Zymosan and lipopolysaccharide decrease gene expression of neuronal nitric oxide synthase in peripheral organs in chicks.

Nitric oxide (NO) is gaseous bioactive molecule that is synthesized by NO synthase (NOS). Inducible NOS (iNOS) expression occurs in response to pathogenic challenges, resulting in the production of large amounts of NO. However, there is a lack of knowledge regarding neuronal NOS (nNOS) and endothelial NOS (eNOS) in birds during pathogenic challenge. Therefore, the present study was conducted to determine the influence of intraperitoneal (IP) injection of zymosan (cell wall component of yeast) and lipopolysaccharide (LPS, a cell wall component of gram-negative bacteria) on NOS expression in chicks (Gallus gallus). Furthermore, the effect of NOS inhibitors on the corresponding behavioral and physiological parameters was investigated. Zymosan and LPS injections induced iNOS mRNA expression in several organs. Zymosan had no effect on eNOS mRNA expression in the organs investigated, whereas LPS increased its expression in the pancreas. Zymosan and LPS decreased nNOS mRNA expression in the lung, heart, kidney, and pancreas. The decreased nNOS mRNA expression in pancreas was probably associated with the NO from iNOS provided that such effect was reproduced by IP injection of sodium nitroprusside, which is a NO donor. Furthermore, pancreatic nNOS mRNA expression decreased following subcutaneous injection of corticosterone. Furthermore, IP injections of a nonspecific NOS inhibitor, NG-nitro-L-arginine methyl ester, and an nNOS-specific inhibitor, 7-nitroindazole, resulted in the significant decreases in food intake, cloacal temperature, and feed passage via the digestive tract in chicks. Collectively, the current findings imply the decreased nNOS expression because of fungal and bacterial infections, which affects food intake, body temperature, and the digestive function in birds.

Expression of neuronal NO synthase α- and ß-isoforms in skeletal muscle of mice.

Knowledge of the primary structure of neuronal NO synthase (nNOS) in skeletal muscle is still conflicting and needs further clarification. To elucidate the expression patterns of nNOS isoforms at both mRNA and protein level, systematic reverse transcription (RT)-PCR and epitope mapping by qualitative immunoblot analysis on skeletal muscle of C57/BL6 mice were performed. The ability of the nNOS isoforms to form aggregates was characterized by native low-temperature polyacrylamide electrophoresis (LT-PAGE). The molecular analysis was focused on the rectus femoris (RF) muscle, a skeletal muscle with a nearly balanced ratio of nNOS α- and ß-isoforms. RT-PCR amplificates from RF muscles showed exclusive exon-1d mRNA expression, either with or without exon-µ. Epitope mapping demonstrated the simultaneous expression of the nNOS splice variants α/µ, α/non-µ, ß/µ and ß/non-µ. Furthermore, immunoblotting suggests that the transition between nNOS α- and ß-isoforms lies within exon-3. In LT-PAGE, three protein nNOS associated aggregates were detected in homogenates of RF muscle and tibialis anterior muscle: a 320 kDa band containing nNOS α-isoforms, while 250 and 300 kDa bands consist of nNOS ß-isoforms that form homodimers or heterodimers with non-nNOS proteins.

Stress during pubertal development affects female sociosexual behavior in mice.

Puberty is a crucial phase for the development of female sexual behavior. Growing evidence suggests that stress during this period may interfere with the development of sexual behavior. However, the neural circuits involved in this alteration remain elusive. Here, we demonstrated in mice that pubertal stress permanently disrupted sexual performance without affecting sexual preference. This was associated with a reduced expression and activation of neuronal nitric oxide synthase (nNOS) in the ventrolateral part of the ventromedial hypothalamus (VMHvl). Fiber photometry revealed that VMHvl nNOS neurons are strongly responsive to male olfactory cues with this activation being substantially reduced in pubertally stressed females. Finally, treatment with a NO donor partially restored sexual performance in pubertally stressed females. This study provides insights into the involvement of VMHvl nNOS in the processing of olfactory cues important for the expression of female sexual behavior. In addition, exposure to stress during puberty disrupts the integration of male olfactory cues leading to reduced sexual behavior.

nNOS in Erbb4-positive neurons regulates GABAergic transmission in mouse hippocampus.

