The role of CRH in behavioral responses to stress.

Corticotropin-releasing hormone (CRH) and urocortin in the central nervous system affect behavior and can enhance behavioral responses to stressors. The action of CRH-related peptides is mediated through multiple receptors that differ markedly in their pharmacological profiles and anatomical distribution. Comparative pharmacology of CRH receptor agonists suggests that CRH, urocortin, sauvagine and urotensin consistently mimic, and CRH receptor antagonists consistently lessen, functional consequences of stressor exposure. Recently, important advances have been made in understanding the CRH system and its role in behavioral responses to stress by the development of specific CRH receptor antagonists, application of antisense oligonucleotides and development of transgenic mice lacking peptides and functional receptors. This review summarizes recent findings with respect to components of the CRH system and their role in stress-induced behavioral responses.

Changes in rat adipocyte and liver glucose metabolism following repeated restraint stress.

Rats exposed to repeated restraint weigh less than controls even 8 weeks after stress. Stress-induced weight loss is lean tissue, but the post-stress difference in weight between control and restrained rats is lean and fat mass. Whole-body glucose clearance is enhanced 1 day after stress, but adipocyte glucose utilization is inhibited and muscle glucose transport is unchanged. The studies described here demonstrated that glucose transport was increased in both restrained and pair-fed rats, but that glycogen synthesis was increased only in restrained rats, which may account for the improved whole-body glucose clearance. Adipocyte glucose transport was inhibited and adipose plasma membrane beta-adrenergic receptor number was increased 1 day post-stress in restrained rats when weight loss was lean tissue, but were not different from control rats 5 days post-stress, when both fat and lean tissue were reduced. Thus, repeated restraint induces reversible changes in adipocyte metabolism that may represent a transition from the catabolic state of stress to a new energetic equilibrium in rats that maintain a reduced body weight for an extended period of time.

The role of CRF receptor subtypes in stress-induced behavioural responses.

The actions of corticotropin-releasing factor (CRF) and CRF-related peptides in the brain and periphery are mediated through multiple receptors. Two CRF receptor subtypes that differ markedly in their pharmacological profiles and anatomical distribution have been identified and characterized. Important advances have been made in understanding CRF and its actions through the development of specific CRF receptor antagonists, application of antisense oligonucleotides, and the production of transgenic mice lacking functional CRF(1) receptors. This chapter describes recent findings with respect to CRF-related peptides and CRF receptors and their role in stress-induced behaviours.

The effects of paradoxical sleep deprivation and valine on spatial learning and brain 5-HT metabolism.

We have previously reported that rapid eye movement sleep deprivation (REMSD), induced by the flower pot technique, causes a deficit in reference spatial memory and increases rates of serotonin (5-HT) metabolism in the brain. In this study we used increased concentrations of dietary valine to inhibit tryptophan (TRP) transport across the blood-brain barrier in an attempt to modify the REMSD-induced increase of 5-HT metabolism. Rats were fed either a control diet or the same diet supplemented to 2% by weight valine, and were allocated to one of three experimental groups: cage control (CC), stress tank control (TC), or REMSD. Reference and working spatial memory of all rats was tested in a Morris water maze on Days 2, 3, and 4. REMSD produced a significant decrement in reference memory on Days 2 and 4, independent of dietary condition. The valine diet had a detrimental effect on the reference memory of TC rats on Day 2 but not Day 4. Measurements made on Day 4 indicated that the valine diet decreased brain TRP only in the CC rats. In contrast, the valine diet did not prevent increases in brain TRP or 5-HT metabolism in REMSD rats, and increased hypothalamic and brain stem TRP concentrations and the hippocampal 5-HIAA/5-HT ratio in TC rats. These results indicate that dietary valine does not prevent REMSD-induced changes in spatial memory or serotonin metabolism, although it does reduce brain TRP in nonstressed rats.

Prevention of stress-induced weight loss by third ventricle CRF receptor antagonist.

