Stress induced alterations in neuronal and inducible nitric oxide synthase in brain structures involved in HPA axis activity

P Rachwalska, A Gadek-Michalska, J Tadeusz, J Spyrka, J Bugajski.

Institute of Pharmacology, Polish Academy of Sciences, Department of Physiology, 31-343, Poland

The purpose of the present study was to determine the role of nitric oxide synthesized by neuronal and inducible nitric oxide synthase (nNOS and iNOS) in response to acute restraint stress in rats.

Experiments were performed on male Wistar rats (6 weeks old, 190–220 g) housed under controlled conditions (12 h light/dark cycle; a constant temperature 22 ± 2°C) with free access to a standard laboratory diet and tap water. Animals were exposed to acute restraint stress in metal tubes for 10 min. Immediately after the restraint or 1 h, 2 h and 3 h later the rats were decapitated and trunk blood was collected and prefrontal cortex, hippocampus and hypothalamus were excised and frozen. Adrenocorticotropic hormone (ACTH), corticosterone and interleukin-1β (IL-1β) levels were determined in plasma using commercially available kits and nNOS, iNOS and IL-1β levels in brain structures samples were analyzed by western blot procedure. Data were analyzed by one-way analysis of variance (n=8 per group) followed by Tukey’s multiple range test which showed statistically significant differences between control and treatment groups.

Restraint stress for 10 min (10\' RS) markedly increased the basal IL-1β content in prefrontal cortex immediately after restraint termination (145 ± 8% of control, **p<0.01). This elevated level substantially decreased 1-2 h later, and significantly raised 3 h later (193 ± 11%, ***p<0.001). In hippocampus IL-1β level was moderately increased in 1 and 2 h, and significantly elevated 3 h (151 ± 14%, **p<0.01) after stress termination. The strongest increase of IL-1β content 1 and 2 h after acute stress cessation appeared in hypothalamus. In prefrontal cortex restraint stress for 10 min decreased moderately constitutive NOS below the control level 1 and 2 h after stress. The changes of iNOS level in that structure closely followed the alterations in nNOS level, a moderate decrease at 1 and 2 h, but there is a significant increase of iNOS 3 h after termination of restraint (161 ± 19%, ***p<0.001). The restraint stress-induced changes of nNOS and iNOS levels in prefrontal cortex were almost identical and parallel in time and magnitude alterations in IL-1ß content. This strongly suggests a functional connection in activation of nitric oxide and IL-1ß in prefrontal cortex during acute stress. Acute restraint stress moderately increased the hypothalamus nNOS level in the whole observation period. The iNOS levels remained unchanged 0-3 h after stress terminations. By contrast, IL-1β content in that structure was significantly elevated 1 h (186 ± 38%, *p<0.05) and 2 h (224 ± 59%, **p<0.01) after stress termination. Neither nNOS nor iNOS levels in hippocampus were significantly affected by restraint stress 0-3 h after its termination and IL-1β level was significantly elevated 3 h after stress termination.

Acute restraint stress induced significant and relatively identical parallel increase in ACTH (from 146.7 ± 8.5 pg/ml in control to 1083.0 ± 66.8 pg/ml in 10\' RS 0h group, ***p<0.001) and corticosterone secretion (from 99.9 ± 26.6 ng/ml to 666.1 ± 22.3 ng/ml, ***p<0.001) immediately after stress termination. These changes completely disappeared 1h later. The plasma IL-1β response was much higher than ACTH and corticosterone response with its peak 1h after cessation of stress (from 27.1 ± 2.2 pg/ml in control to 517.2 ± 123.4 pg/ml in 10\' RS 1h group, **p<0.01). This effect was absent 1h later. Therefore no time and magnitude relations between IL-1ß and pituitary-adrenal response were found during acute psychosocial stress.

This research was supported by a grant POIG 01.01.02-12-004/09-00 ”Depression-Mechanisms-Therapy” financed by European Regional Development Fund.