Maternal aggression in endothelial nitric oxide synthase-deficient mice

Lactating female rodents protect their pups by expressing fierce aggression, termed maternal aggression, toward intruders. Mice lacking the neuronal nitric oxide synthase gene (nNOS-/-) exhibit significantly impaired maternal aggression, but increased male aggression, suggesting that nitric oxide (NO) produced by nNOS has opposite actions in maternal and male aggression. In contrast, mice lacking the endothelial nitric oxide synthase gene (eNOS-/-) exhibit almost no male aggression, suggesting that NO produced by eNOS facilitates male aggression. In the present study, maternal aggression in eNOS-/- mice was examined and found to be normal relative to wild-type (WT) mice in terms of the percentage displaying aggression, the average number of attacks against a male intruder, and the total amount of time spent attacking the male intruder. The eNOS-/- females also displayed normal pup retrieval behavior. Because a significant elevation of citrulline, an indirect marker of NO synthesis, occurs in neurons of the hypothalamus of lactating WT mice in association with maternal aggression, we examined the brains of eNOS-/- females for citrulline immunoreactivity following an aggressive encounter. The aggressive eNOS-/- females exhibited a significant elevation of citrulline in the medial preoptic nucleus and the subparaventricular zone of the hypothalamus relative to unstimulated lactating eNOS-/- females. Taken together, these results suggest that NO produced by eNOS neither facilitates nor inhibits maternal aggression and that NO produced by eNOS has a different role in maternal and male aggression.     

Photoperiod-dependent effects of neuronal nitric oxide synthase inhibition on aggression in Siberian hamsters

Many nontropical species undergo physiological and behavioral adaptations in response to seasonal changes in photoperiod, or day length. In most rodent species, short winter photoperiods reduce testosterone concentrations, which provoke gonadal regression and reduce testosterone-dependent behaviors such as mating and aggression. Seasonally-breeding Siberian hamsters, however, are paradoxically more aggressive in short-days, despite much reduced reproductive activity and testosterone concentrations. Nitric oxide (NO) signaling has been proposed as part of an alternate mechanism underlying this phenomenon. A reduction in neuronal nitric oxide synthase (nNOS), the enzyme responsible for synthesizing NO in the brain, is associated with increased aggression in male short-day hamsters. In the present study, we hypothesized that pharmacological inhibition of nNOS would increase aggressive behavior in long days, but not in short days because nNOS is already reduced. Adult male Siberian hamsters were housed in either long (LD 16:8h) or short (LD 8:16h) photoperiods for 8weeks, then treated with either the selective nNOS inhibitor, 3-bromo-7-nitroindazole (3BrN) or oil vehicle, and subsequently tested for aggression in a resident-intruder test. Treatment with 3BrN increased attack frequency and duration in long days, but had no effect in short days. Short days also reduced testosterone concentrations, without any effect of treatment. These data provide further evidence linking reduced nNOS to elevated short-day aggression and support a role for NO signaling in this phenomenon.     

Nitric oxide-dependent penile erection in mice lacking neuronal nitric oxide synthase.

BACKGROUND: Nitric oxide (NO) has been implicated as a mediator of penile erection, because the neuronal isoform of NO synthase (NOS) is localized to the penile innervation and NOS inhibitors selectively block erections. NO can also be formed by two other NOS isoforms derived from distinct genes, inducible NOS (iNOS) and endothelial NOS (eNOS). To clarify the source of NO in penile function, we have examined mice with targeted deletion of the nNOS gene (nNOS- mice). MATERIALS AND METHODS: Mating behavior, electrophysiologically induced penile erection, isolated erectile tissue isometric tension, and eNOS localization by immunohistochemistry and Western blot were performed on nNOS- mice and wild-type controls. RESULTS: Both intact animal penile erections and isolated erectile tissue function are maintained in nNOS mice, in agreement with demonstrated normal sexual behaviors, but is stereospecifically blocked by the NOS inhibitor, L-nitroarginine methyl ester (L-NAME). eNOS is abundantly present in endothelium of penile vasculature and sinusoidal endothelium within the corpora cavemosa, with levels that are significantly higher in nNOS- mice than in wild-type controls. CONCLUSIONS: eNOS mediates NO-dependent penile erection in nNOS- animals and normal penile erection. These data clarify the role of nitric oxide in penile erection and may have implications for therapeutic agents with selective effects on NOS isoforms.     

