The role of corticotropin-releasing factor and vasopressin in hypoglycemia-induced proopiomelanocortin gene expression in the rat anterior pituitary gland.

In this study, we examined the effect of passive immunization of endogenous corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) on hypoglycemia-induced adrenocorticotropic hormone (ACTH) secretion and determined proopiomelanocortin messenger RNA (POMC mRNA) levels in the anterior pituitary as well as hypothalamic CRF mRNA levels in pentobarbital anesthetized rats. The response of plasma ACTH to hypoglycemia was partially inhibited by the administration of CRF-antiserum (CRF-As) or AVP-antiserum (AVP-As) alone, but was found to be completely abolished by the administration of CRF-As + AVP-As as compared to the response in normal rabbit serum-treated rats. The hypoglycemia-induced POMC mRNA level in the anterior pituitary was completely inhibited by the administration of CRF-As alone and CRF-As + AVP-As, but was not inhibited by AVP-As alone as compared to the response in normal rabbit serum-treated rats. The administration of CRF-As and/or AVP-As did not affect hypoglycemia-induced CRF mRNA levels in the hypothalamus. These results indicate that the synergistic effect of CRF and AVP is important for hypoglycemia-induced ACTH secretion, but CRF is essential and indispensable for hypoglycemia-induced POMC gene expression in the anterior pituitary (AP).     

Reduced expression of the ATRX gene,a chromatin‐remodeling factor,causes hippocampal dysfunction in mice

Mutations of the ATRX gene, which encodes an ATP‐dependent chromatin‐remodeling factor, were identified in patients with α‐thalassemia X‐linked mental retardation (ATR‐X) syndrome. There is a milder variant of ATR‐X syndrome caused by mutations in the Exon 2 of the gene. To examine the impact of the Exon 2 mutation on neuronal development, we generated ATRX mutant (ATRX^ΔE2) mice. Truncated ATRX protein was produced from the ATRX^ΔE2 mutant allele with reduced expression level. The ATRX^ΔE2 mice survived and reproduced normally. There was no significant difference in Morris water maze test between wild‐type and ATRX^ΔE2 mice. In a contextual fear conditioning test, however, total freezing time was decreased in ATRX^ΔE2 mice compared to wild‐type mice, suggesting that ATRX^ΔE2 mice have impaired contextual fear memory. ATRX^ΔE2 mice showed significantly reduced long‐term potentiation in the hippocampal CA1 region evoked by high‐frequency stimulation. Moreover, autophosphorylation of calcium‐calmodulin‐dependent kinase II (αCaMKII) and phosphorylation of glutamate receptor, ionotropic, AMPA 1 (GluR1) were decreased in the hippocampi of the ATRX^ΔE2 mice compared to wild‐type mice. These findings suggest that ATRX^ΔE2 mice may have fear‐associated learning impairment with the dysfunction of αCaMKII and GluR1. The ATRX^ΔE2 mice would be useful tools to investigate the role of the chromatin‐remodeling factor in the pathogenesis of abnormal behaviors and learning impairment. © 2010 Wiley‐Liss, Inc.     

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.     

Reduced attention in mice overproducing corticotropin-releasing hormone

Data from several studies suggest that unrestrained secretion of corticotropin-releasing hormone in the CNS produces several signs and symptoms of depression. Recent evidence indicates that blockade of the CRH receptor 1 reduced depression scores in depressed patients. One of the symptoms that occur is depression is impairment in attentional processes. Whether these impairments are due to alterations in the CRH system are so far unknown. In order to investigate whether overproduction of CRH alters attentional process, transgenic mice overproducing CRH were tested on an operant five choice serial reaction time task, a task which taxes sustained and divided attention. Mutants showed impaired autoshaping. During initial discrimination learning, transgenics performed below wildtype level, but with extended training with long stimulus durations, transgenic animals reached similar accuracy levels as wildtype mice. When animals were tested at shortest stimulus duration (0.5s), a mild but significant impairment in accurate responding emerged in transgenics. This was accompanied by longer correct response latencies, while incorrect latencies did not differ between groups, suggesting attentional impairment in CRH transgenics. Because these animals have been reported to also show increased anxiety-related behaviour, animals were treated with the anxiolytic benzodiazepine diazepam. Diazepam failed to affect accuracy, but transgenic mice showed a stronger behavioural disinhibition. This suggests that the attentional impairment seen in CRH overexpressors is independent of alterations in anxiety-like behaviour. These findings may have implications for understanding the pathophysiology of psychiatric disorders such as depression, where it has been suggested that an overactivity of the CRH system accounts for a variety of symptoms, including hyper-arousal and attentional impairment.     