Neuronal nitric oxide synthase (nNOS, gene name Nos1) orchestrates the synthesis of nitric oxide (NO) within neurons, pivotal for diverse neural processes encompassing synaptic transmission, plasticity, neuronal excitability, learning, memory, and neurogenesis. Despite its significance, the precise regulation of nNOS activity across distinct neuronal types remains incompletely understood. Erb-b2 receptor tyrosine kinase 4 (ErbB4), selectively expressed in GABAergic interneurons and activated by its ligand neuregulin 1 (NRG1), modulates GABA release in the brain. Our investigation reveals the presence of nNOS in a subset of GABAergic interneurons expressing ErbB4. Notably, NRG1 activates nNOS via ErbB4 and its downstream phosphatidylinositol 3-kinase (PI3K), critical for NRG1-induced GABA release. Genetic removal of nNos from Erbb4-positive neurons impairs GABAergic transmission, partially rescued by the NO donor sodium nitroprusside (SNP). Intriguingly, the genetic deletion of nNos from Erbb4-positive neurons induces schizophrenia-relevant behavioral deficits, including hyperactivity, impaired sensorimotor gating, and deficient working memory and social interaction. These deficits are ameliorated by the atypical antipsychotic clozapine. This study underscores the role and regulation of nNOS within a specific subset of GABAergic interneurons, offering insights into the pathophysiological mechanisms of schizophrenia, given the association of Nrg1, Erbb4, Pi3k, and Nos1 genes with this mental disorder.

Stroke Alters the Function of Enteric Neurons to Impair Smooth Muscle Relaxation and Dysregulates Gut Transit.

BACKGROUND: Gut dysmotility is common after ischemic stroke, but the mechanism underlying this response is unknown. Under homeostasis, gut motility is regulated by the neurons of the enteric nervous system that control contractile/relaxation activity of muscle cells in the gut wall. More recently, studies of gut inflammation revealed interactions of macrophages with enteric neurons are also involved in modulating gut motility. However, whether poststroke gut dysmotility is mediated by direct signaling to the enteric nervous system or indirectly via inflammatory macrophages is unknown. METHODS AND RESULTS: We examined these hypotheses by using a clinically relevant permanent intraluminal midcerebral artery occlusion experimental model of stroke. At 24 hours after stroke, we performed in vivo and ex vivo gut motility assays, flow cytometry, immunofluorescence, and transcriptomic analysis. Stroke-induced gut dysmotility was associated with recruitment of muscularis macrophages into the gastrointestinal tract and redistribution of muscularis macrophages away from myenteric ganglia. The permanent intraluminal midcerebral artery occlusion model caused changes in gene expression in muscularis macrophages consistent with an altered phenotype. While the size of myenteric ganglia after stroke was not altered, myenteric neurons from post-permanent intraluminal midcerebral artery occlusion mice showed a reduction in neuronal nitric oxide synthase expression, and this response was associated with enhanced intestinal smooth muscle contraction ex vivo. Finally, chemical sympathectomy with 6-hydroxydopamine prevented the loss of myenteric neuronal nitric oxide synthase expression and stroke-induced slowed gut transit. CONCLUSIONS: Our findings demonstrate that activation of the sympathetic nervous system after stroke is associated with reduced neuronal nitric oxide synthase expression in myenteric neurons, resulting in impaired smooth muscle relaxation and dysregulation of gut transit.

Why is early-onset atrial fibrillation uncommon in patients with Duchenne muscular dystrophy? Insights from the mdx mouse.