Effect of nitric oxide synthesis inhibition on mouse liver and brain metallothionein expression

The role of nitric oxide (NO) production on metallothionein (MT) regulation in the liver and the brain has been studied in mice by means of the administration of nitric oxide synthase (NOS) inhibitors. Mice injected with either the arginine analog N^G-monomethyl-L-arginine (L-NMMA) or the heme binding compound 7-nitro indazole (7-NI) showed consistently increased liver MT-I mRNA and MT-I+II total protein levels, suggesting that NO is involved in the hepatic MT regulation. In agreement with the liver results, in situ hybridization analysis demonstrated a significant upregulation of the brain MT-I isoform in areas such as the cerebrum cortex, neuronal CA1-CA3 layers and dentate gyrus of the hippocampus, and Purkinje cell layer of the cerebellum, in 7-NI treated mice. The same trend was observed for the brain specific isoform, MT-III, but to a much lower extent. The effect of NOS inhibition was also evaluated in a MT-inducing condition, namely during immobilization stress. In both the liver and the brain, stress upregulated the MT-I isoform, and 7-NI significantly reduced or even blunted the MT-I response to stress, suggesting a mediating role of NO on MT-I regulation during stress. Stress also increased the MT-III mRNA levels in some brain areas, an effect blunted by the concomitant administration of 7-NI, which in some areas even decreased MT-III mRNA levels below the saline injected mice. Results in primary culture of neurons and astrocytes demonstrate significant effects of the NOS inhibitors in some experimental conditions. The present results suggest that NO may have some role on MT regulation in both the liver and the brain.     

Prostaglandin E2 inhibits vasotocin-induced osmotic water permeability in the frog urinary bladder by EP1-receptor-mediated activation of NO/cGMP pathway

PGE(2) is a well-known inhibitor of the antidiuretic hormone-induced increase of osmotic water permeability (OWP) in different osmoregulatory epithelia; however, the mechanisms underlying this effect of PGE(2) are not completely understood. Here, we report that, in the frog Rana temporaria urinary bladder, EP(1)-receptor-mediated inhibition of arginine-vasotocin (AVT)-induced OWP by PGE(2) is attributed to increased generation of nitric oxide (NO) in epithelial cells. It was shown that the inhibitory effect of 17-phenyl-trinor-PGE(2) (17-ph-PGE(2)), an EP(1) agonist, on AVT-induced OWP was significantly reduced in the presence of 7-nitroindazole (7-NI), a neuronal NO synthase (nNOS) inhibitor. NO synthase (NOS) activity in both lysed and intact epithelial cells measured as a rate of conversion of l-[(3)H]arginine to l-[(3)H]citrulline was Ca(2+) dependent and inhibited by 7-NI. PGE(2) and 17-ph-PGE(2), but not M&B-28767 (EP(3) agonist) or butaprost (EP(2) agonist), stimulated NOS activity in epithelial cells. The above effect of PGE(2) was abolished in the presence of SC-19220, an EP(1) antagonist. 7-NI reduced the stimulatory effect of 17-ph-PGE(2) on NOS activity. 17-ph-PGE(2) increased intracellular Ca(2+) concentration and cGMP in epithelial cells. Western blot analysis revealed an nNOS expression in epithelial cells. These results show that the inhibitory effect of PGE(2) on AVT-induced OWP in the frog urinary bladder is based at least partly on EP(1)-receptor-mediated activation of the NO/cGMP pathway, suggesting a novel cross talk between AVT, PGE(2), and nNOS that may be important in the regulation of water transport.     

Asymmetric dimethylarginine (ADMA)--a modulator of nociception in opiate tolerance and addiction?

