NMDA-R1 subunit of the cerebral cortex co-localizes with neuronal nitric oxide synthase at pre- and postsynaptic sites and in spines

The majority of nitric oxide's (NO) physiologic and pathologic actions in the brain has been linked to NMDA receptor activation. In order to determine how the NO-synthesizing enzyme within brain, neuronal NO synthase (nNOS), and NMDA receptors are functionally linked, previous studies have used in situ hybridization techniques in combination with light microscopic immunocytochemistry to show that the two are expressed within single neurons. However, this light microscopic finding does not guarantee that NMDA receptors are distributed sufficiently close to nNOS within single neurons to allow direct interaction of the two. Thus, in this study, dual immuno-electron microscopy was performed to determine whether nNOS and NMDA receptors co-exist within fine neuronal processes. We show that nNOS and the obligatory subunit of functional NMDA receptors, i.e. the NMDA-R1, co-exist within dendritic shafts, spines and terminals of the adult rat visual cortex. Axon terminals form asymmetric synaptic junctions with the dually labeled dendrites, suggesting that the presynaptic terminals release glutamate. Axons and dendrites expressing one without the other also are detected. These results indicate that it is possible for the generation of NO to be temporally coordinated with glutamatergic synaptic transmission at axo-dendritic and axo-axonic junctions and that NO may be generated independently of glutamatergic synaptic transmission. Together, our observations point to a greater complexity than previously recognized for glutamatergic neurotransmission, based on the joint versus independent actions of NO relative to NMDA receptors at pre- versus postsynaptic sites.     

Maternal immune activation altered microglial immunoreactivity in the brain of postnatal day 2 rat offspring

Microglia, the resident immune cells of the central nervous system, play critical roles in neurodevelopment, synaptic pruning, and neuronal wiring. Early in development, microglia migrate via the tangential and radial migration pathways to their final destinations and mature gradually, a process that includes morphological changes. Recent research has implicated microglial abnormality in the etiology of schizophrenia. Since prenatal exposure to viral or bacterial infections due to maternal immune activation (MIA) leads to increased risk of schizophrenia in the offspring during adulthood, the present study systematically investigated how MIA induced by polyinosinic:polycytidylic acid (a mimic of viral double‐stranded RNA) affected microglial immunoreactivity along the migration and maturation trajectories in the brains of male and female rat offspring on postnatal day (PND) 2. The immunohistochemistry revealed significant changes in the density of IBA‐1 immunoreactive cells in the corpus callosum, somatosensory cortex, striatum, and the subregions of the hippocampus of the MIA offspring. The male and female MIA offspring displayed markedly altered microglial immunoreactivity in both the tangential and radial migration, as well as maturation, pathways when compared to their sex‐ and age‐matched controls as evidenced by morphology‐based cell counting. Given the important roles of microglia in synaptic pruning and neuronal wiring and survival, these changes may lead to structural and functional neurodevelopmental abnormalities, and so contribute to the functional deficits observed in juvenile and adult MIA offspring. Future research is required to systematically determine how MIA affects microglial migration and maturation in rat offspring.     

Medial prefrontal cortex N‐methyl‐D‐aspartate receptor/nitric oxide/cyclic guanosine monophosphate pathway modulates both tachycardic and bradycardic baroreflex responses

Neural reflex mechanisms, such as the baroreflex, are involved in regulating cardiovascular system activity. Previous results showed that the ventral portion of the medial prefrontal cortex (vMPFC) is involved in modulation only of the cardiac baroreflex bradycardic component. Moreover, vMPFC N‐methyl‐D‐aspartate (NMDA) receptors modulate the bradycardia baroreflex, but the baroreflex tachycardic component has not been investigated. Furthermore, glutamatergic neurotransmission into the vMPFC is involved in activation of the cardiac sympathetic and parasympathetic nervous system. Finally, it has been demonstrated that glutamatergic neurotransmission into the vMPFC can be modulated by the endocannabinoid system and that activation of the CB1 cannabinoid receptor by anandamide, an endocannabinoid, can decrease both cardiac baroreflex bradycardic and tachycardic responses. Thus, there is the possibility that glutamatergic neurotransmission into the vMPFC does not modulate only the cardiac bradycardic component of the baroreflex. Therefore, the present study investigated whether glutamatergic neurotransmission into the vMPFC modulates both cardiac baroreflex bradycardic and tachycardic responses. We found that vMPFC bilateral microinjection of the NMDA receptor antagonist AP7 (4 nmol/200 nl), of a selective inhibitor of neuronal nitric oxide (NO) synthase N‐propyl (0.08 nmol/200 nl), of the NO scavenger carboxy‐PTIO (2 nmol/200 nl), or of the NO‐sensitive guanylate cyclase ODQ (2 nmol/200 nl) decreased the baroreflex activity in unanesthetized rats. Therefore, our results demonstrate the participation of NMDA receptors, production of NO, and activation of guanylate cyclase in the vMPFC in the modulation of both cardiac baroreflex bradycardic and tachycardic responses. © 2013 Wiley Periodicals, Inc.     

