Hypobaric hypoxia modifies constitutive nitric oxide synthase activity and protein nitration in the rat cerebellum

Ischemic hypoxia provokes alterations in the production system of nitric oxide in the cerebellum. We hypothesize that the nitric oxide system may undergo modifications due to hypobaric hypoxia and that may play a role in high altitude pathophysiology. Therefore, changes in the nitric oxide system of the cerebellum of rats submitted to acute hypobaric hypoxia were investigated. Adult rats were exposed for 7 h to a simulated altitude of 8235 m (27000 ft.) and then killed after 0 h or 1, 3, 5 and 10 days of reoxygenation. Nitric oxide synthase calcium-dependent and -independent activity, immunoblotting and immunohistochemistry of neuronal, endothelial, and inducible nitric oxide synthase, and nitrotyrosine were evaluated. Immunoreactivity for neuronal nitric oxide synthase slightly increased in the baskets of the Purkinje cell layer and in the granule cells, after 0 h of reoxygenation, although no changes in neuronal nitric oxide synthase immunoblotting densitometry were detected. Calcium-dependent activity significantly rose after 0 h of reoxygenation, reaching control levels in the following points, and being coincident with a peak of eNOS expression. Nitrotyrosine formation showed significant increments after 0 h and 1 day of reoxygenation. Nitrotyrosine immunoreactivity showed an intracellular location change in the neurons of the cerebellar nuclei and in addition, an appearance of nitration in the soma of the Purkinje cells was detected. No changes in inducible nitric oxide synthase activity, immunoblotting or immunohistochemistry were detected. We conclude that at least part of the nitric oxide system is involved in cerebellum responses to hypobaric hypoxia.     

Nitric oxide system and protein nitration are modified by an acute hypobaric hypoxia in the adult rat hippocampus

Changes in the nitric oxide system of the hippocampus from rats submitted to hypobaric hypoxia were investigated. Adult rats were exposed to a simulated altitude of 8,325 m (27,000 ft) for 7 h and killed after 0 h, 1, 3, 5, 10 and 20 days of reoxygenation. The number of neuronal nitric oxide synthase immunoreactive neurons and their dendritic plexus, as well as neuronal nitric oxide synthase immunoblotting densitometry and calcium-dependent activity increased from 0 h to 3 days of reoxygenation. In addition, endothelial nitric oxide synthase immunoreactivity peaked after 7 h of hypobaric hypoxia. Nitrotyrosine immunoreactivity showed an increase in the pyramidal cells of CA2-CA3 and in glial cells surrounding the blood vessels after 0 h, 1 and 3 days of reoxygenation. Immunoblotting densitometry of 1 of the 2 nitrotyrosine-immunoreactive bands detected also increased after 0 h and 1 day of reoxygenation. Inducible nitric oxide synthase immunoreactivity was found only in some blood vessels after 0 h, 1 and 3 days of reoxygenation, but no changes in inducible nitric oxide synthase activity or immunoblotting were detected. We conclude that transient activation of the nitric oxide system constitutes a hippocampal response to hypobaric hypoxia.     

Estrogenic Induction of NADPH- Diaphorase Activity in the Preoptic Neurons Containing Estrogen Receptor Immunoreactivity in the Female Rat

Nitric oxide and estrogen have been shown to play a critical role in the control of female reproductive function. In order to determine an anatomical relationship between nitric oxide generating neurons and estrogen target neurons, NADPH-diaphorase histochemistry was combined with estrogen receptor immunohistochemistry in the female medial preoptic area. While only a few weakly stained neurons for NADPH-diaphorase were found in ovariectomized control rats, a drastic increase in NADPH-diaphorase activity was observed in the medial preoptic nucleus of estradiol-treated ovariectomized animals. The total number of NADPH-diaphorase neurons in the estradiol-treated group increased three-fold relative to controls, and more than 80% of those neurons contained estrogen receptor-immunoreactivity in their nuclei. Since neuronal NADPH-diaphorase is nitric oxide synthase, the present result suggests that nitric oxide synthase activity can be positively regulated by estradiol in neurons containing estrogen receptor in the female medial preoptic nucleus.     

