Early maternal separation promotes alterations in the thermoregulatory profile of adult Wistar rats.

Stressful lifelong events may influence psychiatric diseases, like depression and anxiety. Interestingly, depressed patients have dysfunction of thermoregulatory cooling mechanisms. Thus, understanding the mechanisms related to the thermoregulatory changes in stress-related pathologies is important to better understand the symptoms and treatments for those diseases. However, the influence of early-life stress on the thermoregulatory profile of adults is unknown. In this study, we aimed to evaluate the thermoregulatory profile of adult male Wistar rats submitted to early-life stress by maternal separation (MS). On postnatal days 2-14, rats were submitted daily to MS for 3 h per day. At 3-4 months of age, anxiety-like behavior was evaluated using the open field test and elevated plus maze, depression-like behavior was evaluated using the forced swim test and thermoregulatory profile were also evaluated. In the behavioral tests, MS animals exhibited anxiety- and depression-like behaviors, and had higher core body temperatures during dark period of the circadian cycle, when compared to controls. In addition, MS animals presented higher hyperthermic and vasoconstriction responses than control animals when exposed to the warmth environment, and engaged in cold-seeking behavior whenever possible to select their preferred ambient temperature. The results suggest that, besides emotional alterations, MS induces a change in the thermoregulatory profile of rats that persists into adulthood.

7-Nitroindazole down-regulates dopamine/DARPP-32 signaling in neostriatal neurons in a rat model of Parkinson's disease.

Neuronal nitric oxide synthase (nNOS) is involved in the regulation of diverse intracellular messenger systems in the brain. Nitric Oxide (NO) contributes to inducing signaling cascades that involve a complex pattern of phosphorylation of DARPP-32 (in Thr-34), which controls the phosphoproteins involved in neuronal activation. However, the role of NO in the pathophysiology of Parkinson's disease (PD) and its effect in striatal neurons have been scarcely explored. In the present work, we investigate the effects of a nitric oxide synthase (NOS) inhibitor, 7-nitroindazole (7-NI) in the nigrostriatal pathway of striatal 6-hydroxydopamine (6-OHDA) lesioned rats. Our quantitative histological findings show that treatment with 7-NI significantly reduced 6-OHDA-induced dopaminergic damage in the dorsolateral striatum and Substantia Nigra pars compacta (SNpc). Moreover, 6-OHDA lesioned rats show a significant increase of nNOS(+) and Phospho-Thr34-DARPP-32(+) cells, accompanied by a consequent decrease of total DARPP-32(+) cells, which suggests an imbalance of NO activity in the DA-depleted striatum, which is also reflected in behavioral studies. Importantly, these effects are reverted in the group treated with 7-NI. These results show a clear link between the state of phosphorylation of DARPP-32 and parkinsonism, which is regulated by nNOS. This new evidence suggests a prominent role for nitric oxide in the neurotransmitter balance within the basal ganglia in the pathophysiology of experimental parkinsonism.

Eag 1, Eag 2 and Kcnn3 gene brain expression of isolated reared rats.

The Eag1 and Eag2, voltage-dependent potassium channels, and the small-conductance calcium-activated potassium channel (Kcnn3) are highly expressed in limbic regions of the brain, where their function is still unknown. Eag1 co-localizes with tyrosine hydroxilase enzyme in the substantia nigra and ventral tegmental area. Kcnn3 deficiency leads to enhanced serotonergic and dopaminergic neurotransmission accompanied by distinct alterations in emotional behaviors. As exposure to stress is able to change the expression and function of several ion channels, suggesting that they might be involved in the consequences of stress, we aimed at investigating Eag 1, Eag2 and Kcnn3 mRNA expression in the brains of rats submitted to isolation rearing. As the long-lasting alterations in emotional and behavioral regulation after stress have been related to changes in serotonergic neurotransmission, expressions of serotonin Htr1a and Htr2a receptors in male Wistar rats' brain were also investigated. Rats were reared in isolation or in groups of five for nine weeks after weaning. Isolated and socially reared rats were tested for exploratory activity in the open field test for 5 min and brains were processed for reverse-transcription coupled to quantitative polymerase chain reaction (qRT-PCR). Isolated reared rats showed decreased exploratory activity in the open field. Compared to socially reared rats, isolated rats showed reduced Htr2a mRNA expression in the striatum and brainstem and reduced Eag2 mRNA expression in all examined regions except cerebellum. To our knowledge, this is the first work to show that isolation rearing can change Eag2 gene expression in the brain. The involvement of this channel in stress-related behaviors is discussed.