We previously reported that rats exposed to repeated restraint (3 h/day for 3 days) experience temporary hypophagia and a sustained reduction in body weight compared with nonrestrained controls. Studies described here determined the involvement of central corticotropin-releasing factor (CRF) receptors in the initiation of this chronic response to acute stress. In experiment 1, Sprague-Dawley rats were fitted with cannulas in the lateral ventricle and infused with 50 micrograms of alphahCRF-(9-41) or saline immediately before restraint on each of the 3 days of restraint. The receptor antagonist inhibited hypophagia and weight loss on day 1 of restraint but not on days 2 and 3. In experiment 2, 10 micrograms of alphahCRF-(9-41) or saline were infused into the third ventricle immediately before each restraint. The receptor antagonist totally blocked stress-induced hypophagia and weight loss. These results demonstrate that CRF receptors located in or near the hypothalamus mediate the acute responses to stress that lead to a permanent change in the hormonal or metabolic processes that determine body weight and body composition.

Effect of repeated stress on body weight and body composition of rats fed low- and high-fat diets.

Exposure to the moderate stressor of 3-h restraint for 3 consecutive days causes a temporary drop in food intake but a permanent reduction in body weight in adult rats. Young rats did not show the same response. Food intake of adult rats exposed to repeated restraint was significantly lower than that of controls for 4 days after the end of stress, and there was no rebound hyperphagia. Body weight remained significantly lower for at least 40 days after stress. When the rats were fed a high-fat diet of 80% chow and 20% vegetable shortening (48% kcal fat, 16% protein), lean body mass accounted for all of the weight loss in stressed rats. When the experiment was repeated with a purified high-fat diet containing corn oil and coconut oil as the source of fat (41% kcal fat, 16% protein), weight loss consisted of both lean and fat tissue. There were no sustained changes in single time point measures of corticosterone, insulin, or leptin that could account for the reduced body weight in these rats.

The role of CRF2 receptors in corticotropin-releasing factor- and urocortin-induced anorexia.

The experiments presented in this study were designed to assess corticotropin-releasing factor (CRF) receptor subtype mediation of CRF- and urocortin (UCN)-induced decrease in food intake. Male Sprague-Dawley rats were treated with antisense and sense oligonucleotides (ON) to CRF2 receptor mRNAs for 36 h and then received an intracerebroventricular (i.c.v.) injection of CRF, UCN (3 micrograms) or saline. Antisense treatment significantly attenuated CRF- and UCN-induced suppression in food intake and HPA activation. Administration of CRF1 receptor antagonist did not affect the decrease in food intake or activation of the HPA axis induced by i.c.v. infusion of 3 micrograms CRF. The data suggest that down-regulation of CRF2 receptors selectively attenuates CRF- and UCN-induced anorexia and hypothalamo-pituitary-adrenocortical activation in rats.

A leptin dose-response study in obese (ob/ob) and lean (+/?) mice.

This experiment determined the amount of leptin required to correct different abnormalities in leptin-deficient ob/ob mice. Baseline food intakes and body weights of lean (+/?) and obese (ob/ob) C57B1/6J mice were recorded for 7 days. An Alzet miniosmotic pump was placed in the peritoneal cavity of each mouse and delivered 0, 1, 2, 5, 10, or 42 microg/day human leptin for 7 days. In ob/ob mice, 2 microg leptin/day reduced food intake and body weight, and increased hypothalamic and brain stem serotonin concentrations. All fat pads were reduced 35-40% by 10 microg leptin/day, and liver weight, lipid, and glycogen decreased. Serum insulin and glucose were reduced in all leptin-treated ob/ob mice, and levels were normalized by 10 microg/day leptin. Low rectal temperatures of ob/ob mice were corrected by 10 and 42 microg/day leptin. These doses also increased brown adipose tissue uncoupling protein expression. The only responses in lean mice were a transient reduction in food intake and weight loss with 10 or 42 microg/day leptin. This study shows enhanced leptin sensitivity in ob/ob mice and suggests that increased temperature and sympathetic activity are indirect responses to high concentrations of protein.

Failure to change exploration or saccharin preference in rats exposed to chronic mild stress.

