Stress-induced hippocampus Npas4 mRNA expression relates to specific psychophysiological patterns of stress response.

Neuronal Per-Arnt-Sim (PAS) domain protein 4 (Npas4) is a key protein that intervenes in GABA synapse scaling and neurotrophicity enhancing. Since GABA and neurotrophicity are implicated in stress response and Npas4-deficient rodents exhibit behavioral alterations, an investigation was designed in rats to verify whether stress-induced spontaneous hippocampus Npas4 mRNA expression would be associated with specific patterns of stress response. The rats were exposed to one of three stressor levels: no stress (CTL, n = 15), exposure to a footshock apparatus (Sham, S, n = 40) and footshock (F, n = 80). After stress exposure the S and F rats were tested in an activity cage, and subsequently in an elevated plus maze (EPM), just prior to the sacrifice. Using cluster analysis, the animals already assigned to a stress level were also distributed into 2 subgroups depending on their Npas4 mRNA levels. The low (L) and high (H) Npas4 expression subgroups were identified in the S and F groups, the CTL group being independent of the Npas4 levels. The Npas4 effect was studied through the interaction between stress (S and F) and Npas4 level (L and H). The biological stress response was similar in H and L rats, except blood corticosterone that was slightly lower in the H rats. The H rats were more active in the actimetry cage and presented higher levels of exploration in the EPM. They also exhibited higher hippocampus activation, as assessed by the c-fos, Egr1 and Arc mRNA levels. Therefore high Npas4 expression favors stress management.

Beneficial effects of a ketamine/atropine combination in soman-poisoned rats under a neutral thermal environment.

Exposure to organophosphorus (OP) compounds, such as pesticides and the chemical warfare agents (soman and sarin), respectively represents a major health problem and a threat for civilian and military communities. OP poisoning may induce seizures, status epilepticus and even brain lesions if untreated. We recently proved that a combination of atropine sulfate and ketamine, a glutamatergic antagonist, was effective as an anticonvulsant and neuroprotectant in mice and guinea-pigs exposed to soman. Since OP exposure may also occur in conditions of heat strain due to climate, wearing of protective gears or physical exercise, we previously demonstrated that ketamine/atropine association may be used in a hot environment without detrimental effects. In the present study, we assess soman toxicity and evaluate the effects of the ketamine/atropine combination on soman toxicity in a warm thermoneutral environment. Male Wistar rats, exposed to 31°C (easily reached under protective equipments), were intoxicated by soman and treated with an anesthetic dose of ketamine combined with atropine sulfate. Body core temperature and spontaneous locomotor activity were continuously monitored using telemetry. At the end of the warm exposure, blood chemistry and brain mRNA expression of some specific genes were measured. In soman-intoxicated animals, metabolic and genic modifications were related to convulsions rather than to soman intoxication by itself. In the warm environment, ketamine/atropine combination did not produce any side-effect on the assessed variables. Furthermore, the ketamine/atropine combination exhibited beneficial therapeutic effects on soman-intoxicated rats such as a limitation of convulsion-induced hyperthermia and of the increase in some blood chemistry markers.

Ketamine does not impair heat tolerance in rats.

Exposure to organophosphorus compounds, either pesticides or chemical warfare agents such as soman or sarin, represents a major health problem. Organophosphorus poisoning may induce seizures, status epilepticus and even brain lesions if untreated. Ketamine, an antagonist of glutamatergic receptors, was recently proved to be effective in combination with atropine sulfate as an anticonvulsant and neuroprotectant in mice and guinea pigs exposed to soman. Organophosphorus exposure may also occur in conditions of contemporary heat exposure. Since both MK-801, a more potent glutamatergic antagonist than ketamine, and atropine sulfate are detrimental for thermoregulation, we evaluated the pathophysiological consequences of ketamine/atropine combinations in a hot environment. Male wistar rats were exposed to 38°C ambient temperature and treated with atropine sulfate and/or ketamine (anesthetic and subanesthetic doses). The abdominal temperature and spontaneous locomotor activity were continuously monitored using telemetry. At the end of heat exposure, blood chemistry and the mRNA expression of some specific genes in the brain were assessed. Unlike MK-801, ketamine did not induce any deleterious effect on thermoregulation in rats. Conversely, atropine sulfate led to heatstroke and depressed the heat-induced blood corticosterone increase. Furthermore, it induced a dramatic increase in Hsp70 and c-Fos mRNA levels and a decrease in IκBα mRNA expression, both suggesting brain aggression. When combined with the anesthetic dose of ketamine, some of the atropine-induced metabolic disturbances were modified. In conclusion, ketamine can be used in hot environment and may even limit some of the biological alterations induced by atropine sulfate in these conditions.

Metyrapone effects on systemic and cerebral energy metabolism.