AIMS: A reduction in both dystrophin and neuronal nitric oxide synthase (NOS1) secondary to microRNA-31 (miR-31) up-regulation contributes to the atrial electrical remodelling that underpins human and experimental atrial fibrillation (AF). In contrast, patients with Duchenne muscular dystrophy (DMD), who lack dystrophin and NOS1 and, at least in the skeletal muscle, have raised miR-31 expression, do not have increase susceptibility to AF in the absence of left ventricular (LV) dysfunction. Here, we investigated whether dystrophin deficiency is also associated with atrial up-regulation of miR-31, loss of NOS1 protein, and increased AF susceptibility in young mdx mice. METHODS AND RESULTS: Echocardiography showed normal cardiac structure and function in 12-13 weeks mdx mice, with no indication by assay of hydroxyproline that atrial fibrosis had developed. The absence of dystrophin in mdx mice was accompanied by an overall reduction in syntrophin and a lower NOS1 protein content in the skeletal muscle and in the left atrial and ventricular myocardium, with the latter occurring alongside reduced Nos1 transcript levels (exons 1-2 by quantitative polymerase chain reaction) and an increase in NOS1 polyubiquitination [assessed using tandem polyubiquitination pulldowns; P < 0.05 vs. wild type (WT)]. Neither the up-regulation of miR-31 nor the substantial reduction in NOS activity observed in the skeletal muscle was present in the atrial tissue of mdx mice. At difference with the skeletal muscle, the mdx atrial myocardium showed a reduction in the constitutive NOS inhibitor, caveolin-1, coupled with an increase in NOS3 serine1177 phosphorylation, in the absence of differences in the protein content of other NOS isoforms or in the relative expression NOS1 splice variants. In line with these findings, transoesophageal atrial burst pacing revealed no difference in AF susceptibility between mdx mice and their WT littermates. CONCLUSION: Dystrophin depletion is not associated with atrial miR-31 up-regulation, reduced NOS activity, or increased AF susceptibility in the mdx mouse. Compared with the skeletal muscle, the milder atrial biochemical phenotype may explain why patients with DMD do not exhibit a higher prevalence of atrial arrhythmias despite a reduction in NOS1 content.

Differential superoxide production in phosphorylated neuronal nitric oxide synthase mu and alpha variants.

Neuronal nitric oxide synthase (nNOS) is regulated by phosphorylation in vivo, yet the underlying biochemical mechanisms remain unclear, primarily due to difficulty in obtaining milligram quantities of phosphorylated nNOS protein; detailed spectroscopic and rapid kinetics investigations require purified protein samples at a concentration in the range of hundreds microM. Moreover, the functional diversity of the nNOS isoform is linked to its splice variants. Also of note is that determination of protein phosphorylation stoichiometry remains as a challenge. To address these issues, this study first expanded a recent genetic code expansion approach to produce phosphorylated rat nNOSµ and nNOSα holoproteins through site-specific incorporation of phosphoserine (pSer) at residues 1446 and 1412, respectively; this site is at the C-terminal tail region, a NOS-unique regulatory element. A quantitative mass spectrometric approach was then developed in-house to analyze unphosphorylated peptides in phosphatase-treated and -untreated phospho-nNOS proteins. The observed pSer-incorporation efficiency consistently exceeded 80%, showing high pSer-incorporation efficiency. Notably, EPR spin trapping results demonstrate that under l-arginine-depleted conditions, pSer1412 nNOSα presented a significant reduction in superoxide generation, whereas pSer1446 nNOSµ exhibited the opposite effect, compared to their unphosphorylated counterparts. This suggests that phosphorylation at the C-terminal tail has a regulatory effect on nNOS uncoupling that may differ between variant forms. Furthermore, the methodologies for incorporating pSer into large, complex protein and quantifying the percentage of phosphorylation in recombinant purified protein should be applicable to other protein systems.

The neuronal nitric oxide synthase expression increases during satellite cell-derived primary myoblasts differentiation.

