Chronic Isolation Stress Affects Subsequent Crowding Stress-Induced Brain Nitric Oxide Synthase (NOS) Isoforms and Hypothalamic-Pituitary-Adrenal (HPA) Axis Responses.

The nitric oxide (NO) pathway in the brain is involved in response to psychosocial stressors. The aim of this study was to elucidate the role of nNOS and iNOS in the prefrontal cortex (PFC), hippocampus (HIP), and hypothalamus (HYPO) during social isolation stress (IS), social crowding stress (CS), and a combined IS + CS. In the PFC, 3 days of CS increased iNOS but not nNOS protein level. In the HIP and HYPO, the levels of nNOS and iNOS significantly increased after 3 days of CS. In the PFC, IS alone (11 days) enhanced iNOS protein level following 3 days of CS and increased nNOS level in the HIP and HYPO after 14 days of CS. By contrast, in the HIP, IS abolished the subsequent CS-induced increase in nNOS in the HIP and strongly elevated iNOS level after 7 days of CS. In the HYPO, prior IS inhibited nNOS protein level induced by subsequent CS for 3 days, but increased nNOS protein level after longer exposure times to CS. Isolation stress strongly upregulated plasma interleukin-1ß (IL-1ß) and adrenocorticotropic hormone (ACTH) levels while corticosterone (CORT) level declined. We show that the modulatory action of the NO pathway and ACTH/CORT adaptation to chronic social isolation stress is dependent on the brain structure and nature and duration of the stressor. Our results indicate that isolation is a robust natural stressor in social animals; it enhances the NO pathway in the PFC and abolishes subsequent social CS-induced NOS responses in the HIP and HYPO.

Chronic social isolation in adaptation of HPA axis to heterotypic stress.

BACKGROUND: Social crowding and isolation are recognized as major stressors and risk factors for development of psychiatric disorders. Chronic isolation stress (IS) and crowding stress (CS) activate neuroendocrine and neurochemical mechanisms, that activate the hypothalamic-pituitary-adrenal (HPA) axis. Changes of the plasma level of interleukin-1ß (IL-1ß), ACTH and corticosterone (CORT) after chronic psychosocial IS and CS were investigated. METHODS: Control rats were kept 5 per cage and not stressed. Stressed groups were subjected to either CS for 3, 7, 14days+restraint stress (RS) or IS for (11days) before this treatment was applied. Crowded rats were remained (24 in one cage) and RS rats were restrained for 10min. Total CORT, ACTH and IL-1ß levels were measured using commercially available kits. RESULTS: Social CS for 3days significantly increased plasma IL-1ß level. Social IS increased plasma IL-1ß level after longer period of subsequent CS 7 and 14days, than ACTH and CORT, after 3 and 7days. Prior IS significantly increased plasma IL-1ß level induced by subsequent combined CS for 3days+acute RS, but significantly or totally inhibited the acute stress-induced increase of plasma IL-1ß level after 7 and 14days of combined stress. IS, by contrast, strongly inhibited the increase of plasma ACTH and CORT level induced by combined CS+acute RS. CONCLUSION: Chronic IS augments the changes of IL-1ß level induced by a longer crowding period than ACTH and CORT. Modulatory action of IL-1ß and pituitary-adrenocortical hormones adaptation to chronic social stress is asynchronous.

Psychosocial stress inhibits additional stress-induced hyperexpression of NO synthases and IL-1ß in brain structures.

BACKGROUND: The aim of this study was to compare the expression of interleukin-1ß (IL-1ß), neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) in the prefrontal cortex (PFC), hippocampus (HIP) and hypothalamus (HT) during chronic crowding (CS) (psychosocial) and restraint (RS) (physico-psychological) stress. Adaptational changes of these stress mediators to a subsequent acute RS, in two models of chronic stress were investigated. METHODS: Rats were crowded (24 in one cage) or restrained in metal tubes for 10min twice a day for 3, 7, and 14 consecutive days and decapitated. For determination of adaptational changes the chronically crowded and restrained rats 24h after the last stress session were subjected to a single 10min RS. The IL-1ß, nNOS and iNOS protein levels in brain structures samples were analyzed by Western blot procedure. RESULTS: Chronic CS for 3days did not markedly change the subsequent acute stress induced expression of nNOS, iNOS and IL-1ß protein level in PFC and iNOS protein level in HT. CS markedly decreased the expression of nNOS, iNOS and IL-1ß in HIP. By contrast, parallel chronic RS, significantly increased the subsequent acute stress-induced expression of iNOS and IL-1ß in PFC and considerably increased iNOS level in HT. CONCLUSION: Chronic psychosocial stress, may protect against possible harmful action of hyperproduction of iNOS and iNOS derived nitric oxide (NO) mainly in PFC and HIP. By contrast, chronic physico-psychosocial stress may strongly potentiate additional stress-induced harmful effects of NOS and IL-1ß hyperproduction.