Thyroxine modulates corticotropin-releasing factor but not arginine vasopressin gene expression in the hypothalamic paraventricular nucleus of the developing Rat

Neonatal rats were daily injected with 100 microg/kg T4 and killed at 4, 8 or 15 days. Circulating corticosterone and corticosteroid binding globulin concentrations increased in 8- and 15-day-old rats after T4 treatment. Plasma adrenocorticotropic hormone (ACTH) concentrations, pituitary ACTH content and pro-opiomelanocortin mRNA expression were unaffected in T4-treated rats. T4 treatment induced an increase in corticotropin-releasing factor (CRF) mRNA expression in the whole population of CRF synthesizing cells of the paraventricular nucleus (PVN) that became significant at day 8 and disappeared at day 15. Double labelling in situ hybridization revealed that CRF gene expression in the CRF+/arginine vasopressin (AVP)+ subpopulation was increased at days 4 and 8 and decreased at day 15. CRF immunoreactivity in the zona externa of the median eminence increased with age but was not affected by the experimental hyperthyroidism. The degree of CRF and AVP colocalization, the concentration of AVP mRNA in the parvo and magnocellular cell bodies of the PVN and the density of immunoreactive AVP in the zona interna or zona externa of the median eminence did not change after T4 treatment. Our data demonstrate that experimental hyperthyroidism accelerates the maturation of hypothalamic CRF gene expression, including in particular in the CRF+/AVP+ subpopulation, during the stress hyporesponsive period. These observations suggest that the physiological peak of plasma thyroxine that occurs between days 8-12 may participate in the maturation of hypothalamic CRF cells.     

A role for corticotropin-releasing factor in the long-term expression of behavioral sensitization to cocaine

Corticotropin-releasing factor (CRF) has been implicated in a number of the behavioral and biochemical effects of cocaine. We recently reported that central injections of CRF produce a potentiated locomotor response in animals that had been given repeated injections of cocaine up to 4 weeks earlier. We now report that with as few as 1 or 3 exposures to cocaine (total of 45 mg/kg, i.p., per day), and a drug-free period of 28 days, i.c.v. injections of CRF (0.5 microg) produce augmented locomotor responses, similar to those induced by cocaine (10 mg/kg, i.p.) itself. In addition, in animals pre-exposed to cocaine for 3 days, pre-treatment with the CRF receptor antagonist, D-Phe CRF(12-41) (1 microg, i.c.v.), blocks the expression of behavioral sensitization to a cocaine challenge after a 28-day drug-free period. These results demonstrate that short-term exposure to cocaine produces a form of long-term sensitization within systems upon which CRF acts and that activation of CRF receptors is importantly involved in the expression of behavioral sensitization to cocaine.     

Interactions of corticotropin-releasing factor with antidepressant and anxiolytic drugs: behavioral studies with pigeons

A number of studies have shown significant interactions between neuronal systems involved with corticotropin-releasing factor (CRF) and either the clinical manifestations of depression and anxiety or the effects of antidepressant or anxiolytic drugs. In the present study, effects of CRF were studied alone and in combination with imipramine and with the sedative-hypnotic/anxiolytic drugs pentobarbital and chlordiazepoxide. Interactions of CRF with the novel, atypical anxiolytic buspirone were also examined. Interactions were evaluated through the use of schedule-controlled responding, responding suppressed by punishment, and drug discrimination procedures using the conditioned key-pecking response of pigeons. Effects of CRF were significantly enhanced when given in combination with imipramine with low noneffective imipramine doses potentiating the rate-reducing effects of CRF. Similarly, in pigeons trained to discriminate imipramine from saline, noneffective doses of CRF shifted the imipramine dose-response curve more than twofold to the left. Low doses of imipramine that produced saline key responding, produced imipramine-key responding when coadministered with CRF. The CRF antagonist alpha-helical CRF9-41 did not alter the rate-decreasing effects of imipramine. Effects of CRF on schedule-controlled responding were, however, antagonized by the administration of chlordiazepoxide and pentobarbital but not by buspirone, suggesting that CRF interacts with the GABA/benzodiazepine receptor mechanism complex but not with those systems involved in mediating the effects of buspirone. These results suggest that CRF interacts in significant ways with specific neurotransmitter systems subserving depression and anxiety.     