Regional alterations of the NO/NOS system in the aging brain: a biochemical, histochemical and immunochemical study in the rat

We have used several approaches (immunohistochemistry and enzyme histochemistry, Western blotting, biochemical assay of Ca(2+)-dependent catalytic activity) in order to detect differences in neuronal nitric oxide synthase (nNOS) expression and activity in various brain regions of young-adult (4-month-old) and aged (28-month-old) rats. In most of the brain regions examined (striatum, neocortex, olfactory cortex and hippocampus) some significant decrease in the density per unit area of nNOS neurons, detected either through immunohistochemistry or enzyme histochemistry, was observed in aged rats. However, only in the striatum and olfactory cortex this was accompanied by a significant decrease of the catalytic activity of the constitutive, Ca(2+)-dependent NOS form. In these two regions, the relative level of expression of nNOS protein was also significantly decreased, as assessed by Western blotting of proteic extracts from young-adult and aged rats. Other observed differences were a paler stain of neurons in some brain areas of the aged rats and differences of cellular compartmentalization of the protein in the same rats, as assessed through confocal microscopy. The present observations demonstrate that the expression and activity of nNOS show regionally-specific alterations in the brain of aged healthy rats, with a trend towards decrease, rather than toward increase as suggested by some previous reports. Therefore, hypotheses implicating nitric oxide increase in brain aging should be reconsidered on the basis of a clear-cut distinction between the physiological and the pathological aspects of the aging process.     

Anteroventral third ventricle (AV3V) lesion affects hypothalamic neuronal nitric oxide synthase (nNOS) expression following water deprivation

Neuronal nitric oxide synthase (nNOS) has been reported to be up-regulated in the hypothalamic supraoptic nucleus (SON) during dehydration which in turn could increase nitric oxide (NO) production and consequently affect arginine vasopressin (AVP) secretion. The anteroventral third ventricle (AV3V) region has strong afferent connections with the SON. Herein we describe our analysis of the effects of an AV3V lesion on AVP secretion, and c-fos and nNOS expression in the SON following dehydration. Male Wistar rats had their AV3V region electrolytically lesioned or were sham operated. After 21 days they were submitted to dehydration or left as controls (euhydrated). Two days later, one group was anaesthetized, perfused and the brains were processed for Fos protein and nNOS immunohistochemistry (IHC). Another group was decapitated, the blood collected for hematocrit, osmolality, serum sodium and AVP plasma level analysis. The brains were removed for measurement of neurohypophyseal AVP content, and the SON was punched out and processed for nNOS detection by western blotting. The AV3V lesion reduced AVP plasma levels and c-fos expression in the SON following dehydration (P<0.05). Western blotting revealed an up-regulation of nNOS in the SON of control animals following dehydration, whereas such up-regulation was not observed in AV3V-lesioned rats (P<0.05). We conclude that the AV3V region plays a role in regulating the expression of nNOS in the SON of rats submitted to dehydration, and thus may affect the local nitric oxide production and the secretion of vasopressin.     

Specific pattern of nitric oxide synthase expression in glial cells after hippocampal injury

In the central nervous system (CNS), nitric oxide (NO) is thought to be involved in a variety of functions including synaptic plasticity, long term potentiation, and neurotoxicity. The aim of the present study was to investigate the expression of nitric oxide synthase (NOS) in the mouse CNS, following surgical injury to the hippocampus. NOS expression was assessed by histochemical detection of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase) activity and immunohistochemistry of the inducible NOS (iNOS). Two days after injury to the CA1 hippocampal field, NADPH-diaphorase activity was detected in pyramidal and granular neurons and also in glial cells in the hippocampus, in contrast to the non-injured one where NADPH-diaphorase staining was observed only in a few interneurons. NADPH-diaphorase histochemistry combined with immunolabelling for GFAP and F4/80 demonstrated that these glial cells were astrocytes and microglia. This pattern of NOS expression is induced specifically after a hippocampal injury since lesion to the prefrontal or cerebellar cortex leads to NOS activity only in monocytes/macrophages like cells. Despite the large expression of NOS detected by NADPH-diaphorase histochemistry after lesioning the hippocampus, immunostaining for iNOS was confined to microglia. The fact that induction of high levels of NOS activity are detected in glial cells after a lesion to the hippocampus could be accounted for by the sensitivity of this structure to a high release of glutamate. GLIA 22:329–337, 1998. © 1998 Wiley-Liss, Inc.     