Expression of nNOS and soluble guanylate cyclase in schizophrenic brain

Recent evidence suggests that nitric oxide (NO) systems are affected in the pathophysiology of schizophrenia. We quantified levels of neuronal NO synthase (nNOS) and soluble guanylate cyclase (sGC) subunit mRNAs in the prefrontal cortex of post-mortem brains from individuals with schizophrenia and controls using real-time quantitative PCR, to determine whether levels of nNOS and sGC subunits are altered in 'schizophrenic' brains. Neuronal NOS expression in the prefrontal cortex was significantly higher in individuals with schizophrenia, whereas no significant changes were found in sGC subunit mRNAs in people with schizophrenia or in controls. Abnormalities of nNOS expression in the brain might contribute to the development of schizophrenia.     

Nitric oxide: a novel link between synaptic and nonsynaptic transmission

Accumulating evidence indicates that nitric oxide (NO) inhibits the function of monoamine transporters. Because the production of NO by neuronal NO synthase (nNOS) is closely related to the activation of NMDA receptors, the level of NO around nNOS-containing synapses reflects the activity of glutamate-mediated neurotransmission. Glutamate participates mainly in synaptic interactions, but with the help of NO, the strength of excitatory input might be nonsynaptically signaled to the surrounding monoaminergic neurons, which can adapt to the changes without receiving glutamatergic input and without synthesizing glutamate receptors. Thus, the effect of NO on transporters represents a new form of interneuronal communication, a nonsynaptic interaction without receptors.     

神经元型一氧化氮合酶在学习记忆过程中的变化和作用

目的　探讨神经元型一氧化氮合酶 (neuronalnitricoxidesynthase,nNOS)及一氧化氮 (nitricoxide ,NO)在学习记忆机制中的相关作用。方法　采用免疫组化方法观察Y迷宫空间辨别学习训练后大鼠不同脑区nNOS表达变化 ,并探讨特异性nNOS抑制剂 7 nitroindozal(7 NI)、钙拮抗剂尼莫通 (nimotop)腹腔注射对大鼠学习获得和记忆再现能力的影响。结果　学习训练后海马各亚区nNOS样神经元数量及染色强度明显增加 ,而皮层和纹状体区则无显著变化 ;7 NI以剂量依赖方式损伤大鼠的学习获得能力 ,但不影响记忆再现 ,尼莫通则对这两种能力均有破坏。结论　提示学习记忆过程可能伴有nNOS合成及活性增加 ,nNOS/NO在学习获得阶段具有重要作用。     

The regulation of NMDA-evoked dopamine release by nitric oxide in the frontal cortex and raphe nuclei of the freely moving rat

The role of nitric oxide (NO) in the N-methyl-D-aspartate (NMDA)-regulated release of dopamine (DA) in the frontal cortex and raphe nuclei of the freely moving rat was measured using in vivo microdialysis. The effects of infusing the NMDA antagonist 2-amino-5-phosphonopentanoic acid (AP5; 100 microM), neuronal nitric oxide synthase (nNOS) inhibitor 7-nitroindazole (7NI; 1 mM) or the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP; 500 microM-5 mM) were studied. The infusion of NMDA caused a significant decrease in DA levels in both regions and these effects were reversed by AP5. AP5 alone was seen to increase DA, indicating that NMDA receptors tonically regulate DA release in these brain regions. 7NI also increased extracellular DA levels when administered alone and reversed the effects of NMDA in both regions. The NO donor SNAP (500 microM(-1) mM) caused a dose-dependent decrease in extracellular DA in the RN. However in the frontal cortex the highest concentration of SNAP caused extracellular dopamine to increase. These results suggest that the regulation of NMDA-evoked DA release by NO occurs in both of the brain regions studied, although the manner in which the regulation occurs seems to differ between the two.     