Tolerance to the cataleptic effect that follows repeated nitric oxide synthase inhibition may be related to functional enzymatic recovery.

Systemic or intra-striatal acute administration of nitric oxide synthase (NOS) inhibitors causes catalepsy in rodents. This effect disappears after sub-chronic treatment. The aim of the present study was to investigate if this tolerance is related to changes in the expression of NOS or dopamine-2 (D2) receptor or to a recovery of NOS activity. Male albino Swiss mice (25-30 g) received single or sub-chronic (once a day for 4 days) i.p. injections of saline or L-nitro-arginine (L-NOARG, 40 mg/kg), a non-selective inhibitor of neuronal nitric oxide synthase (nNOS). Twenty-four hours after the last injection, the animals were killed and their brains were removed for immunohistochemistry assay to detect the presence of nNOS or for 'in-situ' hybridisation study using (35)S-labeled oligonucleotide probe complementary to D2 receptor mRNA. The results were analysed by computerised densitometry. Independent groups of animals received the same treatment, but were submitted to the catalepsy test and had their brain removed to measure nitrite and nitrate (NOx) concentrations in the striatum. Acute administration of L-NOARG caused catalepsy that disappeared after sub-chronic treatment. The levels of NOx were significantly reduced after acute L-NOARG treatment. The decrease in NOx after drug injection suffered a partial tolerance after sub-chronic treatment. The catalepsy time after acute or sub-chronic treatment with L-NOARG was negatively (r = -0.717) correlated with NOx levels. Animals that received repeated L-NOARG injections also showed an increase in the number of nNOS-positive neurons in the striatum. No change in D2 receptor mRNA expression was found in the dorsal striatum, nucleus accumbens and substantia nigra. Together, these results suggest that tolerance to L-NOARG cataleptic effects do not depend on changes in D2 receptors. They may depend, however, on plastic changes in nNOS neurons resulting in partial recovery of NO formation in the striatum.

Nitric oxide synthase inhibition attenuates L-DOPA-induced dyskinesias in a rodent model of Parkinson's disease.

Chronic L-DOPA pharmacotherapy in Parkinson's disease is often accompanied by the development of abnormal and excessive movements known as L-DOPA-induced dyskinesia. Rats with 6-hydroxydopamine lesion of dopaminergic neurons chronically treated with L-DOPA develop a rodent analog of this dyskinesia characterized by severe axial, limb, locomotor and orofacial abnormal involuntary movements. While the mechanisms by which these effects occur are not clear, they may involve the nitric oxide system. In the present study we investigate if nitric oxide synthase inhibitors can prevent dyskinesias induced by repeated administration of L-DOPA in rats with unilateral 6-hydroxydopamine lesion. Chronic L-DOPA (high fixed dose, 100 mg/kg; low escalating dose, 10-30 mg/kg) treatment induced progressive dyskinesia changes. Two nitric oxide synthase inhibitors, 7-nitroindazole (1-30 mg/kg) and NG-nitro-L-arginine (50 mg/kg), given 30 min before L-DOPA, attenuate dyskinesia. 7-Nitroindazolee also improved motor performance of these animals in the rota-rod test. These results suggest the possibility that nitric oxide synthase inhibitors may be useful to treat L-DOPA-induced dyskinesia.

Anxiolytic effects induced by inhibition of the nitric oxide-cGMP pathway in the rat dorsal hippocampus.