Nitric oxide (NO) is generated from l-arginine by NO synthases, of which three forms have been identified: endothelial, inducible and neuronal (eNOS, iNOS and nNOS, respectively). The l-arginine metabolite asymmetric dimethylarginine (ADMA) is a potent, noncompetitive inhibitor of nNOS, while its congener N(G)-monomethyl-l-arginine (l-NMMA) is a less potent, competitive inhibitor. In rat neurons large amounts of ADMA are found, suggesting its importance in modulating neuronal activity. Humans generate approximately 300mumol ( approximately 60mg) ADMA per day. It is released from myelin basic proteins that are highly expressed in neuronal tissue. ADMA is mainly degraded by the action of the enzyme dimethylarginine dimethylaminohydrolase (DDAH), which exists in two isoforms. DDAH1 is highly expressed in brain, suggesting specific function in this area. The presence of nNOS and DDAH1 in brain suggests that ADMA may have specific CNS activity and be more than an unregulated metabolite. Increased NO production-either prior to or concurrently with opioid administration-results in an enhanced rate and extent of development of tolerance to morphine in mice. NO produces an alteration in the mu-opioid receptor that increases constitutive receptor activity. It thereby reduces the ability of a selective mu-opioid agonist to activate the mu-opioid receptor; these in vitro molecular effects occur in a time course consistent with the in vivo development of antinociceptive tolerance in mice. Amongst many other synthetic NOS inhibitors of varying specificity, 7-nitroindazole (7-NI) has been shown to have a high affinity (IC(50) 0.71 microM) to nNOS. Selective blockade of nNOS by 7-NI attenuated morphine withdrawal in opiate dependent rats, suggesting nNOS as a viable target for development of pharmacotherapies. We hypothesize that, by inhibiting nNOS and reducing NO levels, ADMA may decrease mu-opiate receptor constitutive activity, resulting in alteration of the analgesic dose-response curve of morphine.     

Inhibitory effects and spectral interactions of isomeric methoxyindazoles on recombinant nitric oxide synthases.

A series of isomeric methoxyindazoles has been evaluated as inhibitors of purified recombinant neuronal, inducible, and endothelial nitric oxide synthases (NOS). 7-Methoxyindazole (7-MI) was the most active compound of this series and displayed selectivity toward the constitutive neuronal (NOS I) and endothelial (NOS III) NOS isoforms, the inducible NOS II being almost insensitive to this inhibitor. 6-, 5-, and 4-Methoxyindazoles were almost inactive against all three NOS isoforms. Inhibition of NO and citrulline formation catalyzed by neuronal NOS in the presence of 7-MI appeared to be competitive versus both substrate L-arginine (L-arg) and (6R)-5,6,7,8-tetrahydrobiopterin (BH(4)) cofactor. 7-MI only slightly inhibited NADPH oxidase activity and was inactive against the cytochrome c (cyt c) reductase activity of neuronal NOS at concentrations up to 100-fold higher than its IC(50) value for inhibition of citrulline formation. UV/Vis and EPR studies indicated that 7-MI interacts with the oxygenase domain of neuronal NOS (NOS I(oxy)) in an identical manner but with a much lower affinity than 7-nitroindazole (7-NI). These results demonstrate that an indazole derivative bearing an electron-rich substituent in the 7-position is also a NOS I inhibitor and that such a compound presents strong similarities with the mechanism of inhibition of 7-NI.     

Impaired social recognition in male mice with repeated experience of aggression

Social recognition is crucial for many aspects of animal behavior in stabilized population. Preliminary data proposed impairment of social recognition in male mice with long experience of aggression. To check this hypothesis, experiments with male mice with different aggressive experience (during 2 and 20 days) were performed. Two types of losers were used as partners: losers with active defense reactions and losers displaying submissive postures. The enhanced aggressive motivation was found in both groups of aggressors. Mice with short aggressive experience demonstrated intensive attacks toward the active losers and decreased aggression directed to submissive losers. Mice with long aggressive experience did not change their behavior depending on a type of the partner and displayed a high level of aggression as a result of dominant aggressive motivation and impaired social recognition.     

Citrulline immunohistochemistry for demonstration of NOS activity in vivo and in vitro.

Nitric oxide (NO), a biomolecule with major cytotoxic potency, is generated by NO synthases (NOS) utilizing l-arginine as substrate and citrulline is formed as a "side product." In brain tissue, citrulline is considered to be produced exclusively by NOS, due to the incomplete urea cycle in the brain. We aimed to characterize NOS activity by citrulline immunostaining in different cell types of the brain under in situ conditions and in slice and culture experiments. NOS-positive neurons and activated microglial cells were the most prominent citrulline-positive structures. Lack of citrulline immunoreaction in neurons of nNOS knockout mice emphasizes the dependency of citrulline positivity on NOS activity, and likewise there was no citrulline staining after application of the NOS inhibitors 7-nitroindazole and NIL. Interestingly, only a portion of NOS-containing neurons costained for citrulline. The inhibition of argininosuccinate synthetase by alpha-methyl-dl-aspartate increased the number of citrulline-positive cells, apparently due to reduction of the turnover rate of citrulline. Cells positive for NOS but negative for citrulline may indicate that the enzyme is either not activated or inhibited by cellular control mechanisms. The fact that not all citrulline-positive cells were NOS positive may be explained by an insufficient detection sensitivity or by disparate sites of citrulline production and recycling. The present results show that citrulline immunocytochemistry offers a viable and convenient means for studying NOS activity at the single-cell level to elicit its posttranslational control under physiological and pathophysiological conditions.     

