Remote CB1 receptor antagonist administration reveals multiple sites of tonic and phasic endocannabinoid neuroendocrine regulation.

Endogenous cannabinoids (endocannabinoids, eCB) are expressed throughout the body and contribute to regulation of the hypothalamo-pituitary-adrenal (HPA) axis and general stress reactivity. This study assessed the contributions of CB1 receptors (CB1R) in the modulation of basal and stress-induced neural and HPA axis activities. Catheterized adult male rats were placed in chambers to acclimate overnight, with their catheters connected and exteriorized from the chambers for relatively stress-free remote injections. The next morning, the CB1R antagonist AM251 (1 or 2 mg/kg) or vehicle was administered, and 30 min later, rats were exposed to loud noise stress (30 min) or no noise (basal condition). Blood, brains, pituitary and adrenal glands were collected immediately after the procedures for analysis of c-fos and CB1R mRNAs, corticosterone (CORT) and adrenocorticotropin hormone (ACTH) plasma levels. Basally, CB1R antagonism induced c-fos mRNA in the basolateral amygdala (BLA) and auditory cortex (AUD) and elevated plasma CORT, indicating disruption of eCB-mediated constitutive inhibition of activity. CB1R blockade also potentiated stress-induced hormone levels and c-fos mRNA in several regions such as the bed nucleus of the stria terminalis (BST), lateral septum (LS), and basolateral amygdala (BLA) and the paraventricular nucleus of the hypothalamus (PVN). CB1R mRNA was detected in all central tissues investigated, and the adrenal cortex, but at very low levels in the anterior pituitary gland. Interestingly, CB1R mRNA was rapidly and bidirectionally regulated in response to stress and/or antagonist treatment in some regions. eCBs therefore modulate the HPA axis by regulating both constitutive and activity-dependent inhibition at multiple levels.

Acute and chronic effects of ferret odor exposure in Sprague-Dawley rats.

This manuscript describes several behavioral and functional studies evaluating the capacity of ferret odors to elicit a number of acute and long-term responses in male Sprague-Dawley rats. Acute presentation elicits multiple responses, suggesting that ferret odor, likely from skin gland secretions, provides an anxiogenic-like stimulus in this strain of rats. Compared to cat odor, however, ferret odor did not produce rapid fear conditioning, a result perhaps attributable to methodological factors. Inactivation of the olfactory system and medial nucleus of the amygdala, combined with induction of the immediate-early gene c-fos, suggest the necessity of the accessory olfactory system in mediating the effects of ferret odor. Repeated exposures to ferret odor produce variable habituation of neuroendocrine and behavioral responses, perhaps indicative of the lack of control over the exact individual origin or concentration of ferret odor. Ferret odor induces rapid and long-term body weight regulation, thymic involution, adrenal hyperplasia and facilitation of the neuroendocrine response to additional challenges. It is argued that the use of such odors is exquisitely suited to investigate the brain regions coordinating anxiety-like responses and the long-term changes elicited by such stimuli.

Non-associative defensive responses of rats to ferret odor.

Predators and their odors offer an ethologically valid model to study learning processes. The present series of experiments assessed the ability of ferret odor to serve as an unconditioned stimulus and examined behavioral and endocrine changes in male Sprague-Dawley rats with single or repeated exposures in a defensive withdrawal paradigm or in their home cages. Rats exposed to ferret odor avoided the ferret odor stimulus more, exhibited greater risk assessment and displayed higher adrenocorticotropin hormone (ACTH) and corticosterone release compared with control odor exposed rats and these measures did not significantly habituate over repeated exposures. Ferret odor exposure did not show associative conditioning effects during extinction trials. However, rats that were pre-exposed to ferret odor only once, as compared to control and repeatedly exposed rats, displayed a sensitized ACTH and corticosterone response to an additional ferret odor exposure in small cages. These experiments suggest that ferret odor is a highly potent unconditioned stimulus that has long lasting effects on behavior and endocrine responses, and further suggests the independence of habituation and sensitization processes.