Changes in the postnatal development on nitric oxide system induced by serotonin depletion

Serotonin (5-HT) is expressed early during central nervous system (CNS) development and plays an important role during this period. Nitric oxide (NO) is also involved in neuronal development. Morphological and functional relationships between NO and 5-HT, demonstrated as alterations of the nitrergic system, were observed after a 5-HT depletion. It has been hypothesized that NO may be related to the neuronal damage induced by some 5-HT neurotoxins. A parachloroamphetamine (PCA) treatment is able to damage ascending 5-HT fibers proceeding from the dorsal raphe nucleus (DRN) and depletes 5-HT storage in neuronal somata. In order to study the effects of a 5-HT depletion on the nitrergic system during postnatal development, Wistar rat pups were injected subcutaneously twice, on postnatal day (PND) 3 and PND4 with PCA. Neuronal nitric oxide synthase (nNOS) immunoreactivity and NADPH diaphorase reactivity were performed on brain sections from PND5, 7, 12, 19, 29 and 62 animals. After the treatment, we found an increased NADPH-d staining and nNOS immunoreactivity in striatum, frontal cortex and hippocampus along the different studied time periods. Interestingly, the expression of both NO markers was higher when 5-HT depletion was more evident, suggesting a very close relationship between 5-HT and NO systems during postnatal development.     

Immunohistochemical study on the distribution of nitrotyrosine and neuronal nitric oxide synthase in aged rat cerebellum

In the present study, we examined age-related changes in 3-nitrotyrosine (NT) and neuronal nitric oxide synthase (nNOS) in rat cerebellum using immunohistochemistry. No immunoreactivity for NT was found in any layers of adult cerebellar cortex. In aged cerebellar cortex, the most prominent labeling of NT was found in the Purkinje cell layers and molecular layers. In aged cerebellar nuclei, NT immunoreactivity was observed in the surrounding neuropil. In aged rat cerebellum, nNOS immunoreactivity was significantly decreased in the molecular layer, while it was slightly increased in the granular layer. Image analysis showed no significant age-related changes in nNOS immunoreactivity in the cerebellar nuclei. In summary, this report has demonstrated that NT increases with age in the cerebellum, and suggests that NO production by the neuronal form of NOS may not be the rate limiting step in NT formation in the aged brain. Further work is needed to examine the mechanisms underlying the increased immunoreactivity for NT, and the functional implications of this increase.     

Blockade of NMDA receptors and nitric oxide synthesis in the dorsolateral periaqueductal gray attenuates behavioral and cellular responses of rats exposed to a live predator

Innate fear stimulus induces activation of neurons containing the neuronal nitric oxide synthase enzyme (nNOS) in defensive‐related brain regions such as the dorsolateral periaqueductal gray (dlPAG). Intra‐dlPAG administration of nitric oxide synthase (NOS) inhibitors and glutamate antagonists induce anxiolytic‐like responses. We investigated the involvement of nitric oxide (NO) and glutamate neurotransmission in defensive reactions modulated by dlPAG. We tested if intra‐dlPAG injections of the selective nNOS inhibitor, N‐propyl‐L ‐arginine (NP), or the glutamate antagonist, AP7 (2‐amino‐7‐phosphonoheptanoic acid), would attenuate behavioral responses and cellular activation induced by predator exposure (cat). Fos‐like immunoreactivity (FLI) was used as a marker of neuronal functional activation, whereas nNOS immunohistochemistry was used to identify NOS neurons. Cat exposure induced fear responses and an increase of FLI in the dlPAG and dorsal premammillary nucleus (PMd). NP and AP7 attenuated the cat‐induced behavioral responses. Whereas NP tended to attenuate FLI in the dlPAG, AP7 induced a significant reduction in cellular activation of this region. The latter drug, however, increased FLI and double‐labeled cells in the PMd. Cellular activation of this region was significantly correlated with time spent near the cat (r = 0.7597 and 0.6057 for FLI and double‐labeled cells). These results suggest that glutamate/NO‐mediated neurotransmission in the dlPAG plays an important role in responses elicit by predator exposure. Blocking these neurotransmitter systems in this brain area impairs defensive responses. The longer time spent near the predator that follows AP7 effect could lead to an increased cellular activation of the PMd, a more rostral brain area that has also been related to defensive responses. © 2009 Wiley‐Liss, Inc.     