The neuronal nitric oxide synthase (nNOS; encoded by NOS1)-derived nitric oxide (NO) plays an important role in maintaining skeletal muscle mass. In adult skeletal muscle, nNOS localizes to the cell membrane, cytosol, and nucleus, and regulates muscle hypertrophy and atrophy in various subcellular fractions. However, its role in muscle stem cells (also known as muscle satellite cells), which provide myonuclei for postnatal muscle growth, maintenance, and regeneration, remains unclear. The present study aimed to determine nNOS expression in muscle satellite cell-derived primary myoblasts during differentiation and its DNA methylation levels, an epigenetic modification that controls gene expression. Undifferentiated and differentiated satellite cell-derived primary myoblasts were found to express nNOS. Immunohistochemical analysis revealed that nNOS colocalized with Pax7 (satellite cell marker) only in the undifferentiated myoblasts. Furthermore, nNOS immunoreactivity spread to the cytosol of Pax7-negative differentiated myotube-like cells. The level of Nos1µ mRNA, the main isoform of skeletal muscle nNOS, was increased in differentiated satellite cell-derived primary myoblasts compared to that in the undifferentiated cells. However, Nos1 methylation levels remained unchanged during differentiation. These findings suggest that nNOS induction and the appropriate transition of its subcellular localization may contribute to muscle differentiation.

Nos1+ and Nos1- excitatory neurons in the BLA regulate anxiety- and depression-related behaviors oppositely.

BACKGROUND: The basolateral amygdala (BLA) neurons are primarily glutamatergic and have been associated with emotion regulation. However, little is known about the roles of BLA neurons expressing neuronal nitric oxide synthase (nNOS, Nos1) in the regulation of emotional behaviors. METHODS: Using Nos1-cre mice and chemogenetic and optogenetic manipulations, we specifically silenced or activated Nos1+ or Nos1- neurons in the BLA, or silenced their projections to the anterdorsal bed nucleus of the stria terminalis (adBNST) and ventral hippocampus (vHPC). We measured anxiety behaviors in elevated plus maze (EPM) and open-field test (OFT), and measured depression behaviors in forced swimming test (FST) and tail suspension test (TST). RESULTS: BLA Nos1+ neurons were predominantly glutamatergic, and glutamatergic but not GABAergic Nos1+ neurons were involved in controlling anxiety- and depression-related behaviors. Interestingly, by selectively manipulating the activities of BLA Nos1+ and Nos1- excitatory neurons, we found that they had opposing effects on anxiety- and depression-related behaviors. BLA Nos1+ excitatory neurons projected to the adBNST, this BLA-adBNST circuit controlled the expression of anxiety- and depression-related behaviors, while BLA Nos1- excitatory neurons projected to vHPC, this BLA-vHPC circuit contributed to the expression of anxiety- and depression-related behaviors. Moreover, excitatory vHPC-adBNST circuit antagonized the role of BLA-adBNST circuit in regulating anxiety- and depression-related behaviors. CONCLUSIONS: BLA Nos1+ and Nos1- excitatory neuron subpopulations exert different effects on anxiety- and depression-related behaviors through distinct projection circuits, providing a new insight of BLA excitatory neurons in emotional regulation. LIMITATIONS: We did not perform retrograde labeling from adBNST and vHPC regions.

The Role of Macula Densa Nitric Oxide Synthase 1 Beta Splice Variant in Modulating Tubuloglomerular Feedback.

Abnormalities in renal electrolyte and water excretion may result in inappropriate salt and water retention, which facilitates the development and maintenance of hypertension, as well as acid-base and electrolyte disorders. A key mechanism by which the kidney regulates renal hemodynamics and electrolyte excretion is via tubuloglomerular feedback (TGF), an intrarenal negative feedback between tubules and arterioles. TGF is initiated by an increase of NaCl delivery at the macula densa cells. The increased NaCl activates luminal Na-K-2Cl cotransporter (NKCC2) of the macula densa cells, which leads to activation of several intracellular processes followed by the production of paracrine signals that ultimately result in a constriction of the afferent arteriole and a tonic inhibition of single nephron glomerular filtration rate. Neuronal nitric oxide (NOS1) is highly expressed in the macula densa. NOS1ß is the major splice variant and accounts for most of NO generation by the macula densa, which inhibits TGF response. Macula densa NOS1ß-mediated modulation of TGF responses plays an essential role in control of sodium excretion, volume and electrolyte hemostasis, and blood pressure. In this article, we describe the mechanisms that regulate macula densa-derived NO and their effect on TGF response in physiologic and pathologic conditions. © 2023 American Physiological Society. Compr Physiol 13:4215-4229, 2023.

