posterior pituitary gland, the intensity of the eGFP fluorescence did not change after salt loading for 5 days, but increased after 10 days of salt loading. Upon salt loading, significant increases in the plasma AVP concentrations, plasma osmolality and plasma Na+ were observed. Furthermore, there were no significant differences in changes of water intake, food intake, urine volume, urine osmolality, urine Na+ concentrations, and the body weights in both models under normal or salt-loaded conditions. Our results show that the response of the AVP-eGFP fusion gene to chronic salt loading is exaggerated, and humoral responses such as AVP and OXT and the body fluid homeostasis are maintained in AVP-eGFP transgenic rats. The AVP-eGFP transgenic rat gives us a new opportunity to study the dynamics of the AVP system in vivo.     

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.     

The magnocellular arginine-vasopressin mRNA responds differently to food deprivation between the supraoptic and paraventricular nuclei of the hypothalamus in adrenalectomized rats with low corticosterone replacement.

We previously reported that food deprivation significantly decreased arginine-vasopressin (AVP) mRNA levels in the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus and also greatly stimulated the pituitary-adrenocortical system in rats. In this study, we deprived adrenalectomized rats with subcutaneously implanted low-dose corticosterone pellets (ADX + B) of food for 3 days to investigate the involvement of corticosteroid feedback regulation in the food deprivation-induced decrease in AVP mRNA in both the SON and the PVN. The plasma corticosterone levels in these animals were maintained at low levels constantly over 24 h. The ACTH concentration in the morning plasma was markedly increased in the food-deprived ADX + B rats as compared to the fed ADX + B rats. Food deprivation significantly decreased the corticotropin-releasing hormone (CRH) content in the median eminence and increased the CRH and AVP content in the neurointermediate lobe of the pituitary. Semiquantitative in situ hybridization histochemistry revealed that AVP mRNA levels were decreased in the SON but, inversely, increased in magnocellular as well as parvocellular subdivisions of the PVN following food deprivation. These results suggest that: (1) AVP mRNA responds differently to food deprivation between the SON and the PVN; (2) the glucocorticoid feedback can exert on AVP mRNA in the PVN but not in the SON in the food-deprived rats; and (3) food deprivation affects the neurohypophysial levels of CRH and AVP.     

Expression of the arginine vasopressin gene in response to salt loading in oxytocin gene knockout mice

Accumulating evidence suggests that both oxytocin and arginine vasopressin (AVP) are vital components in the regulation of body fluid balance. However, the physiological role of oxytocin and possible cooperative interactions between oxytocin and AVP in sodium balance remain obscure, even though recent studies using oxytocin knockout (OTKO) mice suggested that oxytocin may contribute to the regulation of salt appetite. In the present study, we examined the effects of salt loading (drinking 2% NaCl for 5 days) on the expression of the AVP gene in the paraventricular (PVN) and supraoptic nuclei (SON) of wild-type, OTKO and heterozygous littermates using in situ hybridization histochemistry. In addition, the effects of salt loading on the expression of the oxytocin gene were also examined in wild-type and heterozygous mice. Under the non salt-loaded condition, the levels of AVP mRNA in the PVN and SON of OTKO mice were significantly decreased compared to those in wild-type mice. Nevertheless, the up-regulation of the expression of the AVP gene in response to salt loading was preserved in OTKO mice. The degree of the up-regulation in OTKO mice tended to be greater compared to those in wild-type mice, suggesting compensatory up-regulation of the expression of the AVP gene in OTKO mice after salt loading. The basal levels of oxytocin mRNA in the PVN and SON of heterozygous mice were significantly lower than those in wild-type mice. Salt loading caused an increase of oxytocin mRNA levels in the PVN and SON of both wild-type and heterozygous mice. The ratios of increase of oxytocin mRNA levels were very similar between wild-type and heterozygous mice, suggesting that the single remaining oxytocin gene in heterozygous mice responds normally to an osmotic cue. Finally, salt loading tended to increase the serum concentration of sodium regardless of genotype, and there were no genotype differences in both the control and salt-loaded groups. These results suggest ways in which oxytocin may play a cooperative role together with AVP in the regulation of sodium balance.     

cGMP-induced presynaptic depression and postsynaptic facilitation at glutamatergic synapses in visual cortex