Reduced ultrasonic vocalizations in vasopressin 1b knockout mice

The neuropeptides oxytocin and vasopressin have been implicated in rodent social and affiliative behaviors, including social bonding, parental care, social recognition, social memory, vocalizations, territoriality, and aggression, as well as components of human social behaviors and the etiology of autism. Previous investigations of mice with various manipulations of the oxytocin and vasopressin systems reported unusual levels of ultrasonic vocalizations in social settings. We employed a vasopressin 1b receptor (Avpr1b) knockout mouse to evaluate the role of the vasopressin 1b receptor subtype in the emission of ultrasonic vocalizations in adult and infant mice. Avpr1b null mutant female mice emitted fewer ultrasonic vocalizations, and their vocalizations were generally at lower frequencies, during a resident-intruder test. Avpr1b null mutant pups emitted ultrasonic vocalizations similar to heterozygote and wildtype littermates when separated from the nest on postnatal days 3, 6, 9, and 12. However, maternal potentiation of ultrasonic vocalizations in Avpr1b null and heterozygote mutants was absent, when tested at postnatal day 9. These results indicate that Avpr1b null mutant mice are impaired in the modulation of ultrasonic vocalizations within different social contexts at infant and adult ages.     

Type 1 corticotropin-releasing factor receptor expression reported in BAC transgenic mice: implications for reconciling ligand-receptor mismatch in the central corticotropin-releasing factor system

In addition to its established role in initiating the endocrine arm of the stress response, corticotropin-releasing factor (CRF) can act in the brain to modulate neural pathways that effect coordinated physiological and behavioral adjustments to stress. Although CRF is expressed in a set of interconnected limbic and autonomic cell groups implicated as primary sites of stress-related peptide action, most of these are lacking or impoverished in CRF receptor (CRFR) expression. Understanding the distribution of functional receptor expression has been hindered by the low resolution of ligand binding approaches and the lack of specific antisera, which have supported immunolocalizations at odds with analyses at the mRNA level. We have generated a transgenic mouse that shows expression of the principal, or type 1, CRFR (CRFR1). This mouse expresses GFP in a cellular distribution that largely mimics that of CRFR1 mRNA and is extensively colocalized with it in individual neurons. GFP-labeled cells display indices of activation (Fos induction) in response to central CRF injection. At the cellular level, GFP labeling marks somatic and proximal dendritic morphology with high resolution and is also localized to axonal projections of at least some labeled cell groups. This includes a presence in synaptic inputs to central autonomic structures such as the central amygdalar nucleus, which is implicated as a stress-related site of CRF action, but lacks cellular CRFR1 expression. These findings validate a new tool for pursuing the role of central CRFR signaling in stress adaptation and suggest means by which the pervasive ligand-receptor mismatch in this system may be reconciled.     

Reduced startle reactivity and plasticity in transgenic mice overexpressing corticotropin-releasing hormone.

BACKGROUND: Corticotropin-releasing hormone (CRH) hyperactivity in transgenic mice overexpressing CRH in the brain (CRH-OE(2122)) appears to be associated with chronic stress-like alterations, including increased CRH content in the hypothalamus, changes in hypothalamus-pituitary-adrenal axis regulation, and increased heart rate and body temperature. In the present study, we investigated if sensory information processing of startling auditory stimuli was affected in CRH-OE(2122) mice. METHODS: CRH-OE(2122) mice (on C57BL/6J background) were subjected to a number of procedures probing sensory information processing mechanisms, including the acoustic startle response, habituation, and prepulse inhibition of startle. RESULTS: CRH-OE(2122) mice displayed reduced acoustic startle reactivity and increased motor activity during startle testing compared to wild-type mice. Furthermore, transgenic mice did not show habituation of the startle response after repeated exposure to the auditory stimulus, or habituation across procedures. CRH-OE(2122) mice exhibited robust impairments of prepulse inhibition in two different paradigms. CONCLUSIONS: The results in CRH-OE(2122) mice indicate that chronic CRH hyperactivity is associated with reductions in startle reactivity, habituation, and prepulse inhibition. The latter two abnormalities are also observed in schizophrenia patients. We conclude that chronic CRH excess may reduce behavioral reactivity to environmental stimuli and impair information processing mechanisms.     