Disrupting the nNOS/NOS1AP interaction in the medial prefrontal cortex impairs social recognition and spatial working memory in mice.

The neuronal isoform of nitric oxide synthase (nNOS) and its interacting protein NOS1AP have been linked to several mental disorders including schizophrenia and depression. An increase in the interaction between nNOS and NOS1AP in the frontal cortex has been suggested to contribute to the emergence of these disorders. Here we aimed to uncover whether disruption of their interactions in the frontal cortex leads to mental disorder endophenotypes. Targeting the medial prefrontal cortex (mPFC), we stereotaxically injected wild-type C57BL/6J mice with recombinant adeno-associated virus (rAAV) expressing either full-length NOS1AP, the nNOS binding region of NOS1AP (i.e. NOS1AP396-503), or the nNOS amino-terminus (i.e. nNOS1-133), which was shown to disrupt the interaction of endogenous nNOS with PSD-95. We tested these mice in a comprehensive behavioural battery, assessing different endophenotypes related to mental disorders. We found no differences in anxiety-related and exploratory behaviours. Likewise, social interaction was comparable in all groups. However, social recognition was impaired in NOS1AP and NOS1AP396-503 mice. These mice, as well as mice overexpressing nNOS1-133 also displayed impaired spatial working memory (SWM) capacity, while spatial reference memory (SRM) remained intact. Finally, mice overexpressing NOS1AP and nNOS1-133, but not NOS1AP396-503, failed to habituate to the startling pulses in an acoustic startle response (ASR) paradigm, though we found no difference in overall startle intensity or prepulse inhibition (PPI) of the ASR. Our findings indicate a distinct role of NOS1AP/nNOS/PSD-95 interactions in the mPFC to contribute to specific endophenotypic changes observed in different mental disorders.

Male minipuberty involves the gonad-independent activation of preoptic nNOS neurons.

BACKGROUND: The maturation of the hypothalamic-pituitary-gonadal (HPG) axis is crucial for the establishment of reproductive function. In female mice, neuronal nitric oxide synthase (nNOS) activity appears to be key for the first postnatal activation of the neural network promoting the release of gonadotropin-releasing hormone (GnRH), i.e. minipuberty. However, in males, the profile of minipuberty as well as the role of nNOS-expressing neurons remain unexplored. METHODS: nNOS-deficient and wild-type mice were studied during postnatal development. The expression of androgen (AR) and estrogen receptor alpha (ERα) as well as nNOS phosphorylation were evaluated by immunohistochemistry in nNOS neurons in the median preoptic nucleus (MePO), where most GnRH neuronal cell bodies reside, and the hormonal profile of nNOS-deficient male mice was assessed using previously established radioimmunoassay and ELISA methods. Gonadectomy and pharmacological manipulation of ERα were used to elucidate the mechanism of minipubertal nNOS activation and the maturation of the HPG axis. RESULTS: In male mice, minipubertal FSH release occurred at P23, preceding the LH surge at P30, when balanopreputial separation occurs. Progesterone and testosterone remained low during minipuberty, increasing around puberty, whereas estrogen levels were high throughout postnatal development. nNOS neurons showed a sharp increase in Ser1412 phosphorylation of nNOS at P23, a phenomenon that occurred even in the absence of the gonads. In male mice, nNOS neurons did not appear to express AR, but abundantly expressed ERα throughout postnatal development. Selective pharmacological blockade of ERα during the infantile period blunted Ser1412 phosphorylation of nNOS at P23. CONCLUSIONS: Our results show that the timing of minipuberty differs in male mice when compared to females, but as in the latter, nNOS activity in the preoptic region plays a role in this process. Additionally, akin to male non-human primates, the profile of minipuberty in male mice is shaped by sex-independent mechanisms, and possibly involves extragonadal estrogen sources.