NMDA receptor-dependent nitric oxide and cGMP synthesis in brain hemispheres and cerebellum during reperfusion after transient forebrain ischemia in gerbils: Effect of 7-nitroindazole

In this study, the N-Methyl-D-Aspartate (NMDA) receptor-dependent nitric oxide and cyclic GMP (cGMP) synthesis in the course of reperfusion after 5 min of ischemia in gerbil brain hemispheres and cerebellum were investigated. Moreover, the role of the neuronal isoform of nitric oxide (NO) synthase (nNOS) in liberation of NO in postischemic brain and the involvement of NO in membrane lipoperoxidations activated during reperfusion were evaluated. Enhancement of Ca²⁺/calmodulin-regulated NOS activity and cGMP level in brain hemispheres and in cerebellum during reperfusion was found to be coupled to the activation of the NMDA receptor. cGMP concentration 40% above the control level was observed to persist up to 7 days after ischemia. The amount of conjugated double bounds in membrane lipids and the level of thiobarbituric acid reactive substances were increased exclusively in brain hemispheres, indicating activation of lipid peroxidation. The NMDA receptor antagonist, MK-801, eliminated, and a rather selective nNOS inhibitor, 7-Nitroindazole (7-NI) attenuated, NMDA receptor-evoked enhancement of NOS activity and cGMP level in brain hemispheres and in cerebellum during reperfusion. Moreover, 7-NI decreased significantly membrane lipid peroxidation during the early time of reperfusion. Histological examination demonstrated that 7-NI protects against death a selected population of neuronal cells in CA₁ layer of hippocampus. It is suggested that NMDA receptor dependence of NO release during reperfusion is responsible for the degeneration of some populations of neurons and that the effect is mediated by activation of free radical formation and lipid peroxidation. Moreover, in cerebellum, ischemia-evoked activation of glutamatergic system stimulates NO-dependent signal transmission. Our results indicated that 7-NI has a significant ameliorating effect on biochemical alterations evoked by ischemia, suggesting nNOS inhibitors as a potential therapeutic agents in reperfusion injury. J. Neurosci. Res. 54:681–690, 1998. © 1998 Wiley-Liss, Inc.     

Bed nucleus of the stria terminalis N‐methyl‐D‐aspartate receptors and nitric oxide modulate the baroreflex cardiac component in unanesthetized rats

The bed nucleus of the stria terminalis (BST) plays a tonic role modulating the baroreflex bradycardiac response. In the present study, we verified whether local BST glutamatergic receptors and nitric oxide (NO) system modulate baroreflex bradycardiac responses. Bilateral BST‐ N‐methyl‐D‐aspartate (NMDA) receptor inhibition by treatment with the selective NMDA receptor antagonist LY235959 increased bradycardiac response to mean arterial pressure increases. Treatment with the selective non‐NMDA antagonist NBQX did not affect reflex bradycardia. These results suggest an involvement of local NMDA receptors in the BST‐related tonic inhibitory modulation of baroreflex bradycardiac response. BST treatment with the nonselective NO synthase (NOS) inhibitor L‐NAME or the selective neuronal NOS (nNOS) inhibitor N^ω‐propyl‐L‐arginine increased bradycardiac response, indicating that NO generated by nNOS activation modulates baroreflex. The NO involvement was further reinforced by observation that BST treatment with the NO scavenger carboxy‐PTIO caused an effect similar to that observed after NMDA receptor blockade or treatment with NOS inhibitors. Additionally, it was observed that LY235959 effects on baroreflex bradycardiac response were reverted by BST treatment with the NO‐donor sodium nitroprusside, suggesting an NMDA receptor–NO interaction. Baroreflex bradycardiac responses observed before and after BST treatment with LY235959 or N^ω‐propyl‐L‐arginine were no longer different when animals were pretreated intravenously with the anticholinergic drug homatropine methyl bromide. These results indicate that parasympathetic activation accounts for the effects observed after BST pharmacological manipulation. In conclusion, our data point out that local NMDA and nNOS interaction mediates the tonic inhibitory influence of the BST on the baroreflex bradycardiac response, modulating the parasympathetic cardiac activity. © 2009 Wiley‐Liss, Inc.     

