Orexin expression and function: glucocorticoid manipulation, stress, and feeding studies.

We investigated the effects of glucocorticoid manipulation on orexin-A-induced feeding and prepro-orexin mRNA levels in the lateral hypothalamic area (LHA) of the rat brain. Adrenalectomy (ADX) reduced orexin-A-induced feeding over 4 h by about 60%, compared with shams, an effect that was reversed by corticosterone (CORT) replacement. ADX had no effect on prepro-orexin mRNA levels in the LHA in either the morning or the evening; however, message was up-regulated by CORT in the morning but not the evening. An increased number of emulsion grains per cell in the LHA suggests that this is a specific increase in prepro-orexin mRNA and is not due to an increased number of cells expressing message. Prepro-orexin mRNA levels in the LHA were elevated 4 h after injection of lipopolysaccharide, compared with saline-injected controls. Partial but not complete abolition of orexin-A-induced feeding by ADX suggests that orexin-A-induced feeding may be mediated through glucocorticoid-dependent and glucocorticoid-independent pathways. In the morning increased prepro-orexin mRNA after CORT replacement demonstrates that orexin expression is sensitive to increased concentrations of glucocorticoids. However, the lack of effect of ADX on prepro-orexin mRNA levels suggests that endogenous glucocorticoids are not involved in tonic regulation of basal prepro-orexin expression. Overall our data constitute a body of evidence for an integrated relationship between central orexin expression, stress, glucocorticoid manipulation, and feeding patterns in the rat.

Adrenalectomy further suppresses the NT-3 mRNA response to traumatic brain injury but this effect is not reversed with corticosterone.

Fluid percussion injury (FPI) and in situ hybridisation were used to evaluate the expression of NT-3 mRNA in the hippocampus after traumatic brain injury (TBI) in adrenal-intact and adrenalectomised rats (with or without corticosterone replacement). FPI and adrenalectomy independently significantly reduced the expression of NT-3 mRNA in the dentate gyrus (DG) and CA2 region. The effects of adrenalectomy in the CA2 region were partially reversed with corticosterone. In adrenalectomised animals undergoing FPI, a further significant decrease in NT-3 mRNA was observed in the DG, but this was not reversed by corticosterone. Glucocorticoids may, therefore, play a role in the basal regulation of NT-3 in the hippocampus, but the role of glucocorticoids in the modulation of the NT-3 response to TBI is unclear.

Behavioral, neuroendocrine and neurochemical effects of the imidazoline I2 receptor selective ligand BU224 in naive rats and rats exposed to the stress of the forced swim test.

RATIONALE: There is evidence for alterations in imidazoline(2) (I(2)) receptor density in depressed patients. Selective I(2) receptor ligands modulate central monoamine levels and activate the hypothalamo-pituitary-adrenal (HPA) axis and may have potential as antidepressants. OBJECTIVES: To study the behavioral effects of the selective I(2) receptor ligand BU224 in the rat forced swim test (FST) and its effects on the HPA axis and central monoaminergic responses. METHODS: Rats received saline or BU224 (10 mg/kg IP) 24, 18 and 1 h prior to 15 min exposure to the FST. Saline- and BU224-treated non-stressed groups were included. Time spent immobile, struggling and swimming calmly was measured. Plasma adrenocorticotrophic hormone (ACTH) and corticosterone levels 90 min post-BU224 were measured in addition to tissue levels of monoamines and metabolites in the frontal cortex, hippocampus and hypothalamus. RESULTS: Administration of BU224 significantly reduced immobility and increased mild swimming without affecting struggling. Exposure to the FST significantly increased plasma ACTH and corticosterone levels. BU224 administration also increased ACTH and potentiated the ACTH response to FST with no effect on corticosterone. BU224 administration significantly increased frontal cortex 5-hydroxytryptamine (5-HT) levels and decreased 5-HT turnover in the frontal cortex and hypothalamus of rats exposed to FST. In non-stressed rats, BU224 decreased 5-HT turnover in the hippocampus and hypothalamus and decreased norepinephrine turnover in the frontal cortex. CONCLUSIONS: The selective I(2) receptor ligand BU224 reduces immobility of rats in the FST, indicative of antidepressant-like activity. This effect is accompanied by alterations in HPA axis and central monoaminergic activity.

Opioid peptides endomorphin-1 and endomorphin-2 in the immune system in humans and in a rodent model of inflammation.