The interleukin-enhanced binding factor 3-mediated inhibition of nitric oxide production via phosphoinositide 3-kinase/protein kinase B pathway contributes to central cardiovascular regulation in the rostral ventrolateral medulla in hypertension.

Hypertension is characterized by sympathetic hyperactivity, which is related to the overexcitation of the presympathetic neurons in the rostral ventrolateral medulla (RVLM). Nitric oxide (NO) has been reported to be a vital neuromodulator involved in central cardiovascular regulation. However, the mechanism of interleukin-enhanced binding factor 3 (ILF3) participating in blood pressure (BP) regulation is still unclear. Therefore, this study aims to clarify the role of ILF3 within the rostral ventrolateral medulla (RVLM) in regulating NO in hypertension. It was found that the expression level of ILF3 was significantly increased in the RVLM of spontaneously hypertensive rats (SHR) compared with Wistar-Kyoto (WKY) rats through microarray gene expression analysis, Western blot, and immunofluorescence. Overexpression of ILF3 by injecting constructed adenovirus into the RVLM increased the BP and renal sympathetic nerve activity (RSNA) of the WKY rats, significantly decreasing NO production and neuronal nitric oxide synthase (nNOS) expression. Knockdown of ILF3 in the RVLM of SHR significantly reduced BP but increased NO production and the neuronal nitric oxide synthase (nNOS) expression. Furthermore, it was found that the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway was activated via Western blotting in the RVLM after overexpression of ILF3, whereas it was attenuated after knockdown of ILF3 in SHR. In addition, inhibition of PI3K by intracisternal infusion of the PI-103 attenuated the increase in Akt phosphorylation and decrease in nNOS expression and NO production caused by overexpressing ILF3, which ultimately blunted high BP induced by overexpressing ILF3. Taken together, this current study suggests that ILF3 participates in high BP via reducing NO production in the RVLM through PI3K/Akt pathway.

NOS1 mutations cause hypogonadotropic hypogonadism with sensory and cognitive deficits that can be reversed in infantile mice.

The nitric oxide (NO) signaling pathway in hypothalamic neurons plays a key role in the regulation of the secretion of gonadotropin-releasing hormone (GnRH), which is crucial for reproduction. We hypothesized that a disruption of neuronal NO synthase (NOS1) activity underlies some forms of hypogonadotropic hypogonadism. Whole-exome sequencing was performed on a cohort of 341 probands with congenital hypogonadotropic hypogonadism to identify ultrarare variants in NOS1. The activity of the identified NOS1 mutant proteins was assessed by their ability to promote nitrite and cGMP production in vitro. In addition, physiological and pharmacological characterization was carried out in a Nos1-deficient mouse model. We identified five heterozygous NOS1 loss-of-function mutations in six probands with congenital hypogonadotropic hypogonadism (2%), who displayed additional phenotypes including anosmia, hearing loss, and intellectual disability. NOS1 was found to be transiently expressed by GnRH neurons in the nose of both humans and mice, and Nos1 deficiency in mice resulted in dose-dependent defects in sexual maturation as well as in olfaction, hearing, and cognition. The pharmacological inhibition of NO production in postnatal mice revealed a critical time window during which Nos1 activity shaped minipuberty and sexual maturation. Inhaled NO treatment at minipuberty rescued both reproductive and behavioral phenotypes in Nos1-deficient mice. In summary, lack of NOS1 activity led to GnRH deficiency associated with sensory and intellectual comorbidities in humans and mice. NO treatment during minipuberty reversed deficits in sexual maturation, olfaction, and cognition in Nos1 mutant mice, suggesting a potential therapy for humans with NO deficiency.

Nitric Oxide Linked to mGluR5 Upregulates BDNF Synthesis by Activating MMP2 in the Caudate and Putamen after Challenge Exposure to Nicotine in Rats.