Voluntary freewheel running selectively modulates catecholamine content in peripheral tissue and c-Fos expression in the central sympathetic circuit following exposure to uncontrollable stress in rats.

Modulation of sympathetic drive to the spleen is one potential mechanism whereby physical activity prevents stress-induced splenic immune suppression in rats. The current study tested the hypothesis that voluntary freewheel running reduces peripheral sympathetic drive by modulating stress-induced activity of brain regions synaptically linked to sympathetically innervated peripheral organs, including the adrenals and spleen. To this end, adrenal and splenic catecholamine content and activity of the central sympathetic circuit indexed by c-Fos protein induction, elicited by acute exposure to inescapable tail shock, were measured. Stressor exposure depleted adrenal and splenic norepinephrine content and elicited a robust increase in c-Fos in the brains of sedentary rats. Physical activity status had no effect on adrenal norepinephrine content. Indicative of attenuated sympathetic drive to the spleen, however, 6 weeks of voluntary freewheel running diminished stress-induced splenic norepinephrine depletion, and significantly attenuated stress-induced c-Fos in specific brain regions responsible for sympathetic regulation, including tyrosine hydroxylase-immunoreactive neurons of the locus coeruleus, A5 cell group and rostral ventrolateral medulla. Results suggest that voluntary activity attenuates sympathetic drive to the spleen during stressor exposure by selectively modulating stress-induced activity of the central sympathetic circuit. The attenuation of sympathetic responses observed in this study may be one important mechanism for the protective effect of physical activity against stress-related illness and immunosuppression.

Environmental context and drug history modulate amphetamine-induced c-fos mRNA expression in the basal ganglia, central extended amygdala, and associated limbic forebrain.

The context in which amphetamine is administered modulates its ability to induce both behavioral sensitization and immediate early gene expression. When given in a novel test environment amphetamine produces greater levels of c-fos and arc mRNA expression in many brain regions relative to when it is given in the home cage. The purpose of the current study was to determine if environment and drug history interact to influence amphetamine-induced c-fos mRNA expression. Rats with a unilateral 6-hydroxydopamine lesion were treated for 7 days with saline or 0.5 mg/kg of d-amphetamine (i.v.) in a distinct and relatively novel test environment (Novel), or in their home cage (Home). Following a 10-12-day withdrawal period, a challenge injection of either saline or 0.5 mg/kg d-amphetamine was administered. In situ hybridization histochemistry was used to examine c-fos mRNA expression in several regions of the basal ganglia, the central extended amygdala, and limbic forebrain. In most brain regions amphetamine given in the Novel environment produced greater c-fos mRNA expression than when given it was given at Home, and drug history had no effect on amphetamine-induced c-fos mRNA expression. However, within the subthalamic nucleus, substantia nigra reticulata, and central nucleus of the amygdala prior experience with amphetamine in the Novel but not Home environment enhanced the effect of an amphetamine challenge injection on c-fos mRNA expression. In contrast, there was a decrease in c-fos mRNA expression in amphetamine-pretreated animals, regardless of environmental context, in the ventral portion of the far caudal striatum. Reexposure to an environment previously paired with amphetamine produced a conditioned increase in c-fos mRNA expression in portions of the caudate-putamen, the subthalamic nucleus, the nucleus accumbens shell and a conditioned decrease in c-fos mRNA expression in the central nucleus of the amygdala. We conclude that environmental context and drug history interact to alter the basal ganglia and central extended amygdala circuitry engaged by subsequent exposure to amphetamine, or exposure to an environment previously paired with amphetamine.

Sex differences in activated corticotropin-releasing factor neurons within stress-related neurocircuitry and hypothalamic-pituitary-adrenocortical axis hormones following restraint in rats.