Cytokines, prostaglandins and nitric oxide in the regulation of stress-response systems.

Hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis is accepted as one of the fundamental biological mechanisms that underlie major depression. This hyperactivity is caused by diminished feedback inhibition of glucocorticoid (GC)-induced reduction of HPA axis signaling and increased corticotrophin-releasing hormone (CRH) secretion from the hypothalamic paraventricular nucleus (PVN) and extra-hypothalamic neurons. During chronic stress-induced inhibition of systemic feedback, cytosolic glucocorticoid receptor (GR) levels were significantly changed in the prefrontal cortex (PFC) and hippocampus, both structures known to be deeply involved in the pathogenesis of depression. Cytokines secreted by both immune and non-immune cells can markedly affect neurotransmission within regulatory brain circuits related to the expression of emotions; cytokines may also induce hormonal changes similar to those observed following exposure to stress. Proinflammatory cytokines, including interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) are implicated in the etiologies of clinical depression and anxiety disorders. Prolonged stress responses and cytokines impair neuronal plasticity and stimulation of neurotransmission. Exposure to acute stress and IL-1ß markedly increased IL-1ß levels in the PFC, hippocampus and hypothalamus, as well as overall HPA axis activity. Repeated stress sensitized the HPA axis response to IL-1ß. Inflammatory responses in the brain contribute to cellular damage associated with neuropsychiatric diseases related to stress. Physical, psychological or combined-stress conditions evoke a proinflammatory response in the brain and other systems, characterized by a complex release of several inflammatory mediators including cytokines, prostanoids, nitric oxide (NO) and transcription factors. Induced CRH release involves IL-1, IL-6 and TNF-α, for stimulation adrenocorticotropic hormone (ACTH) release from the anterior pituitary. NO also participates in signal transduction pathways that result in the release of corticosterone from the adrenal gland. NO participates in multiple interactions between neuroendocrine and neuroimmune systems in physiological and pathological processes. Neuronal NO synthase (nNOS) modulates learning and memory and is involved in development of neuropsychiatric diseases, including depression. Nitric oxide generated in response to stress exposure is associated with depression-like and anxiety-like behaviors. In the central nervous system (CNS), prostaglandins (PG) generated by the cyclooxygenase (COX) enzyme are involved in the regulation of HPA axis activity. Prior exposure to chronic stress alters constitutive (COX-1) and inducible (COX-2) cyclooxygenase responses to homotypic stress differently in the PFC, hippocampus and hypothalamus. Both PG and NO generated within the PVN participate in this modulation. Acute stress affects the functionality of COX/PG and NOS/NO systems in brain structures. The complex responses of central and peripheral pathways to acute and chronic stress involve cytokines, NO and PG systems that regulate and turn off responses that would be potentially harmful for cellular homeostasis and overall health.

Influence of chronic stress on brain corticosteroid receptors and HPA axis activity.

