NOS2 expression is restricted to neurons in the healthy brain but is triggered in microglia upon inflammation

Nitric oxide (NO) is a diffusible second messenger with a great variety of functions in the brain. NO is produced by three isoforms of NO synthase (NOS), NOS1, NOS2, and NOS3. Although broad agreement exists regarding the expression of NOS1 and NOS3 in neurons and endothelial cells, the pattern of NOS2 expression is still controversial and remains elusive. We have now generated a novel transgenic mouse that expresses the fluorescent reporter tdTomato and the CRE recombinase under the control of the Nos2 gene regulatory regions. Such tool allows the reliable tracking of NOS2 expression in tissue and further unravels episodes of transient NOS2 expression. Using this transgenic mouse, we show that in the healthy brain, NOS2 is only transiently expressed in neurons scattered in the piriform and entorhinal cortex, the amygdaloid nuclei, the medial part of the thalamus, the hypothalamus, the dentate gyrus, and the cerebellum. NOS2 expression was rarely detected in microglia. We further show that inflammation, induced by intracerebral injection of LPS and IFNγ, triggers transient expression of NOS2 in microglia but not in neurons. This novel transgenic tool has thus allowed us to clarify the NOS2 expression pattern and its differential profile in neurons and microglia in healthy and inflammatory conditions. GLIA 2014;62:956–963     

Nitric oxide as intracellular modulator: internal production of NO increases neuronal excitability via modulation of several ionic conductances

Nitric oxide (NO) has been shown to regulate neuronal excitability in the nervous system, but little is known as to whether NO, which is synthesized in certain neurons, also serves functional roles within NO‐producing neurons themselves. We investigated this possibility by using a nitric oxide synthase (NOS)‐expressing neuron, and studied the role of intrinsic NO production on neuronal firing properties in single‐cell culture. B5 neurons of the pond snail Helisoma trivolvis fire spontaneous action potentials (APs), but once the intrinsic activity of NOS was inhibited, neurons became hyperpolarized and were unable to fire evoked APs. These striking long‐term effects could be attributed to intrinsic NO acting on three types of conductances, a persistent sodium current (I_NaP), voltage‐gated Ca currents (I_Ca) and small‐conductance calcium‐activated potassium (SK) channels. We show that NOS inhibitors 7‐nitroindazole and S‐methyl‐l ‐thiocitrulline resulted in a decrease in I_NaP, and that their hyperpolarizing and inhibiting effects on spontaneous spiking were mimicked by the inhibitor of I_NaP, riluzole. Moreover, inhibition of NOS, soluble guanylate cyclase (sGC) or protein kinase G (PKG) attenuated I_Ca, and blocked spontaneous and depolarization‐induced spiking, suggesting that intrinsic NO controlled I_Ca via the sGC/PKG pathway. The SK channel inhibitor apamin partially prevented the hyperpolarization observed after inhibition of NOS, suggesting a downregulation of SK channels by intrinsic NO. Taken together, we describe a novel mechanism by which neurons utilize their self‐produced NO as an intrinsic modulator of neuronal excitability. In B5 neurons, intrinsic NO production is necessary to maintain spontaneous tonic and evoked spiking activity.     

Reduced activity and protein expression of NOS in R6/2 HD transgenic mice: effects of -NAME on symptom progression

