Angelo Contarino

Corticotropin Releasing Factor Receptor 1–Deficient Mice Display Decreased Anxiety, Impaired Stress Response, and Aberrant Neuroendocrine Development

Corticotropin releasing factor (CRF) is a major integrator of adaptive responses to stress. Two biochemically and pharmacologically distinct CRF receptor subtypes (CRFR1 and CRFR2) have been described. We have generated mice null for the CRFR1 gene to elucidate the specific developmental and physiological roles of CRF receptor mediated pathways. Behavioral analyses revealed that mice lacking CRFR1 displayed markedly reduced anxiety. Mutant mice also failed to exhibit the characteristic hormonal response to stress due to a disruption of the hypothalamic-pituitary-adrenal (HPA) axis. Homozygous mutant mice derived from crossing heterozygotes displayed low plasma corticosterone concentrations resulting from a marked agenesis of the zona fasciculata region of the adrenal gland. The offspring from homozygote crosses died within 48 hr after birth due to a pronounced lung dysplasia. The adrenal agenesis in mutant animals was attributed to insufficient adrenocorticotropic hormone (ACTH) production during the neonatal period and was rescued by ACTH replacement. These results suggest that CRFR1 plays an important role both in the development of a functional HPA axis and in mediating behavioral changes associated with anxiety.

Mice deficient for corticotropin-releasing hormone receptor-2 display anxiety-like behaviour and are hypersensitive to stress

Corticotropin-releasing hormone (Crh) is a critical coordinator of the hypothalamic-pituitary-adrenal (HPA) axis. In response to stress, Crh released from the paraventricular nucleus (PVN) of the hypothalamus activates Crh receptors on anterior pituitary corticotropes, resulting in release of adrenocorticotropic hormone (Acth) into the bloodstream. Acth in turn activates Acth receptors in the adrenal cortex to increase synthesis and release of glucocorticoids. The receptors for Crh, Crhr1 and Crhr2, are found throughout the central nervous system and periphery. Crh has a higher affinity for Crhr1 than for Crhr2, and urocortin (Ucn), a Crh-related peptide, is thought to be the endogenous ligand for Crhr2 because it binds with almost 40-fold higher affinity than does Crh (ref. 2). Crhr1 and Crhr2 share approximately 71% amino acid sequence similarity and are distinct in their localization within the brain and peripheral tissues. We generated mice deficient for Crhr2 to determine the physiological role of this receptor. Crhr2-mutant mice are hypersensitive to stress and display increased anxiety-like behaviour. Mutant mice have normal basal feeding and weight gain, but decreased food intake following food deprivation. Intravenous Ucn produces no effect on mean arterial pressure in the mutant mice.

Mice Deficient for Both Corticotropin-Releasing Factor Receptor 1 (CRFR1) and CRFR2 Have an Impaired Stress Response and Display Sexually Dichotomous Anxiety-Like Behavior

Corticotropin-releasing factor (CRF) and its family of peptides are critical coordinators of homeostasis whose actions are mediated through their receptors, CRF receptor 1 (CRFR1) and CRFR2, found throughout the CNS and periphery. The phenotypes of mice deficient in either CRFR1 or CRFR2 demonstrate the critical role these receptors play. CRFR1-mutant mice have an impaired stress response and display decreased anxiety-like behavior, whereas CRFR2-mutant mice are hypersensitive to stress and display increased anxiety-like behavior. To further elucidate the roles of both CRF receptors and determine their interaction in behaviors, we have generated mice deficient in both CRFR1 and CRFR2. The behavioral phenotype of these mice demonstrates a novel role of the mothers genotype on development of pup anxiety. We have found that although the female double-mutant mice display anxiolytic-like behavior, the male double-mutant mice show significantly more anxiety-like behavior compared with the females. We have also determined that the dams CRFR2 genotype affects the anxiety-like behavior of the male mice, such that a pup born to a heterozygous or mutant dam displays significantly more anxiety-like behavior regardless of that pups genotype. Double-mutant mice also display an even greater impairment of their hypothalamic–pituitary–adrenal axis response to stress than that of the CRFR1-mutant mice. CRF mRNA levels are elevated in CRFR1- and double-mutant mice, and urocortin III and vasopressin mRNA levels are increased in CRFR2- and double-mutant mice. These results indicate that both CRFR1 and CRFR2 have critical roles in gene regulation and the maintenance of homeostasis in response to stress.

Reduced anxiety-like and cognitive performance in mice lacking the corticotropin-releasing factor receptor 1.