BACKGROUND: Disruption of the glucocorticoid negative feedback system evoked in animals by chronic stress can be induced by downregulation of glucocorticoid receptors (GRs) in several brain regions. In the present study, the dynamics of the changes in GRs, in brain structures involved in stress reactions, prefrontal cortex, hippocampus and hypothalamus was compared with the peripheral hypothalamo-pituitary-adrenocortical (HPA) axis hormones response to chronic stress. METHODS: Rats were exposed to 10 min restraint or restrained twice a day for 3, 7 or 14 days, and 24 h after the last stress session exposed to homotypic stress for 10 min. Control rats were not restrained. After rapid decapitation at 0, 1, 2, and 3 h after stress termination, trunk blood for plasma adrenocorticotropic hormone (ACTH) and corticosterone determinations was collected and prefrontal cortex, hippocampus and hypothalamus were excised and frozen. Plasma hormones were determined using commercially available kits and glucocorticoids and mineralocorticoids protein levels in brain structure samples were determined by western blot procedure. RESULTS: Restraint stress alone significantly decreased glucocorticoid receptor (GR) level in prefrontal cortex and hippocampus, and increased mineralocorticoid receptor (MR) level in hypothalamus. Prior repeated stress for 3 days significantly increased GR protein level in hippocampus and diminished that level in hypothalamus in 7 days stressed rats. Acute stress-induced strong increase in plasma ACTH and corticosterone levels decreased to control level after 1 or 2 h, respectively. Prior repeated stress for 3 days markedly diminished the fall in plasma ACTH level and repeated stress for 7 days moderately deepened this decrease. Plasma ACTH level induced by homotypic stress in rats exposed to restraint for 3, 7, and 14 days did not markedly differ from its control level, whereas plasma corticosterone response was significantly diminished. The fast decrease of stress-induced high plasma ACTH and corticosterone levels was accompanied by a parallel decline of GR level only in prefrontal cortex but not in the hippocampus or hypothalamus. CONCLUSIONS: Comparison of the dynamics of changes in plasma ACTH and corticosterone level with respective alterations in GR and MR in brain structures suggests that the buffering effect of repeated stress depends on the period of habituation to stress and the brain structure involved in regulation of these stress response.

Effect of repeated restraint on homotypic stress-induced nitric oxide synthases expression in brain structures regulating HPA axis.

BACKGROUND: Restraint stress (RS) markedly increases interleukin 1-ß (IL-1ß) generation in brain structures involved in hypothalamic-pituitary adrenocortical (HPA) axis regulation. The IL-1ß-induced transient stimulation of HPA axis activity was parallel in time and magnitude to respective changes in regulation of HPA activity. In the present experiment the expression of neuron al and inducible nitric oxide synthase (nNOS and iNOS) were investigated in prefrontal cortex, hippocampus and hypothalamus in response to acute restraint stress in control and prior repeatedly restrained rats. METHODS: Experiments were performed on male Wistar rats which were exposed to 10 min restraint stress or restrained twice a day for 3 days, and 24 h after the last stress period exposed to homotypic stress for 10 min. After rapid decapitation at 0, 1, 2 and 3 h after cessation of stress, trunk blood was collected and prefrontal cortex, hippocampus and hypothalamus were excised and frozen. Interleukin-1ß, adrenocorticotropic hormone (ACTH) and corticosterone (CORT) levels were determined in plasma using commercially available kits and neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) in brain structure samples were analyzed by western blot procedure. RESULTS: Prior repeated restraint stress enhanced the acute restraint stress induced increase in IL-1ß levels in all three structures examined. Restraint stress for 10 min moderately decreased nNOS level in prefrontal cortex in control rats, augmented this level in hippocampus and markedly increased nNOS level in hypothalamus. Restraint itself significantly decreased iNOS level in prefrontal cortex, while it enhanced iNOS level in hippocampus and hypothalamus. Prior restraint stress for 3 days enhanced the nNOS level in prefrontal cortex and hippocampus and did not substantially affect nNOS levels response in hypothalamus. Repeated restraint stress considerably augmented the iNOS levels in both prefrontal cortex, hippocampus and hypothalamus induced by followed homotypic stress. CONCLUSION: These results indicate that during restraint stress nNOS regulate formation of low amount of NO and the high-output generation of NO is effected by inducible isoform of nitric oxide synthase. Prior repeated stress significantly enhances the homotypic stress-induced nNOS and iNOS responses.

Brain nitric oxide synthases in the interleukin-1ß-induced activation of hypothalamic-pituitary-adrenal axis.