The role of nitric oxide on visual-evoked potentials in MPTP-induced Parkinsonism in mice

The present study aimed to elucidate visual evoked potentials (VEP) changes in MPTP induced Parkinson’s disease (PD) and investigate the possible benefical effects of neuronal (n) and inducible (i) nitric oxide synthase (NOS) inhibitors on altered VEPs, lipid peroxidation and apoptosis. 3 months old C57BL/6 mice were randomly divided into 6 groups which included control (C), 7-nitra indazole treated (7-NI), S-methylisothiourea (SMT) treated, 1,2,3,6-tetrahydropyridine (MPTP) treated, 7-NI + MPTP treated and SMT + MPTP treated. Motor activity of mice was evaluated via the pole test. At the end of the experimental period VEPs were recorded, brain and retina tissues were removed for biochemical analysis. Dopaminergic neuron death at substantia nigra (SN) was determined by immunohistochemical analysis of tyrosine hydroxylase (TH). Immunohistochemical staining was also performed to determine iNOS and nNOS in all tissue sections. Mice with experimental PD exhibited decreased motor activity. Dopaminergic cell death at pars compacta of SN (SNpc) was significantly increased in MPTP treated group compared to control. Diminished Parkinsonism symptoms were observed in 7-NI + MPTP and SMT + MPTP groups. Treatment with 7-NI and SMT decreased dopaminergic cell death in MPTP treated mice. Caspase-3 activity, nitrite/nitrate and 4-hydroxynonenal (4-HNE) levels were significantly increased in SN of MPTP treated mice compared to control. Treatment with 7-NI and SMT significantly decreased elevated caspase-3 activity, nitrite/nitrate and 4-HNE levels in SN of MPTP treated mice. No significant difference in above parameters were observed in the retina. VEP latencies were significantly prolonged in MPTP group compared to control group. 7-NI and SMT treatment caused a significant decrease in VEP latencies in MPTP treated mice compared to none treated MPTP group. This data shows that 7-NI and SMT improves prolonged VEP latencies. The protective effects of 7-NI and SMT on VEP alterations can be related to decreased dopaminergic cell death and reduced lipid peroxidation.     

Possible involvement of neuronal nitric oxide synthase enzyme in early-phase isoflurane-induced hypotension in rats

This study was conducted to demonstrate the involvement of nitric oxide synthase (NOS) in the early-phase isoflurane-induced hypotension and to ascertain whether this NOS is neuronal NOS (nNOS) or endothelial NOS (eNOS). Mean arterial pressures (MAPs) were directly measured from the femoral arteries of urethane-anesthetized rats. Isoflurane-induced changes in MAP were monitored in rats following pretreatment with vehicle or one of the following NOS inhibitors: L-N^G-monomethyl-L-arginine (L-NMMA), which is non-selective; L-N^G-nitro arginine (L-NOARG), which is more selective for nNOS and eNOS; and 7-nitroindazole (7-NI), which is selective for nNOS. Exposure to 2% isoflurane in oxygen produced a triphasic reduction in MAP, including an early phase in which mean arterial pressure (MAP) fell by 25-30% during the initial 2½ min. This early hypotensive response, but not subsequent phases, was abolished by i.v. pretreatment with either L-NMMA or L-NOARG. The early-phase hypotension was also significantly attenuated by i.p. pretreatment with 7-NI; however, the blockade was not as complete as with L-NMMA or L-NOARG. Cerebella and aorta were removed from vehicle- and 7-NI pretreated rats and assayed for NOS activity by determining the conversion of [¹⁴C]L-arginine to [¹⁴C]L-citrulline. The 7-NI pretreatment significantly reduced NOS activity in the cerebellum but not the aorta. These findings indicate that the early-phase isoflurane-induced hypotension may involve nNOS as well as eNOS. The nNOS may participate in regulation of isoflurane-induced neuronal release of endogenous opioid peptide, which produces a vasodilation that is dependent on NO derived from an action of eNOS.     