RATIONALE: Conflicting results have been reported regarding the role of the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) pathway in the hippocampus on anxiety modulation. OBJECTIVES: To investigate the effects of intrahippocampal injections of drugs that modify the NO-cGMP pathway in rats submitted to two animal models that are sensitive to anxiolytic drugs, the elevated plus-maze and the Vogel punished licking test. MATERIALS AND METHODS: Male Wistar rats with cannulae aimed at the dentate gyrus of the dorsal hippocampus received microinjections of the NO synthase (NOS) inhibitors N (G)-nitro-L: -arginine methyl ester (LNAME, 15-300 nmol/0.2 microl), N (G)-nitro-L: -arginine (LNOARG, 50-300 nmol/0.2 microl), 7-nitroindazole (7NI, 10-100 nmol/0.2 microl), or the soluble guanylate cyclase inhibitor 1H-oxadiazolo-quinoxalin-1 one (ODQ, 10-100 nmol/0.2 microl), and were submitted to the elevated plus-maze. In a second group, the animals received 7NI, LNAME, or ODQ and were submitted to the Vogel punished licking test. To control for drug-induced changes in locomotor behavior, the animals were submitted to an open arena or to the Rota-rod test. RESULTS: All drugs increased the exploration of the open arms of the elevated plus-maze. They also increased the number of punished licks in the Vogel test, indicating an anxiolytic effect. The anxiolytic effect of LNAME was prevented by previous treatment with L: -arginine (300 nmol/0.2 microl). Except for the lower dose of LNAME (15 nmol), administration of the NOS inhibitors or ODQ did not change exploratory activity in the open field nor cause any gross locomotor impairment in the Rota-rod test. CONCLUSION: The results suggest that NO plays an anxiogenic role in the dentate gyrus of the dorsal hippocampus.

Intracerebroventricular administration of nitric oxide-sensitive guanylyl cyclase inhibitors induces catalepsy in mice.

RATIONALE: Catalepsy is a preclinical test that predicts extrapyramidal symptoms in humans. It models symptoms of acute extrapyramidal side effects induced at the beginning of antipsychotic treatment. Nitric oxide (NO) plays a role in a series of neurobiological functions underlying behavior. For example, inhibition of NO synthesis disrupts rodent exploratory behavior and induces catalepsy. Although several effects mediated by NO involve the activation of soluble guanylyl cyclase (sGC), the transduction mechanism of the catalepsy-inducing effect of NO has not yet been investigated. OBJECTIVES: The study was designed to test if intracerebroventricular (i.c.v.) microinjection of NO-sensitive inhibitors of sGC (NO-sGC) induces catalepsy in mice similar to that induced by NO synthase (NOS) inhibitors. Exploratory behavior was tested in the open field. In addition, the effects of a NOS inhibitor on oxidative metabolites of NO were measured in the striatum. MATERIALS AND METHODS: Drug effects were examined in the hanging-bar test after the following i.c.v. treatments: oxadiazolo-quinoxalin (ODQ, 30-300 nmol) or methylene blue (MB, 3-100 nmol), selective and nonselective sGC inhibitors, respectively, or 7-nitroindazole (7-NI, 3-90 nmol) and G-nitro-L: -arginine methyl ester (L: -NAME, 3-90 nmol), selective and nonselective neuronal NOS inhibitors. To test if the effects were related to interference with the NO system, additional groups received 7-NI (30 nmol), ODQ (100 nmol), or L-NAME (90 nmol) preceded by L: -arginine (L: -arg, 30-100 nmol, i.c.v. 30 min before). A possible interference of ODQ and 7-NI on exploratory behavior was tested in an open field. The concentration of nitrites and nitrates (NO( x )) in striatum homogenates was measured by the Griess reaction. RESULTS: Both NO-sGC and NOS inhibitors induced catalepsy in mice that lasted for at least 2 h. The range of effective doses of these drugs, however, was limited, and the dose-effect curves had an inverted U shape. The cataleptic effect induced by L: -NAME was inversely correlated with NO( x ) products in the striatum. The cataleptic effect of 7-NI and ODQ was prevented by pretreatment with L: -arginine. No drug changed exploratory behavior in the open field. CONCLUSION: This study showed that pharmacological disruption of the endogenous NO-sGC signaling in the central nervous system induces long-lasting catalepsy in mice. Moreover, the cataleptic effect of NOS inhibition correlates with the decrease in NO( x ) products formation in the striatum. The results give further support to the hypothesis that NO plays a role in motor behavior control mediated, at least in part, by cyclic guanosine monophosphate production in the striatum.