Chronic mild stress (CMS) exposes animals to unpredictable stressors. Reduced consumption of sucrose or saccharin solutions by CMS rats has been used as a putative measure of anhedonia, typical of depression. Our objective was to determine whether saccharin consumption and preference and suppression of exploratory and rearing behaviors in the open field were reliable indicators of CMS-induced behavioral depression. In Experiment 1, male Wistar rats subjected to 6 weeks of CMS consumed significantly less food and gained less weight than controls. CMS did not effect saccharin intake, or preference, measured in a two-bottle test with water. CMS rats exposed to a novel open field showed increased exploration and rearing. In a second test, performed immediately after a novel stress of restraint, there were no differences in exploratory or rearing behavior of CMS and control rats. In Experiment 2, CMS was reduced to 3 weeks and rats were single or group housed in their home cages. Open field activity of CMS rats was similar to that in Experiment 1. Saccharin preference of CMS rats was significantly suppressed when tested after 24 hours of water deprivation, but was not different from controls after 5 hours of water deprivation. In the final experiment Sprague Dawley rats behaved the same as Wistar rats in the CMS paradigm. Therefore, the CMS protocol used in these experiments did not induce behaviors indicative of depression but did cause a mild anorexia and weight loss. Saccharin intake of CMS rats was dependent upon their dehydration state and could not be attributed to stress-induced anhedonia.

Effect of restraint stress on food intake and body weight is determined by time of day.

Three experiments were conducted to investigate the effect of restraint stress applied at different times of the light-dark cycle on feeding behavior and body weight of rats. Sprague-Dawley rats were restrained for 3 h in restraining tubes either at the start or the end of the light cycle. There was a significant reduction in food intake on the day of restraint and no change in food intake during a 10-day recovery period in either experiment. Reductions of food intake on the day of restraint were about the same for both restrained groups compared with their controls. When stress was applied in the evening, eating was inhibited during the first 2 h after restraint, whereas in rats restrained in the morning, feeding was suppressed twice: during the 4 h after restraint and during the first 2 h of the dark cycle. Restraint induced a significant weight loss that was greater in the rats stressed in the morning. Neuropeptide Y (NPY) levels determined at the time of food suppression for both experiments (beginning of the dark cycle) revealed an elevation of NPY in the paraventricular nucleus of rats stressed in the morning compared with other groups, but no difference in hypothalamic NPY mRNA expression. Expression of uncoupling protein mRNA in brown adipose tissue and leptin mRNA in epididymal fat, measured at the start of the dark period, was not altered by stress. There was an elevation of dopamine turnover in the hypothalami of rats restrained at the end of light cycle, but not those restrained in the morning. These results show that restraint stress has a greater effect on metabolism and energy balance when it is applied in the morning. Additional studies are needed to elucidate mechanisms involved in the suppression of food intake 9 h after restraint.

Influenza virus infection of mice induces anorexia: comparison with endotoxin and interleukin-1 and the effects of indomethacin.

The effects of infection of mice with influenza virus on ingestive behavior were assessed by both 22-h intake of food pellets, and intake of sweetened milk in a 30-minute access period. Infection with a lethal dose of virus resulted in losses in body weight as well as a reduction in food pellet intake. By contrast, infection with a sublethal dose of virus decreased body weight and food pellet intake to a lesser extent, but did not alter milk intake. Acute intraperitoneal injection of endotoxin (LPS, 0.3-5 micrograms), interleukin-1 alpha (IL-1 alpha, 50-100 ng) or IL-1 beta (100 ng) reduced milk intake, suggesting that the reduction of ingestive behavior may be associated with immune activation in general, and IL-1 in particular Pretreatment of the mice with the cyclooxygenase inhibitor, indomethacin (10 mg/kg SC) substantially attenuated, but did not completely reverse, the reduction in milk intake by LPS and IL-1. However, chronic treatment with indomethacin failed to alter the body weight or the intake of sweetened milk in influenza-infected mice, although there was some attenuation of the reduction in food intake. These results suggest that although IL-1 may play a role in the anorexia caused by influenza virus infection, it is not the only factor involved.

Sleep deprivation by the "flower pot" technique and spatial reference memory.