Women may be more vulnerable to certain stress-related psychiatric illnesses than men due to differences in hypothalamic-pituitary-adrenocortical (HPA) axis function. To investigate potential sex differences in forebrain regions associated with HPA axis activation in rats, these experiments utilized acute exposure to a psychological stressor. Male and female rats in various stages of the estrous cycle were exposed to 30min of restraint, producing a robust HPA axis hormonal response in all animals, the magnitude of which was significantly higher in female rats. Although both male and female animals displayed equivalent c-fos expression in many brain regions known to be involved in the detection of threatening stimuli, three regions had significantly higher expression in females: the paraventricular nucleus of the hypothalamus (PVN), the anteroventral division of the bed nucleus of the stria terminalis (BSTav), and the medial preoptic area (MPOA). Dual fluorescence in situ hybridization analysis of neurons containing c-fos and corticotropin-releasing factor (CRF) mRNA in these regions revealed significantly more c-fos and CRF single-labeled neurons, as well as significantly more double-labeled neurons in females. Surprisingly, there was no effect of the estrous cycle on any measure analyzed, and an additional experiment revealed no demonstrable effect of estradiol replacement following ovariectomy on HPA axis hormone induction following stress. Taken together, these data suggest sex differences in HPA axis activation in response to perceived threat may be influenced by specific populations of CRF neurons in key stress-related brain regions, the BSTav, MPOA, and PVN, which may be independent of circulating sex steroids.

Evidence for a lack of phasic inhibitory properties of habituated stressors on HPA axis responses in rats.

This experiment tested the hypothesis that habituation to repeated stressor exposures is produced by phasic inhibitory influence on the neural circuitry that normally drives the paraventricular nucleus of the hypothalamus and subsequently the adrenocortical hormone response to psychological stress. Such a process would be expected to lower the acute response to a novel stressor when experienced concurrently with a habituated stressor. Rats were exposed to restraint or no stress conditions for 14 consecutive days. On the 15th day, the rats were exposed to the control condition (no stress), acute restraint, loud noise, or restraint and loud noise concurrently. Blood was taken and assayed for ACTH and corticosterone and brains were collected to examine c-fos messenger RNA expression in several brain areas. As predicted, the rats that received the same (homotypic) stressor repeatedly and again on the test day displayed low levels of ACTH and corticosterone, similar to the control conditions (i.e., showed habituation). All rats that received a single novel stressor on the test day, regardless of prior stress history, exhibited high levels of ACTH and corticosterone. The rats that received two novel stressors also displayed high levels of ACTH and corticosterone, but little evidence of additivity was observed. Importantly, when a novel stressor was concurrently given with a habituated stressor on the test day, no reduction of HPA axis response was observed when compared to previously habituated rats given only the novel stressor on the test day. In general, c-fos mRNA induction in several stress responsive brain areas followed the same patterns as the ACTH and corticosterone data. These data suggest that habituation of the adrenocortical hormone response to psychological stressors is not mediated by phasic inhibition of the effector system.

Hypothalamic pituitary adrenal axis responses to low-intensity stressors are reduced after voluntary wheel running in rats.

Regular physical exercise is beneficial for both physical and mental health. By contrast, stress is associated with deleterious effects on health and there is growing evidence that regular physical exercise counteracts some of the effects of stress. However, most previous studies have suggested that prior exercise does not alter the acute hypothalamic pituitary adrenal (HPA) axis responses to stress. The present series of studies provides evidence that in rats, 6 weeks (but not 1 or 3 weeks) of voluntary wheel running reduces the HPA axis responses to lower-intensity stressors such as an i.p. saline injection, exposure to a novel environment or exposure to moderate intensity noise, but not to more intense stressors such as predator odour exposure or restraint. Daily exercise does not appear to be necessary for the reduction in HPA axis responses, with intermittent access (24 h out of each 72-h period) to a running wheel for 6 weeks, resulting in similar decrements in adrenocorticotrophic hormone and corticosterone release in response to 85 dBA noise exposure. Data from in situ hybridisation for c-fos mRNA are consistent with the hypothesis that voluntary exercise results in a decrease in HPA axis responsiveness to a low-intensity stressor at a central level, with no changes in primary sensory processing. Together, these data suggest that 6 weeks of daily or intermittent exercise constrains the HPA axis response to mild, but not more intense stressors, and that this regulation may be mediated at a central level beyond the primary sensory input.