This study was aimed to examine the neuronal and glial response in the hypothalamus and neurohypophysis of rats with streptozotocin-induced diabetes. At various time intervals after induction of diabetes the neurons in the paraventricular- (PVN) and supraoptic- (SON) nucleus showed upregulated arginine vasopressin (AVP) and oxytocin (OXT) immunoexpression, being most pronounced at 2 weeks. Concomitant to this was the hypertrophy of PVN and SON neurons. NMDAR1, which was constitutively and moderately expressed in normal rats, was markedly augmented, being most intense at 4 months. This coincided with the expression of neuronal nitric oxide synthase (nNOS). Contrary to this, the expression of GluR2/3 was progressively downregulated, so that it was hardly detected at 4 months. Both astrocytes and microglia marked by anti-GFAP and OX-42, respectively, appeared activated. In pars nervosa, the projection target of the axon terminals of PVN and SON neurons, massive axons and terminals (Herring bodies) laden with neurosecretions were observed in diabetic rats. Colocalization study showed that the neurosecretions were internalized by activated pituicytes and microglia associated with the axons. The present results suggest that the neurosecretion of PVN and SON neurons is enhanced in diabetes. This is coupled by upregulation of NMDAR1 and nNOS but downregulation of GluR2/3. It is speculated that the glutamate receptors and NO are linked to overactivation of PVN and SON neurons leading ultimately to cell death of some of them. The pituicytes and microglia in pars nervosa would help to modulate the release of neurosecretion.     

Upregulation of corticotropin-releasing hormone gene expression in the paraventricular nucleus of cyclophosphamide-induced cystitis in male rats

We examined the effects of cyclophosphamide (CP)-induced cystitis on the expression of corticotropin-releasing hormone (CRH) mRNA in the paraventricular nucleus (PVN) and the serum levels of adrenocorticotropic hormone (ACTH) using in situ hybridization histochemistry and radioimmunoassay. In addition, the expression of AVP heteronuclear (hn) RNA and neuronal nitric oxide synthase (nNOS) mRNA was also examined in the PVN of a CP-induced cystitis model. We found that the levels of CRH mRNA were significantly increased in the PVN at 2 h after intraperitoneal administration of CP compared to those in saline-treated rats. The CRH mRNA levels in the PVN peaked at 12 h after CP administration and the levels were still significantly higher than those in saline-treated group at 24 h after CP administration. The serum ACTH levels in CP-treated group were also significantly higher compared to those in saline-treated group at any of the time points examined. Unlike previous findings showing upregulation of nNOS mRNA and AVP hnRNA under somatic nociceptive states, the levels of nNOS mRNA and AVP hnRNA were unchanged in the PVN following CP-induced cystitis, visceral nociceptive stimulation. These results suggest that visceral nociceptive stimulation as well as somatic nociceptive stimulation may activate the hypothalamo-pituitary axis but the hypothalamic neuroendocrine responses produced by visceral nociceptive stimulation may be different from those produced by somatic nociceptive stimulation.     

Sequential Exposure to Estrogen and Testosterone (T) and Subsequent Withdrawal of T Increases the Level of Arginine Vasopressin Messenger Ribonucleic Acid in the Hypothalamic Paraventricular Nucleus of the Female Rat

The hypothalamic peptides arginine vasopressin (AVP) and oxytocin (OT) have been implicated as mediators of socio-sexual behaviors in addition to their roles in osmolar homeostasis (AVP), milk ejection and uterine contractility (OT). Within 24  h of parturition, OT and AVP messenger ribonucleic acid (mRNA) levels increase in the hypothalamic paraventricular, and to a lesser degree, the supraoptic nucleus (PVN and SON) of the rat. We previously reported that the prepartum increase in OT mRNA is related to the spontaneous decline in progesterone levels prior to parturition. We also reported that increases in PVN and SON OT mRNA can be induced by exposing the ovariectomized rat to a steroid regimen that mimics the steroid milieu of pregnancy, namely sequential estrogen and progesterone and subsequent progesterone withdrawal. Levels of PVN and SON AVP mRNAs were not affected by progesterone withdrawal in late pregnant rats or the steroid regimen that increased OT mRNA in ovariectomized rats. These observations suggest that other factors, perhaps hormonal, may influence AVP mRNA levels. A decline in testosterone coincident with waning progesterone levels also occurs prepartum. Since peak levels of AVP mRNA prepartum coincide with the prepartum decline in testosterone, we questioned whether declining testosterone levels are important for the increase in AVP mRNA levels.     