Intermittent Hypobaric Hypoxia Induces Neuroprotection in Kainate-Induced Oxidative Stress in Rats

Severe hypoxia induces oxidative stress, which can lead to brain injury. In this study, we wanted to determine whether intermittent hypobaric hypoxia induces oxidative stress in the brain. In adult rats exposed to 380 mmHg in a hypobaric chamber for 3 h/day for 6 days, we determined the levels of malondialdehyde and nitric oxide derivatives in the brain, which indicated that there was no oxidative stress. The levels of N-acetylaspartate indicated that there was no neuronal loss or mitochondrial dysfunction and finally because apoptotic proteins such as caspase-3 and nuclear factor-kappa B (NF-κB) were not activated, apoptosis was probably not induced. The increase in the expression of erythropoietin (EPO) in the brain of rats exposed to hypoxia confirms the efficacy of the method used to induce hypoxia in the brain. Because EPO have antioxidant effects on the brain, the results suggest that intermittent hypoxia can increase the antioxidant capacity of the brain. This effect of intermittent hypoxia was studied using the systemic administration of kainate, as a model of brain oxidative stress. Kainate treatment induces oxidative stress in the brain, which is measured by an increase in lipid peroxidation and nitric oxide. Furthermore, in rats treated with kainate, both caspase-3 and NF-κB activity increased. However, in rats previously exposed to intermittent hypobaric hypoxia, 3 h per day for 6 days, the effect of kainate treatment resulted in the reduction of both oxidative stress and apoptotic activity. This study demonstrates that intermittent hypobaric hypoxia can increase brain antioxidant capacity in rats and induces neuroprotection in kainate-induced oxidative injury.     

Alteration of neuronal nitric oxide synthase activity and expression in the cerebellum and the forebrain of microencephalic rats

Microencephalic rats were obtained through gestational (for the forebrain) or neonatal (for the cerebellum) administration of the DNA-alkylating agent methylazoxymethanol acetate (MAM), which selectively kills dividing cells during neurogenesis. In the microencephalic cerebellum the specific activity of calcium-dependent nitric oxide synthase (NOS) was decreased by 35–40% at 12, 28 and 70 days of age. Other neurochemical markers not related to granule cells (the neuronal population selectively compromised by neonatal MAM treatment), choline acetyltransferase (ChAT) and glutamate decarboxylase (GAD) were not decreased, but actually increased when determined as specific activity. In agreement with the decreased catalytic activity measured in the tube, the expression of neuronal NOS protein was attenuated as judged from immunohistochemistry and Western blotting. In the microencephalic forebrain, the specific calcium-dependent NOS activity measured in homogenates of the whole hemisphere was significantly increased as compared to normal animals. Accordingly, immunohistochemistry for neuronal NOS, as well as NADPH-diaphorase histochemistry revealed an apparent increase in the density of strongly reactive neurons in the underdeveloped cortex and striatum of microencephalic rats. The results reported here demonstrate that permanent alterations of neuronal NOS activity and expression occur when the development of the brain and its neuronal circuits are severely compromised. Furthermore, the permanent downregulation of neuronal NOS in the cerebellum of microencephalic rats may be exploited for the study of the role of NO in mechanisms of synaptic plasticity such as long term depression (LTD).     

Reproductive state changes NADPH-diaphorase staining in the paraventricular and supraoptic nuclei of female rats

It has been demonstrated in guinea pigs that nitric oxide synthase (NOS) activity is increased in late pregnancy in some peripheral tissues and in the cerebellum. To determine whether similar changes would be observed in areas of the brain known to play a role in parturition, staining for NADPH-diaphorase, a histochemical marker of NOS synthase, in the paraventricular (PVN) and supraoptic nuclei (SON) was compared among ovariectomized, virgin and late pregnant rats. The number of cells showing dense staining for NADPH-diaphorase increased in both the SON and PVN in late pregnancy compared to that observed in virgin and ovariectomized females. Thus, changes in reproductive state are associated with changes in NADPH-diaphorase staining in areas of the brain that are intimately involved in the control of reproductive function.     