BACKGROUND: Interleukin-1ß (IL-1ß), the major cytokine involved in activation of hypothalamic-pituitary-adrenal (HPA) axis modulates both central and peripheral components regulating HPA activity. The role of nitric oxide (NO) generated by neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) in brain structures involved in HPA axis regulation has not been elucidated. The aim of the study was to assess the receptor selectivity of IL-1ß stimulatory action on HPA axis and to determine the involvement of nNOS and iNOS in this stimulation. METHODS: Experiments were performed on male Wistar rats which were injected intraperitoneally (ip) with IL-1ß (5 µg/kg) or IL-1 receptor antagonist (IL-1ra) (50 µg/kg or 100 µg/kg) 15 min before IL-1ß. Rats were sacrificed by rapid decapitation 1, 2 or 3 h after IL-1ß administration. Trunk blood for ACTH, corticosterone and IL-1ß determinations was collected and prefrontal cortex, hippocampus and hypothalamus were excised and snap frozen. Western blot analyses were performed and IL-1ß, nNOS and iNOS protein were determined in brain structures samples. RESULTS: IL-1ß significantly increased plasma ACTH, corticosterone and IL-1ß levels during 2 h after ip administration. IL-1 receptor antagonist was able to abolish the stimulatory effect of IL-1ß on plasma ACTH and corticosterone levels and significantly, but not totally, reduced plasma IL-1ß level. The role of NO in prefrontal cortex, hippocampus and hypothalamus in the IL-1ß-induced HPA axis activity alterations was determined by measuring the changes in nNOS and iNOS levels. The highest level of both izoenzymes 1 h following IL-1ß administration decreased in a regular, parallel manner 2 and 3 h later, approaching control values. These changes were almost totally prevented by pretreatment with IL-1 receptor antagonist. In the hypothalamus the IL-1ß-induced initial significant increase of nNOS regularly decreased in a modest rate and remained at significant higher level compared to control values. By contrast, iNOS level gradually increased 2 and 3 h after IL-1ß administration in a significant time-dependent manner. The changes in both NOS izoenzyme levels in hypothalamus were suppressed by pretreatment with IL-1 receptor antagonist. Results also show that a regular and parallel decrease of nNOS in the hypothalamus and prefrontal cortex are parallel in time and magnitude to respective fall in plasma IL-1ß and ACTH levels. CONCLUSION: The present study suggests that the IL-1ß-induced transient stimulation of HPA axis activity is parallel in time and magnitude to the respective changes of nNOS in hypothalamus and prefrontal cortex, the brain structures involved in regulation of HPA axis activity.

Effect of prior stress on interleukin-1ß and HPA axis responses to acute stress.

Interleukin-1ß (IL-1ß) level is modulated during multiple stress reactions both in brain structures involved in hypothalamic-pituitary-adrenal (HPA) axis regulation and peripheral systems. Multiple distinct stressors induce different IL-1ß and HPA axis responses. The purpose of the present study was to determine if the effect of prior repeated restraint stress on IL-1ß levels in prefrontal cortex, hippocampus, hypothalamus and plasma may have an impact on alterations induced in HPA axis responses. Experiments were performed on male Wistar rats which were exposed to 10 min restraint stress twice a day for 3 days. Twenty four hours after the last stress period rats were restrained for 10 min and decapitated at 0, 1, 2 or 3 h after cessation of stress. Control rats were injected ip with saline and some of experimental groups with IL-1ß receptor antagonist (IL-1ra). After rapid decapitation, trunk blood was collected and prefrontal cortex, hippocampus and hypothalamus were excised and frozen. Interleukin-1ß, adrenocorticotropic hormone (ACTH) and corticosterone (CORT) levels were determined in plasma using commercially available kits and IL-1ß levels in brain structures samples were analyzed by western blot procedure. Repeated restraint for 3 days alone did not alter resting plasma levels of IL-1ß, and moderately augmented plasma ACTH and CORT levels and IL-1ß content in brain structures 24 h after the last restraint. IL-1ß antagonist abolished the increase in plasma levels of IL-1ß, ACTH and CORT as well as IL-1ß in brain structures in response to repeated stress and also reduced these changes induced by 10 min stress. This suggests the selectivity of IL-1ß receptors in central and peripheral mechanisms modulating the stress-induced HPA axis responses. These results indicate that repeated stress markedly increases IL-1ß production in brain structures involved in HPA axis regulation. The present results support the role of brain and peripheral IL-1ß in adaptation of HPA response during prolonged stress.

Interleukin-1 (IL-1) in stress-induced activation of limbic-hypothalamic-pituitary adrenal axis.

Proinflammatory cytokine interleukin-1 (IL-1) produced during psychological and immunological stress, plays a significant role in the neuroendocrine and stress responses. Brain IL-1 is an important mediator in stress-induced stimulation of the limbic-hypothalamic-pituitary-adrenal axis and secretion of ACTH and corticosterone. This review aims to describe some signaling pathways between the limbic-hypothalamic-pituitary structures during prolonged stress responses including their sensitization and adaptation. Interleukin-1 represents an important central component operating in neurochemical and immune network for efficient coping in preventing stress-associated psycho- and neuropathology.