This study investigated whether paradoxical, or rapid eye movement (REM), sleep deprivation (SD) affected spatial memory. SD was induced in male Wistar rats by housing them on small platforms over water. They fell into the water if they lost muscle tone. Controls were either housed in tanks with large platforms (TC) or in normal cages (CC). All rats had free access to food and water. Each day they were tested in a place-learning set task using a Morris water maze. The rats were released from 6 different starting points (sets) and allowed 2 min to find a submerged platform. Two trials were conducted from each starting point. SD caused a significant decrement in performance in Trial 1 from Day 2. By Day 4, when distance swum to find the platform was plotted against set, area under the curve was doubled in SD compared to that in TC and CC rats, indicating a significant impairment in reference spatial memory. There was no consistent effect on working memory, indicated by Trial 2. SD caused weight loss and increased serum corticosterone compared to that in CC rats. There were no differences in concentrations of hypothalamic, hippocampal, or cortical catecholamines or their metabolites. Serotonin metabolism was elevated in the hypothalamus and hippocampus in SD rats. These results indicate that SD induced in rats housed on small platforms causes a substantial impairment of reference memory. The memory deficit may not be specific to SD because the rats are physically stressed and lose some nonREM sleep when housed in these conditions.

Participation of central kappa-opioid receptor in arrhythmogenesis.

The effect of kappa receptors agonists and antagonists was studied in the model of epinephrine induced arrhythmias. Kappa receptor agonists U-50,488 and [D-Ala2]-Dynorphin A (1-13) administered I.C.V. potentiate the arrhythmogenic effect of epinephrine. The effect of U-50,488 was completely blocked by kappa receptor antagonist, nor-binaltorphine. Administration of N-cholinergic receptor inhibitor, hexamethonium, prevented pro-arrhythmic effects of U-50,488 and [D-Ala2]-Dynorphin A (1-13). The data support the hypothesis that central kappa opioid receptors play an important role in the arrhythmogenesis.

CRF receptor antagonist attenuates immobilization stress-induced norepinephrine release in the prefrontal cortex in rats.

Neuroanatomical, neurophysiological, and behavioral studies suggest that brain stem nucleus locus coeruleus (LC) plays an important role in stress response. The present study was designed to clarify, whether infusion of CRF antagonist, alpha hCRF, into LC could attenuate or block stress-induced changes in norepinephrine (NE) concentrations in microdialysates collected from the medial prefrontal cortex (PFM). Rats were implanted with a bilateral cannulae assembly aimed in the LC and a microdialysis probe (4 mm active membrane length) into the LC. Immobilization of animals significantly increased the concentration of NE in microdialysates from PFM to a maximum of 170.8 +/- 12.8% of the baseline ten minutes after the onset of stressor. Concentration of NE in dialysates remained significantly elevated for the next 40 min. Infusion of alpha hCRF into the LC significantly attenuated stress-induced increase in PFM NE concentration in samples collected at 10, 20, 30, and 50 min after the onset of immobilization. Infusion of alpha hCRF alone (no immobilization) did not change concentrations at any time during sample collection. These results are consistent with other studies and suggest that stress can facilitate NE release in the PFM through the activation of the CRF system in the brain.

The role of cytokines in the behavioral responses to endotoxin and influenza virus infection in mice: effects of acute and chronic administration of the interleukin-1-receptor antagonist (IL-1ra).

Following infection with influenza virus, animals display decreased locomotor activity and feeding behavior and loss of body weight. It has been suggested that these effects may be mediated by cytokines, such as interleukin-1 (IL-1), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha), induced by the infection. To assess the potential role of IL-1, we tested the ability of a naturally occurring IL-1-receptor antagonist (IL-1ra) to antagonize the changes in feeding behavior induced by IL-1, endotoxin (lipopolysaccharide, LPS), and infection with influenza virus. Feeding behavior was assessed by measuring the daily intake of food pellets and sweetened milk in a 30-min period. Acute injection of IL-1 beta decreased milk intake, but mouse IL-6 and mouse TNF-alpha did not. However, TNF-alpha decreased food pellet intake slightly, especially when it was injected at the beginning of the dark phase. The reductions in milk intake induced by mouse IL-1 beta were largely prevented by IL-1ra pretreatment (100 micrograms/mouse i.p.). The LPS-induced reductions in milk intake were attenuated, but not blocked, by IL-1ra treatment (300 micrograms/mouse). LPS still induced significant decrements in the presence of the antagonist. In influenza virus-infected mice, IL-1ra was administered either by repeated subcutaneous (s.c.) injections, or by continuous s.c. infusion from osmotic minipumps. These IL-1ra treatments produced small, but statistically significant, attenuations of the depression in milk and food pellet intake in the virus-infected mice. In several experiments, IL-1ra treatment increased the survival of influenza virus-infected mice. Thus the attenuation of the hypophagia may have been caused by this IL-1ra-induced increase in survival. The results suggest that IL-1 contributes to sickness behavior induced by LPS and influenza virus infection, but it is not the only factor involved.