Inhibition of neuronal nitric oxide synthase by 7-nitroindazole attenuates acute lung injury in an ovine model

Nitric oxide (NO) has been shown to play a major role in acute lung injury (ALI) after smoke inhalation. In the present study, we developed an ovine sepsis model, created by exposing sheep to smoke inhalation followed by instillation of bacteria into the airway, that mimics human sepsis and pneumonia. We hypothesized that the inhibition of neuronal NO synthase (nNOS) might be beneficial in treating ALI associated with this model. Female sheep (n = 26) were surgically prepared for the study and given a tracheostomy. This was followed by insufflation of 48 breaths of cotton smoke (40 degrees C) into the airway of each animal and subsequent instillation of live Pseudomonas aeruginosa [5 x 10(11) colony forming units (CFU)] into each sheep's lung. All sheep were mechanically ventilated using 100% O2. Continuous infusion of 7-nitroindazole (7-NI), an nNOS inhibitor, NG-monomethyl-l-arginine (l-NMMA), a nonspecific NOS inhibitor, or aminoguanidine (AG), an inducible NOS inhibitor, was started 1 h after insult. The administration of 7-NI improved pulmonary gas exchange (PaO2/FiO2; where PaO2 is arterial PO2 and FiO2 is fractional inspired oxygen concentration) and pulmonary shunt fraction and attenuated the increase in lung wet-to-dry weight ratio seen in the nontreated sheep. Histologically, 7-NI prevented airway obstruction. The increase in airway blood flow after injury in the nontreated group was significantly inhibited by 7-NI. The increase in plasma concentration of nitrate and nitrite (NOx) was inhibited by 7-NI as well. Posttreatment with l-NMMA improved the pulmonary gas exchange, but AG did not. The results of the present study show that nNOS may be involved in the pathogenesis of ALI after smoke inhalation injury followed by bacterial instillation in the airway.     

In vitro and in vivo evidences that antioxidant action contributes to the neuroprotective effects of the neuronal nitric oxide synthase and monoamine oxidase-B inhibitor, 7-nitroindazole

The neuronal nitric oxide synthase (nNOS) inhibitor, 7-nitroindazole (7-NI) is neuroprotective against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonism. Monoamine oxidase (MAO)-B inhibitory action partially contributes to this effect. We tested the hypothesis that 7-NI could be a powerful hydroxyl radical (OH) scavenger, and interferes with oxidative stress caused by MPTP. We measured OH, reduced glutathione (GSH), as well as superoxide dismutase (SOD) and catalase activities in the nucleus caudatus putamen and substantia nigra of Balb/c mice following MPTP and/or 7-NI administration. The nNOS inhibitor caused dose-dependent inhibition in the production of OH in (i) Fenton-like reaction employing ferrous citrate in a cell-free system in test tubes, (ii) in isolated mitochondrial preparation in presence of MPP+, and (iii) in the striatum of mice systemically treated with MPTP. An MPTP-induced depletion of GSH in both the nuclei was blocked by 7-NI, which was dose-dependent (10-50mg/kg), but independent of MAO-B inhibition. The nNOS-mediated recovery of GSH paralleled attenuation of MPTP-induced depletion of striatal dopamine. MPTP-induced increase in the activities of striatal or nigral SOD and catalase were significantly attenuated by 7-NI treatment. These results suggest potent antioxidant action of 7-NI in its neuroprotective effects against MPTP-induced neurotoxicity.     

Mice: progesterone stimulates aggression in pregnancy-terminated females

Three experiments were conducted in order to assess the role of progesterone (P) in the aggressive behavior displayed by late pregnant Rockland-Swiss mice toward adult male intruders. In Experiment 1, hysterectomy on the 15th day of gestation reduced the aggressive behavior normally displayed by pregnant mice toward male intruders. In Experiment 2, Silastic implants of P stimulated aggression in hysterectomized mice but did not fully restore the behavior to the level of fighting normally observed in pregnant animals. In Experiment 3, aggressive behavior in P-treated hysterectomized animals was inhibited by simultaneous exposure to estradiol (E). Also, treatment with E alone did not stimulate aggression in hysterectomized mice. While pregnancy-induced aggression is promoted by P, other neuroendocrine factors may act in concert with the steroid to fully stimulate aggression in gravid mice.     