Previous work found that dietary l-arginine alters symptom progression in mice transgenic for Huntington’s disease (HD), and that cerebral blood flow (CBF) is abnormal in early stage HD patients. Both of these findings potentially implicate nitric oxide (NO) and its converting enzyme, nitric oxide synthase (NOS), in HD. The current experiment found that both NOS enzymatic activity and neuronal NOS (nNOS) protein expression were reduced (P<0.05) in R6/2 HD transgenic mice compared to non-HD controls (CON). Conversely, inducible NOS (iNOS) protein expression was not significantly different between groups. The changes in nNOS were accompanied by changes in protein expression of calmodulin kinase II (CaMKII) (P<0.05) and calmodulin kinase IV (CaMKIV) (P<0.05). Protein expression of 3-nitrotyrosine (3-NT), a marker for the neurotoxin peroxynitrite, was slightly increased in non-drug treated HD and was accompanied by increased immunostaining of 3-NT in cells adhering to the vasculature and choroid plexus. Mice that received the broad-spectrum NOS inhibitor N^g-nitro- -arginine methyl ester hydrochloride ( -NAME) via their drinking water had reduced NOS enzyme activity. NOS activity varied as a function of -NAME dose, was virtually eliminated in the 500-mg/l groups, and correlated (P<0.05) with the behavioral scores as revealed by regression and correlation analyses. High dose -NAME (500 mg/l) accelerated symptom onset in HD transgenics. These results support the hypothesis that nNOS activity and NO production are abnormal in HD, this in the setting of a more global dysregulation of calcium protein expression. Taken collectively with earlier data from our laboratory demonstrating abnormal CBF findings in early-stage HD patients, these results suggest that abnormalities in NOS function may significantly contribute to the neurodegeneration found in HD.     

Effects of tetrahydrobiopterin on cerebral infarction after transient focal ischemia in rats

Abstract

Objectives: The present study investigated the effects of tetrahydrobiopterin (BH4) on cerebral infarction after transient focal ischemia in rats.

Methods: Focal ischemia (1·5 hours) was created in male Sprague-Dawley rats (250-280 g) by middle cerebral artery occlusion. Some rats were treated with 20 mg/kg tetrahydrobiopterin by intraperitoneal injection 30 minutes before reperfusion. At 2, 6, and 12 hours of reperfusion, the brains were harvested for the nitric oxide synthase (NOS) activity and nitric oxide (NO) level assays. At 12 hours of reperfusion, the brains were harvested for infarct size measurement.

Results: NOS activity and NO level were all augmented after reperfusion. BH4 treatment significantly further increased NOS activity and NO level. Cerebral infarct size was significantly bigger in BH4 treatment group compared to that in no treatment group.

Conclusions: The data indicate that BH4 enhances cerebral infarction after transient focal ischemia in rats, through NOS and NO pathway.     

Nitric oxide regulates the firing rate of neuronal subtypes in the guinea pig ventral cochlear nucleus

The gaseous free radical, nitric oxide (NO) acts as a ubiquitous neuromodulator, contributing to synaptic plasticity in a complex way that can involve either long term potentiation or depression. It is produced by neuronal nitric oxide synthase (nNOS) which is presynaptically expressed and also located postsynaptically in the membrane and cytoplasm of a subpopulation of each major neuronal type in the ventral cochlear nucleus (VCN). We have used iontophoresis in vivo to study the effect of the NOS inhibitor L‐NAME (L‐NG‐Nitroarginine methyl ester) and the NO donors SIN‐1 (3‐Morpholinosydnonimine hydrochloride) and SNOG (S‐Nitrosoglutathione) on VCN units under urethane anaesthesia. Collectively, both donors produced increases and decreases in driven and spontaneous firing rates of some neurones. Inhibition of endogenous NO production with L‐NAME evoked a consistent increase in driven firing rates in 18% of units without much effect on spontaneous rate. This reduction of gain produced by endogenous NO was mirrored when studying the effect of L‐NAME on NMDA(N‐Methyl‐D‐aspartic acid)‐evoked excitation, with 30% of units showing enhanced NMDA‐evoked excitation during L‐NAME application (reduced NO levels). Approximately 25% of neurones contain nNOS and the NO produced can modulate the firing rate of the main principal cells: medium stellates (choppers), large stellates (onset responses) and bushy cells (primary‐like responses). The main endogenous role of NO seems to be to partly suppress driven firing rates associated with NMDA channel activity but there is scope for it to increase neural gain if there were a pathological increase in its production following hearing loss.     