Medial prefrontal cortex NMDA receptors and nitric oxide modulate the parasympathetic component of the baroreflex

The ventral portion of the medial prefrontal cortex (vMPFC) is involved in the modulation of the parasympathetic component of the baroreflex. In the present study, we verified the effect of blockade of vMPFC glutamatergic receptors and nitric oxide synthases (NOS) on the parasympathetic component of baroreflex in awake rats. Bilateral microinjection of the non-selective ionotropic glutamate antagonist kynurenic acid (KYN) into the vMPFC caused a shift of the threshold of reflex bradycardia toward higher pressures in response to increases in mean arterial pressure (MAP) caused by intravenous infusion of phenylephrine, thus indicating a tonic facilitatory influence action of vMPFC glutamate receptors on the parasympathetic component of the baroreflex. The effect of blockade of vMPFC-NMDA receptors by AP7 was similar to that observed after KYN, suggesting mediation via NMDA receptors. Pretreatment with the NOS inhibitor L-NAME or the specific neural NOS (nNOS) N(omega)-propyl-l-arginine microinjected in the vMPFC caused a shift of the reflex threshold toward higher pressures that was similar to that observed after blockade of NMDA receptors, thus indicating participation of the NO/NMDA-receptor pathway in the vMPFC modulation of the parasympathetic component of the baroreflex. In conclusion, our data indicate that glutamatergic neurotransmission in the vMPFC has a tonic facilitatory influence on the parasympathetic component of the baroreflex. Because local treatment with either the nNOS inhibitor N(omega)-propyl-l-arginine or the specific NMDA antagonist AP7 had similar effects on the baroreflex, it is also suggested that this modulation involves an NMDA-NO interaction within the vMPFC.     

In vivo modulation of nitric oxide concentration dynamics upon glutamatergic neuronal activation in the hippocampus

Nitric oxide (^?NO) is a labile endogenous free radical produced upon glutamatergic neuronal activity in hippocampus by neuronal nitric oxide synthase (nNOS), where it acts as a modulator of both synaptic plasticity and cell death associated with neurodegeneration. The low CNS levels and fast time dynamics of this molecule require the use of rapid analytical methods that can more accurately describe its signaling in vivo. This is critical for understanding how the kinetics of ^?NO‐dependent signaling pathways is translated into physiological or pathological functions. In these studies, we used ^?NO selective microelectrodes coupled with rapid electrochemical recording techniques to characterize for the first time the concentration dynamics of ^?NO endogenously produced in hippocampus in vivo following activation of ionotropic glutamate receptors. Both L ‐glutamate (1–100 mM) and N‐methyl‐D ‐aspartate (NMDA; 0.01–5 mM) produced transient, dose‐dependent increases in extracellular ^?NO concentration. The production of ^?NO in the hippocampus by glutamate was decreased by the nNOS inhibitor 7‐NI. Intraperitoneal administration of the NMDA receptor blocker, MK‐801, and the inhibitor of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoazolepropionic acid (AMPA) receptor, NBQX, applied locally greatly attenuated glutamate‐evoked overflow of ^?NO. Thus, ^?NO overflow elicited by activation of glutamate receptors appeared to result from an integrated activation of ionotropic glutamate receptors, both of the NMDA and AMPA receptors subtypes. Additionally, distinct concentration dynamics was observed in the trisynaptic loop with stronger and longer lasting effects of glutamate activation on ^?NO overflow seen in the CA1 region as compared with the dentate gyrus. Overall, the results provide a quantitative and temporal basis for a better understanding of ^?NO activity in the rat hippocampus. © 2010 Wiley‐Liss, Inc.     