Corticotropin-releasing factor (CRF) has been hypothesized to be involved in the pathophysiology of anxiety, depression, cognitive and feeding disorders. Two distinct CRF receptor subtypes, CRFR1 and CRFR2, are thought to mediate CRF actions in the CNS. However, the role for each receptor subtype in animal models of neuropsychiatric disorders remains to be determined. Using CRFR1 deficient mice, the present study investigated the functional significance of this CRF receptor subtype in anxiety-like and memory processes. CRFR1 knockout mice displayed an increased exploratory behavior in both the Elevated Plus-maze (EPM) and the Black and White (B-W) test box models of anxiety, indicating an anxiolytic-like effect of the CRFR1 gene deletion. In contrast, during the retrieval trial of a two-trial spatial memory task wild type mice made more visits to and spent more time in the novel arm as opposed to the two familiar ones of a Y-maze apparatus. No increase in the level of exploration of the novel arm by the CRFR1 deficient mice was observed. This indicates that CRFR1 knockout mice are impaired in spatial recognition memory. These results demonstrate that genetic deletion of the CRFR1 receptor can lead to impairments in anxiety-like and cognitive behaviors, supporting a critical role for this receptor in anxiety and cognitive biological processes.

Urocortin-deficient mice show hearing impairment and increased anxiety-like behavior

Urocortin is a member of the corticotropin-releasing hormone peptide family and is found in many discrete brain regions. The distinct expression pattern of urocortin suggests that it influences such behaviors as feeding, anxiety and auditory processing. To better define the physiological roles of urocortin, we have generated mice carrying a null mutation of the urocortin gene. Urocortin-deficient mice have normal basal feeding behavior and stress responses, but show heightened anxiety-like behaviors in the elevated plus maze and open-field tests. In addition, hearing is impaired in the mutant mice at the level of the inner ear, suggesting that urocortin is involved in the normal development of cochlear sensory-cell function. These results provide the first example of a function for any peptidergic system in hearing.

Dissociation of Locomotor Activation and Suppression of Food Intake Induced by CRF in CRFR1-Deficient Mice

ABSTRACT Corticotropin-releasing factor (CRF) systems are involved in locomotor and feeding behaviors. Two distinct CRF receptor subtypes, CRFR1 and CRFR2, are thought to mediate CRF actions in the central nervous system. However, the role for each receptor in locomotor activity and feeding remains to be determined. Using CRFR1 null mutant mice, the present study examined the functional significance of this receptor in ambulation and feeding. CRF treatment of wild-type mice resulted in increased levels of locomotion whereas no change was observed in CRFR1-deficient mice as compared to vehicle-treated mutant mice. In contrast, CRF decreased food-water intake in both wild type and CRFR1-deficient mice equally. These results support an important role for CRFR1 in mediating CRF-induced locomotor activation, whereas other receptor subtypes, likely CRFR2, may mediate the appetite-suppressing effects of CRF-like peptides.

Lack of reward and locomotor stimulation induced by heroin in μ-opioid receptor-deficient mice

The micro-opioid receptor is the main substrate mediating opiate reward. Multiple micro-opioid receptor subtypes have been postulated to underlie opiate actions. Animals treated with antisense oligonucleotides targeting specific micro-opioid receptor exons show differential sensitivity to morphine versus heroin. The present work examined the rewarding and locomotor activating effects of heroin in mutant mice with a disrupted exon 2 of the micro-opioid receptor. Heroin (1-3 mg/kg) produced significant place preferences and stimulated locomotor activity in wild-type mice, whereas it had no effect in micro-opioid receptor-deficient mice. In contrast, treatment with cocaine (10-30 mg/kg) produced comparable place preferences and locomotor activation in both wild-type and micro-opioid receptor-deficient mice, thus providing evidence that the mutant mice are able to show drug-induced effects in the two behavioral paradigms used here. These results support an essential role for the micro-opioid receptor in the rewarding and locomotor activating effects of heroin.