Reduced programmed cell death in brains of serotonin transporter knockout mice

Serotonin (5-HT) is known to reduce apoptosis and in rodent models of brain ischemia. Modulation of programmed cell death during neural development was assessed in early postnatal brains of serotonin transporter (5-HTT) knockout mice, characterized by elevated extracellular 5-HT levels. The number of apoptotic cells visualized at postnatal day-1 (P1) by ISEL+ or TUNEL staining was significantly reduced in the striatum, thalamus/hypothalamus, cerebral cortex and hippocampus of 5-HTT knockout mice, compared to wild type and heterozygote mice, with differences displaying an increasing fronto-caudal gradient and regional specificity. These findings underscore 5-HT roles in the regulation of programmed cell death during brain development, and spur interest into pharmacological interventions aimed at relieving pathological apoptosis by potentiating serotoninergic neurotransmission.     

Intermale aggression in corticotropin-releasing factor receptor 1 deficient mice

The anxiogenic neuropeptide, corticotropin-releasing factor (CRF), has a complex effect on intermale aggression. CRF receptor 1 (CRFR1) is the primary receptor for CRF and in this study, we examined in detail isolation-induced intermale aggression in CRFR1 deficient mice. All mice contained a mixed 50:50 inbred/outbred background to improve aggressive performance. Mice were isolated for 4 weeks prior to 2 consecutive days of aggression testing using the resident-intruder paradigm. Mice were also tested for anxiety on the elevated plus maze. Relative to littermate wild-type (WT) controls, CRFR1-mutant mice exhibited normal levels of intermale aggression over the 2 test days in terms of percentage showing aggression, number of attacks, time aggressive, and latency to first attack. In terms of sites of attacks on intruders, CRFR1-deficient mice attacked the ventral portion of the mid-section (including belly) significantly less frequently than WT males on test day 1, but these differences did not reach significance on test day 2. No other differences in sites of attacks were observed. Tail rattling also did not differ between groups. Importantly, KO males showed decreased anxiety relative to WT mice (consistent with previous reports) as evidenced by spending significantly more time on the open arms and significantly less time on the closed arms of the elevated plus maze. Plus maze performance did not correlate with any measure of levels of aggression, suggesting a dissociation between altered levels of anxiety and aggressive performance. Taken together, the results suggest that the activation CRFR1 is not necessary for the normal production of isolation-induced intermale aggression.     

Role of arginine vasopressin and corticotropin-releasing factor in mediating alcohol-induced adrenocorticotropin and vasopressin secretion in male rats bearing lesions of the paraventricular nuclei

In male rats, lesions of the paraventricular nucleus (PVN) of the hypothalamus attenuate, but do not abolish, adrenocorticotropin (ACTH) secretion in response to acute alcohol injection. As the PVN is the major source of corticotropin-releasing factor (CRF) in the median eminence, this observation suggests that extra-PVN brain regions, and/or ACTH secretagogues other than CRF (e.g. arginine vasopressin (AVP)), mediate ACTH stimulation by alcohol. This hypothesis was tested by examining the effect of AVP immunoneutralization in PVN-lesioned (PVNx) rats. Removal of endogenous AVP diminished alcohol-evoked ACTH secretion in both sham-operated and PVNx animals, indicating that AVP from outside the PVN partially mediates the hypothalamic-pituitary-adrenal (HPA) axis response to alcohol. This led us to determine whether alcohol might also regulate AVP steady-state gene expression in the supraoptic nucleus (SON) and PVN, and/or CRF mRNA in the PVN and the central nucleus of the amygdala (AMY). In the magnocellular portion of the PVN, sham-operated animals showed significantly increased PVN levels of both CRF and AVP mRNAs 3 h after alcohol. In the SON, alcohol administration tended to decrease AVP gene expression in PVNx rats, while the drug increased AVP mRNA levels in the SON of sham-operated rats. AMY levels of CRF mRNA were unaffected by these manipulations. Finally, since the regulation of alcohol-induced AVP mRNA levels in the SON appeared to depend on the presence of the PVN, we measured peripheral levels of AVP in both sham-operated and PVNx animals after injection of vehicle or alcohol. Although AVP decreased in all groups, alcohol depressed AVP secretion to a greater extent in PVNx animals, suggesting that AVP systems are more sensitive to inhibition in the absence of the PVN. Our results demonstrate that although AVP of PVN origin may participate in regulating the stimulatory effect to AVP on ACTH secretion, AVP from areas other than the PVN also plays a role. Additionally, regulation of both AVP gene expression in the SON and secretion in the systemic circulation are altered in rats bearing lesions of the PVN.     