Increased nitric oxide synthase in the vasculature of the epaulette shark brain following hypoxia.

Epaulette sharks inhabiting reef platforms are exposed to hypoxic and hyperoxic cycles. The adaptive mechanisms used to prevent neurological damage during these cycles have not been examined. Nitric oxide has a neuroprotective role in some hypoxia-tolerant species. We examined epaulette brains following a severe experimental hypoxic regimen (0.39 mgO2/l for 2 h) and compared nitric oxide synthase (NOS) expression with that in normoxic controls using NADPH-diaphorase staining. Intense NOS activity occurred in microvasculature following exposure to a severely hypoxic environment in contrast to the low levels seen in controls. We established for the first time that the epaulette shark was hypoxia-tolerant because there was no delayed phase of neuronal apoptosis. Enhanced NOS production in response to hypoxia may cause vasodilation, which would maintain the appropriate metabolic environment for continued neuronal survival during exposure to hypoxia.     

Age-Dependent Induction of Nitric Oxide Synthase Activity in Facial Motoneurons after Axotomy

The facial nerve was transected in rats at different postnatal ages, from birth to early adulthood. NADPH-diaphorase histochemistry was performed to analyze the induction of nitric oxide synthase, the synthetic enzyme of the free radical nitric oxide, in injured facial motoneurons. In addition,in situnick-end labeling of DNA fragmentation (TUNEL technique) was performed after axotomy at birth, to verify the occurrence of apoptosis in the damaged facial motoneurons. A striking age-dependency was found in the induction of nitric oxide synthase activity in axotomized facial motoneurons. NADPH-diaphorase positivity was not detectable in these neurons 1 and 2 days after axotomy at birth, when apoptotic changes were evident and marked. In addition, NADPH-diaphorase staining was hardly detectable in the facial nucleus 4 days after axotomies at birth, when extensive motoneuron loss was evident. NADPH-diaphorase positivity was instead induced in the facial motoneurons axotomized from the end of the first postnatal week to adulthood, when the nerve cell loss was less severe than in newborns. However, the time course of the enzyme activity induction varied considerably in relation to the animals' age. These findings are discussed in relation to the role of nitric oxide in motoneuron death or protective response to injury and of oxidative stress in neurodegeneration.     

Comparative Distribution of Nitric Oxide Synthase (NOS) in Pancreas of the Dog and Rat: Immunocytochemistry of Neuronal Type NOS and Histochemistry of NADPH-Diaphorase

We investigated the localization of nitric oxide synthase in the pancreas of the dog in comparison to the rat by the methods of immunocytohemistry using antineuronal type nitric oxide synthase serum and histochemistry using NADPH-diaphorase activity. In both species, the most intense staining was observed in neuronal cell bodies and fibers in the pancreas and nitric oxide synthase immunoreactivity was completely colocalized with NADPH-diaphorase activity. However, there were differences of the distribution between the two species. In the dog pancreas, immuno- and NADPH-diaphorase-positive nerve fibers were numerous around pancreatic ducts and moderate around the arteries and the acini but few in the islets. In contrast, in the rat pancreas, immuno- and diaphorase-positive fibers were fewer around the pancreatic ducts and acini and more abundant in the islets. The expression ratio of NADPH-diaphorase in intrapancreatic ganglion cell bodies that were scattered in the interlobular connective tissue was low to moderate (28.1% in the right lobe, 49.5% in the left lobe) in the dog, while the ratio in rat pancreas was very high in both lobes of the pancreas (about 86%). Except for neuronal staining, weak NADPH-diaphorase-positive reactions were detected in the vascular endothelial cells of the pancreas in both species. In rat islet cells, weak neuronal type nitric oxide synthase immunoreactivity was observed; however, in dog islet cells, no immunoreactivity was detected. These results suggest that nitric oxide in the pancreas is derived from vascular endothelium and neuronal tissue in both species and that the neuronal nitrergic regulation of the exocrine and endocrine pancreas is different between the species.     