Endomorphin (EM)-1 and EM-2 are tetrapeptides with high affinity and selectivity for the micro-opioid receptor. We have utilized specific radioimmunoassays to characterize EM-1 and EM-2 in immune tissues from normal human subjects and from rats with adjuvant arthritis (AA). PBLs from three normal human subjects contained 248, 13, and 303 pg EM-1 per 100 million cells, whereas EM-2 was measured in two subjects at 69 and 588 pg per 100 million cells. In AA rats, EM-1 (but not EM-2) contents in the spleen and thymus were elevated compared with levels in tissues from non-AA controls. EM-1 was detectable in five of eight samples of synovial tissue from inflamed hind paws, whereas EM-2 was detectable in two of eight synovial extracts. Neither EM-1 nor EM-2 were detectable in synovial tissue from non-AA rats. To our knowledge, this is the first report of endomorphins in normal human immune cells. Increased endomorphin expression or uptake in peripheral tissues in a rodent model of chronic inflammation provides potential for endomorphins to selectively modulate chronic inflammation in mammals.

Hypothalamo-pituitary-adrenal axis and chronic immune activation.

Corticosteroids have potent immunosuppressive and anti-inflammatory effects. Although corticosteroids are an important weapon in the clinical arsenal for treating inflammatory episodes, the mechanisms underlying the actions and regulation of endogenous corticosteroids remain obscure. In the late 1980s and early 1990s, a hypothesis was proposed that suggested that susceptibility to autoimmune disease was linked to a hypoactive hypothalamo-pituitary-adrenal (HPA) axis. It was further suggested that this defect in regulation of the HPA axis was situated at the level of the hypothalamus. This compelling hypothesis directly linked control of the HPA axis with susceptibility to disease rather than just severity of inflammation. The initial findings acted as a stimulus to further research, and over the next decade the hypothesis was tested. Recent studies suggest that the original hypothesis is in need of modification and that susceptibility is more complex and requires the involvement of more than a single parameter. These data are discussed together with recent developments concerning regulation of the HPA in disease in preclinical models and patients with rheumatoid arthritis. The latter studies in patients with rheumatoid arthritis provide evidence for the existence of a subpopulation of these patients with altered negative feedback regulation of the HPA axis.

Imidazoline2 (I2) receptor- and alpha2-adrenoceptor-mediated modulation of hypothalamic-pituitary-adrenal axis activity in control and acute restraint stressed rats.

Central noradrenaline regulates the activity of the hypothalamic-pituitary-adrenal (HPA) axis and the neuroendocrine response to stress. alpha2-adrenoceptors and imidazoline2 (I2) receptors modulate the activity of the central noradrenergic system. The present set of experiments investigated the role of alpha2-adrenoceptors and I2 receptors in the regulation of HPA axis activity under basal conditions and during exposure to the acute psychological stress of restraint. Three separate experiments were carried out in which rats were given an i.p. injection of either saline vehicle, the combined alpha2-adrenoceptor antagonist and I2 receptor ligand idazoxan (10 mg/kg), the selective I2 receptor ligand BU224 (2.5 or 10 mg/kg) or the selective alpha2-adrenoceptor antagonist RX821002 (2.5 mg/kg) with or without restraint stress. Drugs were administered immediately prior to restraint of 60 min duration. Blood was sampled pre-injection, 30, 60 and 240 min post-injection and plasma corticosterone was measured by radioimmunoassay. In experiment 1, idazoxan increased plasma corticosterone levels in naive animals and potentiated the corticosterone response to acute restraint stress. In experiment 2, BU224 administration increased plasma corticosterone levels in a dose-related manner in naive rats. The results of experiment 3 indicated that RX821002 also elevated plasma corticosterone levels in naive rats, however, only BU224 potentiated the corticosterone response to restraint stress. These studies suggest that both alpha2-adrenoceptors and I2 receptors play a role in modulating basal HPA axis activity and that I2 receptors may play a more important role than alpha2-adrenoceptors in modulating the HPA axis response to the acute psychological stress of restraint.

Differential regulation of proopiomelanocortin (POMC) mRNA expression in hypothalamus and anterior pituitary following repeated cyanamide with ethanol administration.

BACKGROUND/AIM: We have investigated proopiomelanocortin (POMC) mRNA expression in the arcuate nucleus of the hypothalamus (ARC) and the anterior lobe of the pituitary (AL) following repeated cyanamide-ethanol reaction (CER). METHODS: Adult male Sprague-Dawley rats (250-290 gr) were housed in a temperature and humidity controlled environment with free access to food and water. Four experimental groups were used as follows: saline (as control), cyanamide alone, ethanol alone and ethanol with cyanamide. The animals recived daily intraperitoneal injections (i.p.) of cyanamide (10 mg/kg, 60 min before ethanol dosing) with or without ethanol (1g/kg) for 5 consecutive days, and were sacrificed 60 min after the last dosing of ethanol. The results were presented as the mean +/- SEM for each group. All groups within each deta set were compared by one-way ANOVA followed by Fisher PLSD test for multiple comparisons. A value of p < 0.05 was considered significant. RESULTS: The POMC mRNA levels in ARC were significantly decreased with cyanamide compared to the control and ethanol alone (p < 0.05 and p < 0.05 respectively), but increased in AL following repeated CER. CONCLUSION: We speculate that this differential regulation of POMC mRNA expression may be partially involved in the preventive effects on alcohol intake in response to CER.