Relationships among estrogen receptor, oxytocin and vasopressin gene expression and social interaction in male mice

The incidence of social disorders such as autism and schizophrenia is significantly higher in males, and the presentation more severe, than in females. This suggests the possible contribution of sex hormones to the development of these psychiatric disorders. There is also evidence that these disorders are highly heritable. To contribute toward our understanding of the mechanisms underlying social behaviors, particularly social interaction, we assessed the relationship of social interaction with gene expression for two neuropeptides, oxytocin (OT) and arginine vasopressin (AVP), using adult male mice. Social interaction was positively correlated with: oxytocin receptor (OTR) and vasopressin receptor (V1aR) mRNA expression in the medial amygdala; and OT and AVP mRNA expression in the paraventricular nucleus of the hypothalamus (PVN). When mice representing extremes of social interaction were compared, all of these mRNAs were more highly expressed in high social interaction mice than in low social interaction mice. OTR and V1aR mRNAs were highly correlated with estrogen receptor α (ERα) mRNA in the medial amygdala, and OT and AVP mRNAs with estrogen receptor β (ERβ) mRNA in the PVN, indicating that OT and AVP systems are tightly regulated by estrogen receptors. A significant difference in the level of ERα mRNA in the medial amygdala between high and low social interaction mice was also observed. These results support the hypothesis that variations of estrogen receptor levels are associated with differences in social interaction through the OT and AVP systems, by upregulating gene expression for those peptides and their receptors.     

Serotonergic stimulation of corticotropin-releasing hormone and pro-opiomelanocortin gene expression

The neurotransmitter serotonin (5-HT) stimulates adrenocorticotropic hormone (ACTH) secretion from the anterior pituitary gland via activation of central 5-HT1 and 5-HT2 receptors. The effect of 5-HT is predominantly indirect and may be mediated via release of hypothalamic corticotropin-releasing hormone (CRH). We therefore investigated the possible involvement of CRH in the serotonergic stimulation of ACTH secretion in male rats. Increased neuronal 5-HT content induced by systemic administration of the precursor 5-hydroxytryptophan (5-HTP) in combination with the 5-HT reuptake inhibitor fluoxetine raised CRH mRNA expression in the paraventricular nucleus (PVN) by 64%, increased pro-opiomelanocortin (POMC) mRNA in the anterior pituitary lobe by 17% and stimulated ACTH secretion five-fold. Central administration of 5-HT agonists specific to 5-HT1A, 5-HT1B, 5-HT2A or 5-HT2C receptors increased CRH mRNA in the PVN by 15-50%, POMC mRNA in the anterior pituitary by 15-27% and ACTH secretion three- to five-fold, whereas a specific 5-HT3 agonist had no effect. Systemic administration of a specific anti-CRH antiserum inhibited the ACTH response to 5-HTP and fluoxetine and prevented the 5-HTP and fluoxetine-induced POMC mRNA response in the anterior pituitary lobe. Central or systemic infusion of 5-HT increased ACTH secretion seven- and eight-fold, respectively. Systemic pretreatment with the anti-CRH antiserum reduced the ACTH responses to 5-HT by 80% and 64%, respectively. It is concluded that 5-HT via activation of 5-HT1A, 5-HT2A, 5-HT2C and possibly also 5-HT1B receptors increases the synthesis of CRH in the PVN and POMC in the anterior pituitary lobe, which results in increased ACTH secretion. Furthermore, the results indicate that CRH is an important mediator of the ACTH response to 5-HT.     

Cardiac dynamics in corticotropin-releasing factor receptor subtype-2 deficient mice

The action of corticotropin-releasing factor (CRF) is mediated by two recently identified receptors, CRFR1 and CRFR2, that differ with respect to their anatomical distribution and pharmacologic ligand-binding properties. Here we show by an analysis of circadian heartbeat interval fluctuations that CRFR2-deficiency in mice does not interfere with the dynamical mechanisms underlying the control of heart rate. Hence, intact CRFR2 would not constitute an indispensable requirement of physiologic cardiac rhythm regulation. However, both CRFR2 knockout (-/-) and wildtype control (+/+) mice showed altered dynamical properties of cardiac interbeat fluctuations in contrast to homogenetic inbred strains of mice (C57BL/6N and C57BL/6J). The results stress the impact of genetic background and support the generalized notion that transgenic 129/Sv-derived knockout mice exhibit altered cardiac dynamics which is interpreted to reflect an attenuation of neuroautonomic sympatho-vagal antagonism.