Nitric oxide (NO) linked to glutamate receptors in the caudate and putamen (CPu) regulates neuroadaptation after drug exposure. Matrix-metalloproteinase (MMP), a Ca2+-dependent zinc-containing endopeptidase, increases mature brain-derived neurotrophic factor (BDNF) synthesis after drug exposure in the brain. The present study determined that NO synthesis linked to metabotropic glutamate receptor subtype 5 (mGluR5) stimulation after challenge exposure to nicotine activates MMP, which upregulates BDNF synthesis in the CPu. Subcutaneous injection of challenge nicotine (1.0 mg/kg) after repeated injections of nicotine (1.0 mg/kg/day) for 14 days and 7 days of nicotine withdrawal increased MMP2 activity and BDNF expression in the CPu of rats. These increases were prevented by the bilateral intra-CPu infusion of the mGluR5 antagonist, MPEP (0.1 nmol/side), the IP3 receptor antagonist, xestospongin C (0.004 nmol/side) or the neuronal nitric oxide synthase (nNOS) and NO inhibitor, Nω-propyl (0.1 nmol/side) prior to the challenge nicotine. Furthermore, bilateral intra-CPu infusion of the MMP2 inhibitor, OA-Hy (1 nmol/side) prevented the challenge nicotine-induced increase in the expression of BDNF. These findings suggest that elevation of NO synthesis linked to mGluR5 potentiates BDNF synthesis via activation of MMP2 after challenge exposure to nicotine in the CPu of rats.

TRPV1 channels in nitric oxide-mediated signalling: insight on excitatory transmission in rat CA1 pyramidal neurons.

Nitric oxide (NO) is a fascinating signalling molecule implicated in a plethora of biological functions, especially at the synaptic level. Exploring neurotransmission in the hippocampus could be instrumental in the individuation of putative targets for nitric-oxide mediated neuromodulation, especially in terms of the potential repercussions on fundamental processes i.e. synaptic plasticity and excitability-related phenomena. Among these targets, endovanilloid signalling constitutes an object of study since Transient Receptors Vanilloid type 1 (TRPV1) channels possess a NO-sensitive gate modulating its activation. Also, NO has been referred to as a mediator for numerous endocannabinoid effects. Notwithstanding, the linkage between TRPV1 and NO systems in neuromodulation still remains elusive. To this end, we aim at investigating the involvement of TRPV1 in nitric oxide-mediated influence on hippocampal processes. Electrophysiological whole-cell recordings in CA1 pyramidal neurons were applied to evaluate excitatory neurotransmission in rat brain slices. Indeed, miniature excitatory postsynaptic currents (mEPSCs) were analysed upon pharmacological manipulation of TRPV1 and NO signalling pathways. In detail, only the administration of the specific TRPV1 exogenous agonist - capsaicin - reduced the frequency and amplitude of mEPSC similarly to the inhibitor of neuronal nitric oxide synthase (nNOS), 7-nitroindazole (7NI). In contrast, capsazepine, TRPV1 antagonist, does not influence excitatory transmission. The combined TRPV1 activation and nNOS blockade confirm the presence of a putative common mechanism. When we administered the endovanilloid-endocannabinoid ligand, i.e. anandamide, we unveiled a potentiation of neurotransmission that was selectively reverted by 7NI. Our data suggest that nitric oxide influences TRPV1 hippocampal signalling since these channels are not constitutively active, but can be "on-demand" activated to modulate excitation in CA1 pyramidal neurons, and that this effect is linked to nitric oxide production.

Female sexual behavior is disrupted in a preclinical mouse model of PCOS via an attenuated hypothalamic nitric oxide pathway.