The effect of <Emphasis Type="Italic">l</Emphasis>-deprenyl on tissue mRNA expressions of NOS isoforms and NO levels in an experimental diabetes mellitus model

Summary Diabetes and aging share some common mechanisms in their pathogenesis and diabetics are more prone to diseases of the elderly. Seeking for therapies likely to be proposed in the synchronised treatment of aging and diabetes is of great interest and l-deprenyl, a selective monoamine oxidase (MAO-B) inhibitor, is a possible candidate with its antioxidant, antiapoptotic and neuroprotective properties. Tissue MAO, NO and mRNA expression of nitric oxide (NO) synthase (NOS) isoforms were assessed in streptozotocin (STZ)-induced diabetic rats to evaluate the effect of l-deprenyl treatment. Twelve weeks of treatment had no significant effect on NO levels. Four-weeks treatment decreased tissue MAO activities and caused a decrease in expression of NOS-2 and NOS-3 in heart tissue of both controls and diabetics, and a decrease of liver NOS-3 expression in controls (p < 0.05). l-Deprenyl, causing a decrease in tissue NOS expressions, might be of benefit by protecting the organism from the toxic radical effects of NO.     

Chronic alcohol consumption induces an overproduction of NO by nNOS‐ and iNOS‐expressing myenteric neurons in the murine small intestine

Background There are indications that alterations in the nitric oxide (NO) system of relaxation mediate gastrointestinal motor disturbances induced by chronic alcohol consumption (CAC). As CAC is known to inhibit the motility of the mouse small intestine, we investigated in this model if CAC affects basal NO synthesis by myenteric neurons and which NOS isoforms are involved. Methods The instantaneous NO synthesis of individual neurons was optically measured in whole‐mount preparations loaded with the NO synthesis indicator DAF‐FM, and the expression of nNOS, iNOS and eNOS was determined by immunohistochemistry. Key Results The DAF‐FM recordings showed that CAC induced an increase in neuronal NO synthesis (absolute fluorescence: control 34 ± 12; CAC 140 ± 56; mean ± SD; P < 0.0004). Neurons of control mice expressed the nNOS (29 ± 3% of total) and iNOS (28 ± 1%) isoforms. eNOS expression was observed in <0.5% of the neurons. Chronic alcohol consumption caused an increase in the proportion of iNOS‐expressing neurons (to 33 ± 5%; P < 0.01) and a decrease in nNOS‐expressing neurons (to 22 ± 3%; P < 0.0001), without altering the proportion of NO‐producing neurons (control 55 ± 13%; CAC 56 ± 11%; P = 0.82). Conclusions & Inferences Chronic alcohol consumption induces a marked increase in NO synthesis by jejunal myenteric neurons, accompanied by an up‐regulation of iNOS‐expressing neurons and a downregulation of nNOS neurons. We conclude that the overproduction of NO may be a direct cause of gastrointestinal motility disturbances.     

The neuronal apoptotic death in global cerebral ischemia in gerbil: Important role for sodium channel modulator

Global ischemia was induced in gerbil by bilateral occlusion of the common carotid arteries for 5 min. Sodium ionophore monensin or sodium channel blocker tetrodotoxin (TTX) was administered at doses of 10 μg/kg, i.p., 30 min before ischemia induction; the dose was repeated after 22 hr. Subsequently, brain infarct occurred, determined at 24 hr after occlusion. Large, well‐demarcated infarcts were observed in both hemispheres, an important observation because it critically influences the interpretation of the data. Because nitric oxide (NO) production is thought to be related to ischemic neuronal damage, we examined increases in Ca²⁺ influx, which lead to the activation of nitric oxide synthase (NOS). Then we evaluated the contributions of neuronal NOS, endothelial NOS, and inducible NOS to NO production in brain cryosections. The cytosolic release of apoptogenic molecules like cytochrome c and p53 were confirmed after 24 hr of reflow. TUNEL (terminal deoxynucleotidyl transferase dUTP nick‐end labeling) labeling detected the apoptotic cells, which were confirmed in neuron‐rich cell populations. After 24 hr, all the ischemic changes were amplified by monensin and significantly attenuated by TTX treatment. Additionally, the nesting behavior and histological outcomes were examined after 7 day of reflow. The neuronal damage in the hippocampal area and significant decrease in nesting scores were observed with monensin treatment and reduced by TTX pretreatment after day 7 of reflow. To our knowledge, this report is the first to highlight the involvement of the voltage‐sensitive Na⁺ channel in possibly regulating in part NO system and apoptosis in a cytochrome c–dependent manner in global ischemia in the gerbil, and thus warrants further investigation. © 2008 Wiley‐Liss, Inc.