Chronic Intermittent Hypobaric Hypoxia Ameliorates Renal Vascular Hypertension Through Up-regulating NOS in Nucleus Tractus Solitarii

Chronic intermittent hypobaric hypoxia(CIHH)is known to have an anti-hypertensive effect, which might be related to modulation of the baroreflex in rats with renal vascular hypertension(RVH). In this study, RVH was induced by the 2-kidney-1-clip method(2 K1 C) in adult male Sprague-Dawley rats. The rats were then treated with hypobaric hypoxia simulating 5000 m altitude for 6 h/day for 28 days. The arterial blood pressure(ABP), heart rate(HR), and renal sympathetic nerve activity(RSNA) were measured before and after microinjection of L-arginine into the nucleus tractus solitarii(NTS) in anesthetized rats.Evoked excitatory postsynaptic currents(eEPSCs) and spontaneous EPSCs(sEPSCs) were recorded in anterogradely-labeled NTS neurons receiving baroreceptor afferents. We measured the protein expression of neuronal nitric oxide synthase(nNOS) and endothelial NOS(eNOS) in the NTS. The results showed that the ABP in RVH rats was significantly lower after CIHH treatment. The inhibition of ABP, HR, and RSNA induced by L-arginine was less in RVH rats than in sham rats, and greater in the CIHHtreated RVH rats than the untreated RVH rats. The eEPSC amplitude in NTS neurons receiving baroreceptor afferents was lower in the RVH rats than in the sham rats and recovered after CIHH. The protein expression of nNOS and e NOS in the NTS was lower in the RVH rats than in the sham rats and this decrease was reversed by CIHH. In short, CIHH treatment decreases ABP in RVH rats via upregulating NOS expression in the NTS.     

Neuronal nitric oxide synthase is localized in extrinsic nerves regulating perireceptor processes in the chemosensory nasal mucosae of rats and humans

Nitric oxide synthase is the enzyme responsible for the production of the free radical gas nitric oxide, which has been implicated as an intercellular messenger in both the central and peripheral nervous systems. Immunoreactivity for nitric oxide synthase is often coincident with the histochemical demonstration of NADPH-diaphorase activity. Using an antibody to the neuronal form of nitric oxide synthase and a histochemical technique for NADPH-diaphorase, we have compared the localization of immunoreactivity and histochemical reaction product in the nasal mucosae of rats and humans. Immunoreactivity for neuronal nitric oxide synthase was localized in the extrinsic perivascular innervation of the olfactory and vomeronasal mucosae of rats and in the olfactory mucosa of humans. In the rat nasal mucosa, specific groups of glands were also innervated; the density of nitrinergic innervation varied among them, with vomeronasal glands and posterior glands of the nasal septum being the most densely innervated. In contrast, NADPH-diaphorase activity was present in olfactory, vomeronasal, and septal organ receptor neurons in rats and in olfactory receptor neurons in humans as well as in numerous nerve fibers, glands, and surface epithelial cells. The localization of neuronal nitric oxide synthase in extrinsic perivascular and periglandular nerve fibers suggests that nitric oxide may modulate the perireceptor processes of local blood flow and mucus secretion that influence the access to and clearance of chemical stimuli from rat and human chemosensory mucosae. © Wiley-Liss, Inc.     

Increase in nitric oxide synthase and NADPH-diaphorase in the adrenal gland of streptozotocin-diabetic Wistar rats and its prevention by ganglioside

Levels of nitric oxide synthase (NOS) and NADPH-diaphorase in adrenal glands of streptozotocin-diabetic rats of 8 and 12 weeks' duration compared with control rats were assessed with histochemical and biochemical techniques. Adrenal glands from streptozotocin-diabetic rats of 8 weeks' duration treated with ganglioside were examined also. In the adrenal medulla of 8-weeks- and 12-weeks-diabetic rats, NOS-immunoreactive nerve fibres were increased and decreased, respectively; additional NOS-immunoreactive and NADPH-diaphorase stained cells, which appeared to be cortical cells, were located in medulla and cortex compared with controls. Increased intensity in NADPH-diaphorase staining of the cortical cells of diabetic rats was observed also. Ganglioside treatment of the 8-weeks-diabetic rats prevented the diabetic-induced increase in NOS-immunoreactive nerve fibres. Also, it reduced most of the increase in the NOS-immunoreactive and NADPH-diaphorase stained cells and the intensity of NADPH-diaphorase staining of cortical cells. With biochemical assay, a significant increase in NOS activity was found in the adrenal glands from 8-weeks-diabetic rats, and this increase was reduced by ganglioside treatment in four out of six diabetic rats.In summary, streptozotocin-induced diabetes causes an initial increase in the levels of NOS and NADPH-diaphorase in the adrenal gland of rat, which was prevented by ganglioside treatment.     