Women with polycystic ovary syndrome (PCOS) frequently experience decreased sexual arousal, desire, and sexual satisfaction. While the hypothalamus is known to regulate sexual behavior, the specific neuronal pathways affected in patients with PCOS are not known. To dissect the underlying neural circuitry, we capitalized on a robust preclinical animal model that reliably recapitulates all cardinal PCOS features. We discovered that female mice prenatally treated with anti-Müllerian hormone (PAMH) display impaired sexual behavior and sexual partner preference over the reproductive age. Blunted female sexual behavior was associated with increased sexual rejection and independent of sex steroid hormone status. Structurally, sexual dysfunction was associated with a substantial loss of neuronal nitric oxide synthase (nNOS)-expressing neurons in the ventromedial nucleus of the hypothalamus (VMH) and other areas of hypothalamic nuclei involved in social behaviors. Using in vivo chemogenetic manipulation, we show that nNOSVMH neurons are required for the display of normal sexual behavior in female mice and that pharmacological replenishment of nitric oxide restores normal sexual performance in PAMH mice. Our data provide a framework to investigate facets of hypothalamic nNOS neuron biology with implications for sexual disturbances in PCOS.

Inhibition of neuronal nitric oxide synthase protects against hippocampal neuronal injuries by increasing neuropeptide Y expression in temporal lobe epilepsy mice.

Neuronal nitric oxide synthase (nNOS) plays a pivotal role in the pathological process of neuronal injury in the development of epilepsy. Our previous study has demonstrated that nitric oxide (NO) derived from nNOS in the epileptic brain is neurotoxic due to its reaction with the superoxide radical with the formation of peroxynitrite. Neuropeptide Y (NPY) is widely expressed in the mammalian brain, which has been implicated in energy homeostasis and neuroprotection. Recent studies suggest that nNOS may act as a mediator of NPY signaling. Here in this study, we sought to determine whether NPY expression is regulated by nNOS, and if so, whether the regulation of NPY by nNOS is associated with the neuronal injuries in the hippocampus of epileptic brain. Our results showed that pilocarpine-induced temporal lobe epilepsy (TLE) mice exhibited an increased level of nNOS expression and a decreased level of NPY expression along with hippocampal neuronal injuries and cognition deficit. Genetic deletion of nNOS gene, however, significantly upregulated hippocampal NPY expression and reduced TLE-induced hippocampal neuronal injuries and cognition decline. Knockdown of NPY abolished nNOS depletion-induced neuroprotection and cognitive improvement in the TLE mice, suggesting that inhibition of nNOS protects against hippocampal neuronal injuries by increasing neuropeptide Y expression in TLE mice. Targeting nNOS-NPY signaling pathway in the epileptic brain might provide clinical benefit by attenuating neuronal injuries and preventing cognitive deficits in epilepsy patients.

Diabetes Mellitus Alters the Immuno-Expression of Neuronal Nitric Oxide Synthase in the Rat Pancreas.

Nitric oxide is generated from nitric oxide synthase following hyperglycemia-induced oxidative stress during the course of diabetes mellitus (DM). We examined the temporal immuno-expression of neuronal nitric oxide synthase (nNOS) in the pancreas of diabetic and non-diabetic rats using immunohistochemical, immunofluorescence and western blot techniques 12 h, 24 h, 1 week, 2 weeks, 1, 8 and 15 months after induction of DM. nNOS co-localized with pancreatic beta cells but disappears 12 h after the onset of DM. In contrast, the nNOS content of pancreatic nerves increased significantly (p < 0.001) 24 h after the induction of DM, and decreased sharply thereafter. However, nNOS-positive ganglion cells were observed even 15 months post-diabetes. ROS increased by more than 100% two months after the onset of DM compared to non-diabetic control but was significantly (p < 0.000001) reduced at 9 months after the induction of DM. The pancreatic content of GSH increased significantly (p < 0.02) after 9 months of DM. Although, TBARS content was significantly (p < 0.009; p < 0.002) lower in aged (9 months) non-diabetic and DM rats, TBARS rate was markedly (p < 0.02) higher 9 months after the induction of DM when compared to younger age group. In conclusion, nNOS is present in pancreatic beta cell, but disappears 12 h after the onset of diabetes. In contrast, the tissue level of nNOS of pancreatic nerves increased in the first week of diabetes, followed by a sharp reduction. nNOS may play important roles in the metabolism of pancreatic beta cell.