Role of the corticotropin‐releasing factor receptor type 2 in the control of food intake in mice: a meal pattern analysis

The actions of corticotropin‐releasing factor (CRF) and related peptides are mediated by two receptors (CRF1 and CRF2). The respective role of each subtype in the control of food intake remains poorly known. In the present study, we examined the quantity and microstructure of ingestive behavior of knockout (KO) mice lacking CRF2 receptors and their wild‐type (WT) littermates. Under basal conditions, CRF2 KO mice showed increased nocturnal food intake, evident as an increased zenith in circadian cosinor analysis of food intake. Microstructure analysis revealed that this greater food intake reflected increased meal size, rather than meal frequency, suggesting a decreased satiating value of food. Following acute restraint stress, CRF2 KO mice showed an intact immediate anorectic response with increased latency to eat and decreased meal size. However, CRF2 deletion abolished the prolonged phase of restraint‐induced anorexia. CRF2 KO mice did not differ from WT controls in feeding responses to food deprivation or injection of ghrelin receptor agonists. Independent of genotype, food deprivation increased food intake, with dramatic changes in meal size, meal frequency, water : food ratio and eating rate. Acyl‐ghrelin or BIM‐28131, a potent ghrelin analog, dose‐dependently stimulated food intake by increasing meal size (ghrelin, BIM‐28131) and meal number (BIM‐28131), while slowing the average eating rate (BIM‐28131) similarly in WT and KO mice. These results suggest that the CRF2 receptor is involved in the control of meal size during the active phase of eating and following acute exposure to stress.

The expression of neuropeptide-induced excessive grooming behavior in dopamine D1 and D2 receptor-deficient mice

Grooming behavior in rodents has long been related to dopamine receptors in the brain. However, the relative contribution of dopamine D1-like receptors (D1 and D5) and D2-like receptors (D2, D3 and D4) in this behavior has not been established yet. Spontaneous novelty-induced grooming (as assessed with a 30-min sampling test) was reduced in knockout mice lacking the dopamine D1, receptor. Furthermore, the intracerebroventricular (i.c.v.) injection of small quantities of oxytocin, prolactin or the adrenocorticotrophic hormone 1-24 fragment, ACTH-(1-24) was followed by a diminished level of novelty-induced excessive grooming. These neuropeptides caused a sustained increase in grooming level of control animals (wild type). Interestingly, the i.c.v. injection of beta-endorphin enhanced novelty-induced grooming to a level similar in control and knockout mice. The systemic administration of the dopamine D2 receptor antagonist, sulpiride did not suppress the residual grooming activity shown by animals injected with oxytocin, prolactin or ACTH-(1-24), and did not change the behavioral expression of those injected with beta-endorphin. In contrast, the systemic administration of the opioid receptor antagonist, naloxone, totally suppressed the residual grooming activity of oxytocin-, prolactin- or ACTH-(1-24)-injected mice and of those treated with beta-endorphin. In contrast with the behavioral deficit observed in dopamine D1 receptor-deficient mice, dopamine D2 receptor-null animals showed a normal expression of spontaneous novelty-induced grooming and a high level of grooming activity induced by i.c.v. injection of oxytocin, prolactin, ACTH-(1-24) or beta-endorphin. Again, the peripheral injection of naloxone was followed by a suppression of neuropeptide-induced excessive grooming in these animals. These data suggest that dopamine D1 receptors are involved in the expression of novelty-induced grooming in mice. In contrast, dopamine D2 receptors seem not to be important for the expression of this behavior. Furthermore, neuropeptide-enhanced grooming involves dopamine D1, but not dopamine D2 receptors. However, neurotransmitters other than dopamine (e.g., endorphins) may play a supplementary role in neuropeptide-enhanced grooming in mice.

Conditioned place preference: no tolerance to the rewarding properties of morphine

Abstract The effect of repeated morphine administration on conditioned place preference (CPP) using a novel treatment schedule, i.e., drug treatment was always contingent with the conditioned environmental stimuli, was investigated. We also examined whether changes in the μ- and κ-opioid receptor binding occurred in the brain of morphine-treated animals. Intraperitoneal (i.p.) administration of morphine (2 and 10 mg/kg) induced a place preference after 8 daily conditioning trials (4 morphine injections on alternate trials), the level of preference being the same with the two doses of the opiate. No change in place preference was observed in the morphine-treated rats at 2 mg/kg, when animals were further trained up to a total of 32 conditioning trials (16 morphine injections). Conversely, after 20 conditioning trials (10 morphine injections), a stronger CPP response developed in the morphine-treated rats at 10 mg/kg. Signs of morphine withdrawal were never detected in morphine-treated rats during the experiment. Loss of body weight (index of opiate dependence) was not observed either 24 h or 48 h after the last morphine administration. μ- and κ-opioid receptor density and affinity were not affected by repeated morphine administrations at either dose. The results demonstrate that no tolerance develops to the rewarding properties of morphine. Indeed, a sensitisation effect may occur at increasing doses of the opiate. Furthermore, changes in the rewarding effect of morphine are not dependent upon alterations in opioid receptors involved in the reinforcing mechanisms.