The emerging role of glutamate in the pathophysiology and treatment of schizophrenia

OBJECTIVE: Research has implicated dysfunction of glutamatergic neurotransmission in the pathophysiology of schizophrenia. This review evaluates evidence from preclinical and clinical studies that brain glutamatergic neurotransmission is altered in schizophrenia, may affect symptom expression, and is modulated by antipsychotic drugs. METHOD: A comprehensive review of scientific articles published over the last decade that address the role of glutamate in the pathophysiology of schizophrenia was carried out. RESULTS: Glutamatergic neurons are the major excitatory pathways linking the cortex, limbic system, and thalamus, regions that have been implicated in schizophrenia. Postmortem studies have revealed alterations in pre- and postsynaptic markers for glutamatergic neurons in several brain regions in schizophrenia. The N-methyl-D-aspartic acid (NMDA) subtype of glutamate receptor may be particularly important as blockade of this receptor by the dissociative anesthetics reproduces in normal subjects the symptomatic manifestations of schizophrenia, including negative symptoms and cognitive impairments, and increases dopamine release in the mesolimbic system. Agents that indirectly enhance NMDA receptor function via the glycine modulatory site reduce negative symptoms and variably improve cognitive functioning in schizophrenic subjects receiving typical antipsychotics. CONCLUSIONS: Dysfunction of glutamatergic neurotransmission may play an important role in the pathophysiology of schizophrenia, especially of the negative symptoms and cognitive impairments associated with the disorder, and is a promising target for drug development.     

Nitric Oxide Production in the Striatum and Cerebellum of a Rat Model of Preterm Global Perinatal Asphyxia

Encephalopathy due to perinatal asphyxia (PA) is a major cause of neonatal morbidity and mortality in the period around birth. Preterm infants are especially at risk for cognitive, attention and motor impairments. Therapy for this subgroup is limited to supportive care, and new targets are thus urgently needed. Post-asphyxic excitotoxicity is partially mediated by excessive nitric oxide (NO) release. The aims of this study were to determine the timing and distribution of nitric oxide (NO) production after global PA in brain areas involved in motor regulation and coordination. This study focused on the rat striatum and cerebellum, as these areas also affect cognition or attention, in addition to their central role in motor control. NO/peroxynitrite levels were determined empirically with a fluorescent marker on postnatal days P5, P8 and P12. The distributions of neuronal NO synthase (nNOS), cyclic guanosine monophosphate (cGMP), astroglia and caspase-3 were determined with immunohistochemistry. Apoptosis was additionally assessed by measuring caspase-3-like activity from P2-P15. On P5 and P8, increased intensity of NO-associated fluorescence and cGMP immunoreactivity after PA was apparent in the striatum, but not in the cerebellum. No changes in nNOS immunoreactivity or astrocytes were observed. Modest changes in caspase-3-activity were observed between groups, but the overall time course of apoptosis over the first 11 days of life was similar between PA and controls. Altogether, these data suggest that PA increases NO/peroxynitrite levels during the first week after birth within the striatum, but not within the cerebellum, without marked astrogliosis. Therapeutic benefits of interventions that reduce endogenous NO production would likely be greater during this time frame.     

Temporal changes in expression of neuronal nitric oxide synthase mRNA in the rat hippocampus associated with kainate-induced seizures

We studied the temporal changes in expression of neuronal nitric oxide (NO) synthase (nNOS) mRNA in the hippocampus of rats treated with kainic acid by use of in situ hybridization technique. Intraperitoneal injection of 10 mg kg-1 kainic acid decreased expression of nNOS mRNAs in the dentate gyrus and CA3 region of the hippocampus at 3 h and 8 h and increased it in the dentate gyrus and CA1 at one week after treatment. Although our previous study indicated that administration of kainic acid increased NO generation in the rat hippocampus, present results suggest that the injection of kainic acid results in differential regulation of nNOS mRNA and NO formation in the rat hippocampus.     