Inhibition of nNOS reduces ischemic cell death through down-regulating calpain and caspase-3 after experimental stroke

In vitro nitric oxide (NO) regulates calpain and caspase-3 activation, and in vivo neuronal nitric oxide synthase (nNOS), calpain and caspase-3 participate in the ischemic brain injury. Our objective was to investigate whether nNOS was involved in the ischemic brain injury through activating calpain and caspase-3 during experimental stroke. Rats received 1-h ischemia by intraluminant filament, and then reperfused for 23 h (R 23 h). nNOS inhibitor 7-nitroindozale (7-NI, 50 mg/kg) was administrated intraperitoneally 5 min before ischemia. Our data showed that treatment with 7-NI markedly reduced neurological deficits, the brain swelling, and the infarct volume at R 23 h. Enzyme studies revealed significant suppression of the activities of m-calpain and caspase-3 in penumbra and core, and the activities of μ-calpain in penumbra, but not in core, in 7-NI-treated rats versus vehicle-treated rats. Western blot analysis demonstrated that 7-NI markedly increased the levels of MAP-2 and spectrin in penumbra and core compared with vehicle-treated rats. Histopathological studies displayed that 7-NI significantly reduced the necrotic cell death in penumbra and core, and apoptotic cell death in penumbra, but not in core. These data demonstrate the involvement of NO produced by nNOS in the ischemic neuronal injury through affecting the activation of calpain and caspase-3 in penumbra and core after experimental stroke, which provides a new perspective on possible mechanisms of action of nNOS inhibition in cerebral ischemia.     

Acute and conditioned hypoxic tolerance augmented by endothelial nitric oxide synthase inhibition in mice.

To identify a possible role for nitric oxide (NO) in acute hypoxic tolerance (HT) we measured hypoxic survival time (HST), effect of hypoxic conditioning (HC), and survival following hypoxic conditioning while blocking or mimicking the action of nitric oxide synthase (NOS). To inhibit NOS, CD-1 mice were given supplemental endogenous NOS inhibitor asymmetrical dimethylarginine (ADMA) or a synthetic NOS inhibitor N(omega)-nitro-L-arginine (L-NNA), both of which nonselectively inhibit three of the isoforms of NOS [inducible (iNOS), neuronal (nNOS), and endothelial NOS (eNOS)]. ADMA (10 mg/kg i.p.) or saline vehicle was given 5 min before HST testing. L-NNA was given orally at 1 g/l in drinking water with tap water as the control for 48 h before testing. Both ADMA and L-NNA significantly increased HST and augmented the HC effect on HST. Neither the nNOS selective inhibitor 7-nitroindazole (7-NI) nor the iNOS selective inhibitor N-{[3-(aminomethyl)phenyl]methyl}-enthanimidamide (1400W) had a statistically significant effect on HST or HT. The NO donor, 3-morpholinosydnoeimine, when given alone did not significantly decrease HT, but it did mitigate the increased HT effect of L-NNA. These data confirm that acute hypoxic conditioning increases HT and that NOS inhibition by endogenous (ADMA) and a synthetic NOS inhibitor (L-NNA) further increases HT, whereas iNOS and nNOS inhibition does not, suggesting that it is the inhibition of eNOS that mediates enhancement of HT.     

Neuronal NOS Inhibitor and Conventional Antidepressant Drugs Attenuate Stress-induced Fos Expression in Overlapping Brain Regions