Corticotropin-releasing factor receptor antagonist infused into the locus coeruleus attenuates immobilization stress-induced defensive withdrawal in rats.

It has been proposed that corticotropin-releasing factor (CRF) released during stress in the region of the locus coeruleus (LC) induces changes in behavior that are typical indices of anxiety. The experiments tested the ability of a CRF antagonist, alpha hCRF9-41, to attenuate stress-induced defensive withdrawal in rats. 1 microgram of alpha hCRF in 300 nl was infused bilaterally in the LC of rats 10 min prior to 30 min immobilization. The apparatus consisted of a small chamber set on one side of a 1 m open field, into which the rat was placed to start the test. Restraint induced defensive withdrawal in rats familiar with the apparatus and significantly increased latency time to emerge from the chamber, total time and mean time spent in the chamber. Infusion of alpha hCRF into the LC prior to restraint significantly decreased total and mean time spent in the chamber comparing to stressed animals. These results are consistent with anatomical, electrophysiological and neurochemical evidence that CRF receptors located in, or close to, the LC region influence behaviors induced by stress.

Peripheral administration of interleukin-1 increases extracellular concentrations of norepinephrine in rat hypothalamus: comparison with plasma corticosterone.

Peripheral administration of interleukin-1 (IL-1) activates the hypothalamo-pituitary-adrenocortical (HPA) axis and also activates norepinephrine (NE) metabolism in the hypothalamus. Because there is evidence that hypothalamic NE can activate CRF secretion, it has been proposed that changes in hypothalamic noradrenergic activity are instrumental in the IL-1-induced activation of the HPA axis. We have examined this hypothesis by assessing the release of hypothalamic NE using in vivo microdialysis following intravenous (IV) or intraperitoneal (IP) injection of human IL-1 beta and comparing the responses with those in plasma corticosterone. The results indicate that extracellular concentrations of NE in the hypothalamus increased following IV and IP administration of IL-1 beta. The elevation was more rapid following IV IL-1 beta, and reached a peak at 1 h, whereas the slower and smaller increase following IP IL-1 beta did not reach a peak until 2 h. The results were similar in both anesthetized and unanesthetized rats. In unanesthetized rats, plasma corticosterone increased shortly following IV or IP IL-1 beta administration, but the peak concentration following IV IL-1 beta was significantly earlier (around 1 h) than that following IP IL-1 beta (around 2 h). The hypothalamic noradrenergic responses followed a pattern similar to that of plasma corticosterone with either route of IL-1 beta administration, but they were not identical. The results are consistent with the possibility that a central noradrenergic mechanism mediates the activation of the HPA axis by peripherally administered IL-1 beta. However, appreciable differences in the time courses of the responses may indicate the involvement of other factors.

Corticotropin-releasing factor administered into the locus coeruleus, but not the parabrachial nucleus, stimulates norepinephrine release in the prefrontal cortex.

Previous studies have indicated that intracerebroventricular application of corticotropin-releasing factor (CRF) activates noradrenergic neurons in the brain stem locus coeruleus (LC) and norepinephrine (NE) metabolism in several brain regions. To assess whether CRF has direct effects on LC noradrenergic neurons, CRF was infused into the LC and concentrations of NE and its metabolites were measured in microdialysates collected from the medial prefrontal cortex (PFM). Infusion of 100 ng of CRF into the LC significantly increased dialysate concentrations of NE and of its catabolite MHPG in the ipsilateral PFM, whereas no significant changes were observed following infusion of artificial CSF. No response was observed when the infusions of CRF occurred outside of the LC, including those in the parabrachial nucleus. Although CRF administered into the LC slightly increased dialysate concentrations of NE in the contralateral PFM, this effect was not statistically significant. The effect of CRF injected into the LC on dialysate NE was prevented by combination with a 10-fold excess of the CRF antagonist alpha-helical CRF9-41, indicating some specificity in the response. These results are consistent with anatomical and electrophysiological evidence suggesting that CRF may directly activate noradrenergic neurons in or close to the LC.