Involvement and role of the hypothalamo-pituitary-adrenal (HPA) stress axis in animal models of chronic pain and inflammation.

Hypothalamo-pituitary-adrenal (HPA) axis changes have been reported in several disease states, including major depressive disorder, rheumatoid arthritis, multiple sclerosis and various other conditions associated with chronic pain. These observations suggest that stress and the HPA axis may play important roles in the pathology of these diseases. In order to contribute to a better understanding of the role that chronic stress may play in human pathology, this review article explores the involvement of the HPA axis in those animal models of chronic pain and inflammation that entail persistent rather than intermittent stress.

Protective effect of prior acute immune challenge, but not footshock, on inflammation in the rat.

Previous studies have revealed that a single exposure to an acute stress or acute immune stimulus can produce long-lasting changes in the activity and responsiveness of the hypothalamo-pituitary-adrenal (HPA) axis. The HPA axis is believed to be an important component in determining the susceptibility and severity of inflammation in autoimmune disease models such as adjuvant-induced arthritis (AA). In the present study we have tested the hypothesis that a single exposure to either footshock or lipopolysaccharide (LPS) 3 weeks prior to adjuvant injection can alter susceptibility to AA. Changes in HPA axis parameters were also determined. The results demonstrated that prior exposure to LPS conferred resistance to inflammation in AA, which was not related to a delay in onset of inflammation but rather an alteration in susceptibility. In contrast, prior exposure to the acute stress of footshock did not alter susceptibility. HPA axis parameters were increased in adjuvant-injected rats whether inflammation was present or not. These data suggest that prior exposure to acute immune stimuli, but not to acute footshock stress, may alter susceptibility to inflammation in the rat AA model. These changes in susceptibility do not appear to be solely mediated by increases in HPA axis activity, which were apparent in all AA groups irrespective of the presence of inflammation.

Effects of acetaldehyde on c-fos mRNA induction in the paraventricular nucleus following ethanol administration.

AIMS: The effect of acetaldehyde on c-fos mRNA expression in the paraventricular nucleus (PVN) of the rat was examined using in situ hybridization histochemistry. METHODS: Increases in acetaldehyde concentrations were induced using cyanamide (a potent inhibitor of aldehyde dehydrogenase), in the presence of two different doses of ethanol. Concentrations of blood ethanol and acetaldehyde were determined by head space gas chromatography. RESULTS: Neither cyanamide alone nor the low dose of ethanol (1 g/kg) alone increased c-fos expression in the PVN. However, the combination of cyanamide and low dose ethanol resulted in a significant and maximal increase in c-fos mRNA in the PVN. High dose ethanol (3 g/kg) resulted in a significant increase in c-fos mRNA. This stimulation also appeared maximal as there was no further increase in c-fos expression in the presence of cyanamide. CONCLUSIONS: These data suggest that acetaldehyde accumulation in blood has an important stimulatory effect on c-fos expression in the PVN at low ethanol concentrations. Furthermore, this stimulation of c-fos mRNA appears to be an either/or response: not activated in response to low dose ethanol, but maximally to high dose ethanol. These data also provide further evidence for a dissociation between the activation of c-fos and corticotrophin-releasing factor (CRF) mRNA in the PVN, as we have previously demonstrated that this dose of cyanamide alone is sufficient to evoke a sustained increase in plasma corticosterone and an increase in CRF mRNA.

High alcohol preferring (HAP) and low alcohol preferring (LAP) rats show altered proopiomelanocortin (POMC) messenger RNA expression in the arcuate nucleus.

AIMS AND METHODS: We have investigated proopiomelanocortin (POMC) and neuropeptide Y (NPY) gene expression in the arcuate nucleus of the hypothalamus comparing high alcohol preference (HAP) rats and low alcohol preference (LAP) rats under basal conditions using in situ hybridization histochemistry. RESULTS: A significantly higher expression of POMC mRNA was observed in HAP rats compared with LAP rats. In contrast, no difference in NPY mRNA expression was observed between the two rat lines. CONCLUSIONS: These data suggest that an endogenous opioid system may contribute to alcohol preference in HAP/LAP rat lines.