Recent evidence indicates that the administration of inhibitors of neuronal nitric oxide synthase (nNOS) induces antidepressant-like effects in animal models such as the forced swimming test (FST). However, the neural circuits involved in these effects are not yet known. Therefore, this study investigated the expression of Fos protein, a marker of neuronal activity, in the brain of rats submitted to FST and treated with the preferential nNOS inhibitor, 7-nitroindazole (7-NI), or with classical antidepressant drugs (Venlafaxine and Fluoxetine). Male Wistar rats were submitted to a forced swimming pretest (PT) and, immediately after, started receiving a sequence of three ip injections (0, 5, and 23 h after PT) of Fluoxetine (10 mg/kg), Venlafaxine (10 mg/kg), 7-NI (30 mg/kg) or respective vehicles. One hour after the last drug injection the animals were submitted to the test session, when immobility time was recorded. After the FST they were sacrificed and had their brains removed and processed for Fos immunohistochemistry. Independent group of non-stressed animals received the same drug treatments, or no treatment (naïve). 7-NI, Venlafaxine or Fluoxetine reduced immobility time in the FST, an antidepressant-like effect. None of the treatments induce significant changes in Fos expression per se. However, swimming stress induced significant increases in Fos expression in the following brain regions: medial prefrontal cortex, nucleus accumbens, locus coeruleus, raphe nuclei, striatum, hypothalamic nucleus, periaqueductal grey, amygdala, habenula, paraventricular nucleus of hypothalamus, and bed nucleus of stria terminalis. This effect was attenuated by 7-NI, Venlafaxine or Fluoxetine. These results show that 7-NI produces similar behavioral and neuronal activation effects to those of typical antidepressants, suggesting that these drugs share common neurobiological substrates.     

Changes of [<Superscript>3</Superscript>H]Muscimol, [<Superscript>3</Superscript>H]Flunitrazepam and [<Superscript>3</Superscript>H]MK-801 Binding in Rat Brain by Prolonged Ventricular Infusion of 7-Nitroindazole

In the present study, we have investigated the effects of prolonged inhibition of nitric oxide synthase (NOS) by infusion of neuronal NOS (nNOS) inhibitor, 7-nitroindazole (7-NI), to examine modulation of NMDA and GABA_A receptor binding in rat brain. The duration of sleeping time was significantly increased by the pre-treatment with 7-NI (100 mg/kg) 30 min before pentobarbital (40 mg/kg) treatment in rats. However, the duration of pentobarbital-induced sleep was shortened by the prolonged infusion of 7-NI into cerebroventricle for 7 days. We have investigated the effect of NOS inhibitor on NMDA and GABA_A receptor binding characteristics in discrete areas of brain regions by using autoradiographic techniques. The GABA_A receptors were analyzed by quantitative autoradiography using [³H]muscimol and [³H]flunitrazepam binding, and NMDA receptor binding was analyzed by using [³H]MK-801 binding in rat brain slices. Rats were infused with 7-NI (500 pmol/10 l/ h, i.c.v.) for 7 days, through pre-implanted cannula by osmotic minipumps. The levels of [³H]muscimol were markedly elevated in cortex, caudate putamen, and thalamus while the levels of [³H]flunitrazepam binding were only elevated in cerebellum by NOS inhibitor. However, there was no change in the level of [³H]MK-801 binding except decreasing in the thalamus. These results show that the prolonged inhibition of NOS by 7-NI-infusion highly elevates [³H]muscimol binding in a region-specific manner and decreases the pentobarbital-induced sleep.     

Oxidative stress in the rats brain capillaries in sepsis--the influence of 7-nitroindazole

As a part of blood-brain barrier, brain capillaries participate in pathophysiological events during systemic inflammation. We investigated the effects of 7-nitroindazole (7-NI), selective neuronal nitric oxide synthase (NOS) inhibitor, to oxidative status (OS) of brain capillaries. Adult Wistar rats were randomized at groups: control group (CG) (sham operated), sepsis group (GS) (cecal ligation and perforation with inoculation of Escherichia coli (ATCC 25922), 7-NI group (G7-NI), (30 mg/kg b/w i.p.) and 7-NI + sepsis group (G7-NIS), (7-NI was applied 30 minutes before operation). Lipid peroxidation index (LPI), nitrite concentration, superoxide dismutase (SOD) activity and superoxide anion (O2*-) content were determined 3, 6, 24 and 48 hour in each group. Cerebral capillaries were separated from non-vascular brain tissue using sucrose gradient. Compared to controls, LPI, nitrite and O2*- increased at SG. In the G7-NIS, LPI reached control values at the 24th and 48th hour, while nitrite were decreased at the 3rd and 24th hour, compared to controls. In the same group, O2*- decreased at the 3rd, 6th and 24th hour, although SOD showed variable activity. The systematic nNOS inhibition with 7-NI forces OS on early terms of sepsis, but lately it contributes to the normalization of OS in cerebral capillaries.