NADPH-diaphorase activity and Fos expression in brain nuclei following nitroglycerin administration

Organic nitrates are considered nitric oxide donors in that they have been shown to form nitric oxide in vitro and in vivo. Nitroglycerin is an organic nitrate which possesses peculiar activities mediated, to some extent, by the central nervous system via the noradrenergic system. Previous reports have shown that systematic nitroglycerin is able to induce Fos expression in brain nuclei which are known to contain also the nitric oxide synthesizing enzyme. Neuronal NADPH-diaphorase has been shown to be a nitric oxide synthase. Thus, in this study we used NADPH-diaphorase histochemistry to evaluate the distribution of Fos-immunoreactive cells within neurons which contain nitric oxide synthase. The data obtained showed co-localization of Fos with NADPH-diaphorase activity in numerous neurons of the paraventricular and supraoptic nuclei of the hypothalamus. In the brainstem, a few neurons were doubly labeled for Fos and NADPH-diaphorase activity, but NADPH-diaphorase positive fibers and Fos-immunoreactive neurons were consistently co-distributed in the locus coeruleus, parabrachial nucleus, nucleus tractus solitarius and spinal trigeminal nucleus caudalis. These findings demonstrate that nitroglycerin administration activates a selective group of neurons which are a source of nitric oxide or which are in close proximity with neuronal processes containing nitric oxide synthase, and suggest that the nitric oxide synthesizing pathway may be involved at various levels in the central effect of nitroglycerin.     

Colocalization of nitric oxide synthase and NADPH-diaphorase in cultured myenteric neurones.

Nitric oxide synthase immunoreactivity and NADPH-diaphorase activity were examined in explant culture preparations of the myenteric plexus from beneath the taenia coli of the guinea-pig caecum. Nitric oxide synthase immunoreactive neurones formed approximately one third of the total neuronal population. NADPH-diaphorase positive neurones, demonstrated histochemically, constituted a similar proportion of the total number of neurones. Immunocytochemistry and NADPH-diaphorase histochemistry performed on the same preparations revealed that all nitric oxide synthase immunoreactive neurones expressed NADPH-diaphorase activity. This histochemical evidence is consistent with the view that nitric oxide may act as a regulatory agent in the guinea-pig caecum.     

Preferential interneuron survival in the transition zone of 3-NP-induced striatal injury in rats

Histology, immunohistochemistry, and Western blotting were used to characterize the changes in morphology, distribution pattern, and marker protein expression of striatal interneurons in the transition zone of striatal injury induced by 3-NP. The 3-NP treatment in rats yielded movement, motor coordination, and cognitive dysfunction. The 3-NP-induced lesion core was unvaryingly in the dorsolateral striatum, with a transition zone of lesser damage around the lesion core, in which medium-sized neurons were significantly decreased in abundance, but larger neurons survived. In both the transition zone and the lesion core, many TUNEL-positive cells negative for the interneuron markers were detected, indicating widespread projection neuron death. Immunohistochemical staining for the four interneuron types (parvalbuminergic, cholinergic, calretinergic, and neuropeptide Y-neuronal nitric oxide synthase cocontaining) showed that few immunolabeled interneurons were observed in the lesion core, but interneuron perikarya showed no evident loss in the transition zone. Consistently with this, Western blotting showed that the five interneuron protein markers were significantly decreased in the striatum after 3-NP treatment. Transition-zone calretinergic and neuropeptide Y-neuronal nitric oxide synthase-cocontaining interneurons, however, possessed more processes and varicosities than normal. These results show that, although striatal interneurons survive in the transition zone after 3-NP-mediated striatal injury, they have enhanced marker protein levels in their processes.     

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.