Cold and novel environment stress affects AVP mRNA in the paraventricular nucleus, but not the supraoptic nucleus: An in Situ hybridization study

The distribution and area of label for arginine vasopressin (AVP) mRNA or peptides were studied in rats exposed to cold or novel environments. In situ hybridization histochemistry was employed to detect AVP mRNA in hypothalamic frozen sections with a 45-mer photobiotinylated oligonucleotide probe. The storage of the peptide in both the hypothalamus and the pituitary was determined by immunohistochemistry. Label for mRNA or peptide was then quantified by the Cue-3 color image analysis system. Exposure to 4°C for 30 min caused a 3.5-fold increase in the label for AVP mRNA in the paraventricular nucleus (PVN) compared with that of control rats. This was correlated with a 2-fold elevation in serum ACTH. In addition, rats exposed to 30 min of a novel, thermoneutral (24°C) environment showed a 1.2- to -2.3-fold enhancement of the label for AVP mRNA in the PVN. In contrast, no changes were seen in the supraoptic nucleus (SON) following exposure to either cold or novel environments. Furthermore, neither stress caused significant changes in the storage of AVP peptide in the PVN, SON, median eminence, and posterior lobe of pituitary. This in vivo study demonstrates that PVN and SON neurons respond differentially to cold and novel environment exposures. The elevation of serum ACTH is correlated with the increased level of label for AVP mRNA in the rat hypothalamus, which suggests that AVP may play a role in the regulation of pituitary—adrenal responses to cold and novel environment stresses.     

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.     

Response of Arginine Vasopressin-Enhanced Green Fluorescent Protein Fusion Gene in the Hypothalamus of Adjuvant-Induced Arthritic Rats

Arginine vasopressin (AVP) and corticotrophin-releasing hormone (CRH) in the parvocellular neurosecretory cells of the paraventricular nucleus (PVN) play a major role in activating the hypothalamic-pituitary-adrenal axis, which is the main neuroendocrine response against the many kinds of stress. We examined the effects of chronic inflammatory/nociceptive stress on the expression of the AVP-enhanced green fluorescent protein (eGFP) fusion gene in the hypothalamus, using the adjuvant arthritis (AA) model. To induce AA, the AVP-eGFP rats were intracutaneously injected heat-killed Mycobacterium butyricum (1 mg/rat) in paraffin liquid at the base of their tails. We measured AVP, oxytocin and corticosterone levels in plasma and changes in eGFP and CRH mRNA in the hypothalamus during the time course of AA development. Then, we examined eGFP fluorescence in the PVN, the supraoptic nucleus (SON), median eminence (ME) and posterior pituitary gland (PP) when AA was established. The plasma concentrations of AVP, oxytocin and corticosterone were significantly increased on days 15 and 22 in AA rats, without affecting the plasma osmolality and sodium. Although CRH mRNA levels in the PVN were significantly decreased, eGFP mRNA levels in the PVN and the SON were significantly increased on days 15 and 22 in AA rats. The eGFP fluorescence in the SON, the PVN, internal and external layers of the ME and PP was apparently increased in AA compared to control rats. These results suggest that the increases in the concentrations of ACTH and corticosterone in AA rats are induced by hypothalamic AVP, based on data from AVP-eGFP transgenic rats.     

Hypotension preferentially induces c-fos immunoreactivity in supraoptic vasopressin neurons.

Immunoreactivity to Fos protein (Fos-IR) was detected in rat hypothalamic neurons within 1 h of onset of hemorrhage by withdrawing 4-5 ml of blood, which lowered the arterial blood pressure to 50-70 mm Hg. About 70% of vasopressin (AVP)-containing neurons in the supraoptic nucleus (SON) and 20% in the paraventricular nucleus (PVN) expressed Fos-IR. In contrast, 5% of oxytocin (OXY)-containing neurons in the SON and < 1% in PVN were Fos-IR. Intravenous infusion of the vasodilating agent, nitroprusside, which lowered the blood pressure to levels comparable to that attained by hemorrhage, induced Fos-IR in greater than 65% of AVP-containing neurons in the SON, while relatively few AVP neurons in the PVN were Fos positive. These results suggest that hemorrhage or hypotension preferentially induces c-fos expression in supraoptic AVP-containing neurons.     

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.     

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.