Metyrapone is a cytochrome P(450) inhibitor that protects against ischemia- and excitotoxicity-induced brain damages in rodents. This study examines whether metyrapone would act on energy metabolism in a manner congruent with its neuroprotective effect. In a first investigation, the rats instrumented with telemetric devices measuring abdominal temperature, received i.p. injection of either metyrapone or saline. One hour after injection, their blood and hippocampus were sampled. Hippocampus metabolite concentrations were measured using (1)H high-resolution magic angle spinning-magnetic resonance spectroscopy ((1)H HRMAS-MRS). The hippocampus levels in phosphorylated mammalian target of rapamycin (mTOR) and adenosine monophosphate-activated protein kinase (AMPK) were measured by Western Blot analysis and those of c-fos and HSP70-2 mRNA were quantified by RT-PCR. In a second investigation, the rats received the same treatment and were sacrificed 1h after. The functioning of mitochondria was immediately studied on their whole brain. Metyrapone provoked a slight hypothermia which was correlated to the increase in blood glucose concentration. Metyrapone also increased blood lactate concentrations without modifying hippocampus lactate content. In the hippocampus, metyrapone decreased γ-aminobutyric acid (GABA) and glutamate levels but increased glutamine and N-acetyl-aspartate contents (NAA). Phosphorylated mTOR and AMPK and the c-fos and HSP70-2 mRNA levels were similar between treatment groups. Metyrapone did not modify blood corticosterone levels. Mitochondrial oxygen consumption was similar in both groups whatever the substrate used. These metabolic modifications, which take place without modifying blood glucocorticoid levels, are consistent with the neuroprotective properties of metyrapone as demonstrated in animal models.

Single administration of metyrapone modifies sleep-wake patterns in the rat.

Metyrapone is a glucocorticoid synthesis inhibitor largely used to study glucocorticoid involvement in stress and memory processes. Metyrapone also acts as a stressor and therefore might modify sleep/wake patterns. However, its effects on rat sleep are unknown. We equipped 8 rats for telemetric assessment of EEG and EMG. They received first a saline injection and 2days later a 150mg/kg metyrapone injection. Metyrapone provoked immediately a waking effect together with a 3-h decrease in slow-wave sleep (SWS) and a 5-h decrease in rapid eye movement sleep (REM sleep). Thereafter, the rats exhibited homeostatic compensation through SWS and REM sleep rebounds recovering totally the sleep debt. The finding that metyrapone modified sleep patterns is important to consider for stress and memory studies using metyrapone.

The relationship between locomotion and heat tolerance in heat exposed rats.

Low spontaneous locomotor activity (SA) represents a thermoregulatory behaviour that aims at improving heat tolerance. However, high SA is observed during heat exposure. We hypothesized that high SA could be associated to brain dysfunction. Eighty male Sprague-Dawley rats were heat exposed for 90-min under a continuous assessment of SA and abdominal temperature (T(abd)) using telemetry. The time course analysis showed two SA peaks. The first one was related to exposure to novel environment, the second to heat. The maximal SA level reached in the second peak served to distribute the rats into three groups (LOW, MED and HIGH). In each SA pattern group, heat tolerance was estimated from T(abd) values. At the end of heat exposure, frontal cortex activation was assessed using c-fos, Hsp70 and IkappaBalpha mRNA expressions. The LOW rats exhibited the lowest T(abd), a slight increase in c-fos and Hsp70 mRNA expressions and a robust increase in IkappaBalpha mRNA expression. The HIGH rats exhibited the highest T(abd) and a robust increase in c-fos and Hsp70 mRNA expressions without any change in IkappaBalpha mRNA expression. The c-fos and Hsp70 mRNA expressions were positively correlated to the highest SA levels occurring 45 min before sacrifice, suggesting that high SA and frontal cortex activation are related. In conclusion, high SA is associated to decreased heat tolerance and frontal cortex activation. It may represent a marker of inadequate stress reaction.

Metyrapone blunts stress-induced hyperthermia and increased locomotor activity independently of glucocorticoids and neurosteroids.

Metyrapone, a cytochrome P(450) inhibitor used to inhibit corticosterone synthesis, triggers biological markers of stress and also reduces stress-induced anxiety-like behaviors. To address these controversial effects, 6 separate investigations were carried out. In a first set of investigations, abdominal temperature (T(abd)), spontaneous locomotor activity (A(S)) and electroencephalogram (EEG) were recorded in freely moving rats treated with either saline or 150 mg kg(-1) metyrapone. An increase in T(abd) and A(S) occurred in saline rats, while, metyrapone rats exhibited an immediate decrease, both variables returning to basal values 5h later. Concomitantly, the EEG spectral power increased in the gamma and beta 2 bands and decreased in the alpha frequency band, and the EMG spectral power increased. This finding suggests that metyrapone depressed stress-induced physiological response while arousing the animal. In a second step, restraint stress was applied 5h after injection. Metyrapone significantly blunted the stress-induced T(abd) and A(S) rise, without affecting the brain c-fos mRNA increase. Corticosterone (5 and 40 mg kg(-1)) injected concomitantly to metyrapone failed to reverse the observed metyrapone-induced effects in T(abd) and A(S). Finasteride (50 mg kg(-1)), which blocks neurosteroid production, was also unable to block these effects. In conclusion, metyrapone acutely reduced stress-induced physiological response in freely behaving rats independently from glucocorticoids and neurosteroids.