Either nitric oxide or nerve growth factor is required for dorsal root ganglion neurons to survive during embryonic and neonatal development

During early embryonic (E12) development almost all dorsal root ganglion (DRG) neurons express the neuronal isoform of nitric oxide synthase (nNOS). At this stage, the axons of these neurons are rudimentary and have not made contact with peripheral tissue targets. As their axons establish contact with peripheral targets such as the skin, the number of neurons expressing nNOS decrease that correspond to increased immunoreactivity for nerve growth factor (NGF) in the skin, and its high affinity receptor, tyrosine kinase A (trkA) in both skin and DRG neurons. During late postnatal development, very few DRG neurons express nNOS; however, axotomy or NGF deprivation of cultured DRG neurons induce nNOS and NOS blockade causes neuronal death. In contrast, NGF-deprived embryonic and neonatal DRG neurons die by apoptosis, while NOS blockade has no effect. Overall, these observations suggest that NGF and nitric oxide (NO) interact during embryonic and postnatal development to facilitate neuronal selection and survival. The roles of NO, NGF and its receptor trkA in DRG neurons during different stages of development are discussed.     

Severe alcohol-induced neuronal deficits in the hippocampus and neocortex of neonatal mice genetically deficient for neuronal nitric oxide synthase (nNOS)

Alcohol can severely damage the developing brain, and neuronal loss is a critical component of this injury. Thus, identification of molecular factors that ameliorate alcohol-induced neuronal loss is of great importance. Previous in vitro work has demonstrated that nitric oxide (NO) protects neurons against alcohol toxicity. We tested the hypothesis that neonatal mice carrying a null mutation for neuronal nitric oxide synthase (nNOS), the enzyme that synthesizes NO in neurons, have an increased vulnerability to alcohol-induced neuronal loss in the neocortex and hippocampus. Wildtype mice and nNOS-/- mice received ethanol (0.0, 2.2, 3.3, or 4.4 g/kg) daily over postnatal days (P) 4-9 and were sacrificed on P10. The number of hippocampal CA1 and CA3 pyramidal cells, dentate gyrus granule cells, and neocortical neurons were determined using stereological methods. Alcohol pharmacokinetics did not differ between wildtype and nNOS-/- strains. Alcohol induced dose-dependent reductions in all four neuronal populations, and the losses were substantially more severe in the nNOS-/- mice than in wildtype. Furthermore, the threshold dose of alcohol to induce cell death was lower in the nNOS-/- mice than in the wildtype mice for all neuronal populations. While nNOS deficiency worsened alcohol-induced neuronal losses, the magnitude of this exacerbation varied among brain regions and depended on alcohol dose. These results demonstrate that nNOS deficiency decreases the ability of developing neurons in vivo to survive the toxic effects of alcohol and strengthen the hypothesis that NO exerts a neuroprotective effect against alcohol toxicity in the developing brain.     

nNOS expression in reactive astrocytes correlates with increased cell death related DNA damage in the hippocampus and entorhinal cortex in Alzheimer's disease

The immunocytochemical distribution of the neuronal form of nitric oxide synthase (nNOS) was compared with neuropathological changes and with cell death related DNA damage (as revealed by in situ end labeling, ISEL) in the hippocampal formation and entorhinal cortex of 12 age-matched control subjects and 12 Alzheimer's disease (AD) patients. Unlike controls, numerous nNOS-positive reactive astrocytes were found in AD patients around beta-amyloid plaques in CA1 and subiculum and at the places of clear and overt neuron loss, particularly in the entorhinal cortex layer II and CA4. This is the first evidence of nNOS-like immunoreactivity in reactive astrocytes in AD. In contrast to controls, in all but one AD subject, large numbers of ISEL-positive neuronal nuclei and microglial cells were found in the CA1 and CA4 regions and subiculum. Semiquantitative analysis showed that neuronal DNA fragmentation in AD match with the distribution of nNOS-expressing reactive astroglial cells in CA1 (r = 0.74, P < 0.01) and CA4 (r = 0.58, P < 0.05). A portion of the nNOS-positive CA2/CA3 pyramidal neurons was found to be spared even in the most affected hippocampi. A significant inverse correlation between nNOS expression and immunoreactivity to abnormally phosphorylated tau proteins (as revealed by AT8 monoclonal antibody) in perikarya of these CA2/3 neurons (r = -0.85, P < 0.01) suggests that nNOS expression may provide selective resistance to neuronal degeneration in AD. In conclusion, our results imply that an upregulated production of NO by reactive astrocytes may play a key role in the pathogenesis of AD.