Acute and delayed restraint stress-induced changes in nitric oxide producing neurons in limbic regions.

RATIONALE: Microinjection into the dentate gyrus of the hippocampus of N(omega)-nitro-l-arginine methyl ester hydrochloride (l-NAME), a nitric oxide synthase (NOS) inhibitor, induces antinociceptive effect 5 days after a single restraint episode. The mechanisms of this stress-antinociceptive modulatory effect have not been investigated but may involve plastic changes in the hippocampal formation (HF). OBJECTIVE: The objective of the present study was to investigate possible mechanisms of the stress-modulating effect on antinociception induced by NOS inhibition in the hippocampus. We analyzed the effects of restraint stress on neuronal NOS (nNOS) expression and nicotinamide adenine dinucleotide phosphate-diaphorase histochemical activity (NADPH-d) in the HF and related brain regions. METHODS: Male Wistar rats (n=6-11/group) were submitted to a single (acute stress) or repeated (5 days) episodes of 2-h restraint. Control animals remained in their home cages being all animals daily handled during this period. In the fifth day, animals received unilateral microinjection of l-NAME (150 nmol/0.2 microl) or saline (control) into the dentate gyrus of the dorsal hippocampus (DG). Immediately before and after drug microinjection tail-flick reflex latency or hotplate licking reaction was measured. Animals were killed i. immediately; ii. 5 days after acute stress; or iii. after repeated stress. NADPH-d and nNOS expression were quantified in the HF, caudate-putamen, secondary somatosensorial, entorhinal and piriform cortices and amygdaloid complex. RESULTS: Five days after one or five restraint episodes l-NAME microinjection into the DG elicited antinociceptive effect (analysis of variance [ANOVA], P<0.05). Acute restraint stress induced a significant increase in the density of neurons expressing NADPH-d and nNOS in the amygdaloid nuclei. nNOS expression increased also in the DG and piriform cortex. Five days after a single or repeated restraint stress there was an additional increase in NADPH-d- and nNOS-positive neurons in CA1, CA3, and entorhinal cortex. No changes were seen in non-limbic regions such as the caudate-putamen and secondary somatosensorial cortex. CONCLUSION: The results confirm that the dorsal hippocampus participates in the modulation of stress consequences. They also show that a single stress episode causes acute changes in nitric oxide system in the amygdala complex and delayed modifications in the HF. The delayed (5 days) antinociceptive effect of NOS inhibition in the HF after a single restraint episode suggests that those latter modifications may have functional consequences. It remains to be tested if the acute amygdala and delayed hippocampal changes are causally related.

Comparison of neurokinin SP with diazepam in effects on memory and fear parameters in the elevated T-maze free exploration paradigm.

The elevated T-maze was combined with a free exploration protocol, which, in contrast to the conventional procedure, dispenses with handling of the animals during the experimental sessions. This allows measurement of fear indexes derived from the elevated plus-maze as well as assessment of acquisition of open arm avoidance and open arm escape in one continuous session. Retention of the different fear-responses is measured 72 h later without drug treatment. In order to assess the effects of two known anxiolytics in this paradigm, rats received an IP injection of diazepam (1 to 4 mg/kg), substance P (5 to 500 microg/kg) or vehicle (1 ml/kg) and were tested on the T-maze for 5 min. Diazepam elevated open arm activity, indicative of an anxiolytic effect. The drug also increased the latency to escape from the open arms, but did not significantly affect acquisition of open arm avoidance. During the retention trial, diazepam in higher doses impaired the performance of both fear-responses, suggestive of an anterograde amnesic effect. Substance P did not influence acquisition and retention of open arm avoidance and escape. However, in high doses, the peptide increased the sojourn time in the central arena of the maze, indicating reduced fear and, hence, a dissociation between anxiolytic and amnesic effects. The present findings demonstrate that the elevated T-maze free exploration paradigm is sensitive to anxiolytic and memory-modulating effects of drugs.