Control of voltage-gated potassium channel Kv2.2 expression by pyruvate-isocitrate cycling regulates glucose-stimulated insulin secretion.

Recent studies have shown that the pyruvate-isocitrate cycling pathway, involving the mitochondrial citrate/isocitrate carrier and the cytosolic NADP-dependent isocitrate dehydrogenase (ICDc), is involved in control of glucose-stimulated insulin secretion (GSIS). Here we demonstrate that pyruvate-isocitrate cycling regulates expression of the voltage-gated potassium channel family member Kv2.2 in islet ß-cells. siRNA-mediated suppression of ICDc, citrate/isocitrate carrier, or Kv2.2 expression impaired GSIS, and the effect of ICDc knockdown was rescued by re-expression of Kv2.2. Moreover, chronic exposure of ß-cells to elevated fatty acids, which impairs GSIS, resulted in decreased expression of Kv2.2. Surprisingly, knockdown of ICDc or Kv2.2 increased rather than decreased outward K(+) current in the 832/13 ß-cell line. Immunoprecipitation studies demonstrated interaction of Kv2.1 and Kv2.2, and co-overexpression of the two channels reduced outward K(+) current compared with overexpression of Kv2.1 alone. Also, siRNA-mediated knockdown of ICDc enhanced the suppressive effect of the Kv2.1-selective inhibitor stromatoxin1 on K(+) currents. Our data support a model in which a key function of the pyruvate-isocitrate cycle is to maintain levels of Kv2.2 expression sufficient to allow it to serve as a negative regulator of Kv channel activity.

Corticotropin-releasing factor-binding protein (CRF-BP) inhibits CRF- and urotensin-I-mediated activation of CRF receptor-1 and -2 in common carp.

Corticotropin-releasing factor-binding protein (CRF-BP) is considered a key determinant for CRF receptor (CRF-R) activation by CRF and several related peptides. Earlier studies have shown that the CRF system is highly conserved in gene structures throughout evolution, yet little is known about the evolutionary conservation of its biological functions. Therefore, we address the functional properties of CRF-BP and CRF-Rs in a teleost fish (common carp; Cyprinus carpio L.). We report the finding of two similar, yet distinct, genes for both CRF-R1 and CRF-R2 in this species. The four receptors are differentially responsive to CRF, urotensin-I (UI), sauvagine, and urocortin-2 (Ucn-2) and -3 (Ucn-3) as shown by luciferase assays. In vitro, carp CRF-BP inhibits CRF- and UI-mediated activation of the newfound CRF-Rs, but its potency to do so varies between receptor and peptide ligand. This is the first paper to establish the functionality and physiological interplay between CRF-BP, CRF-Rs and CRF-family peptides in a teleostean species.

A triple urocortin knockout mouse model reveals an essential role for urocortins in stress recovery.

Responding to stressful events requires numerous adaptive actions involving integrated changes in the central nervous and neuroendocrine systems. Numerous studies have implicated dysregulation of stress-response mechanisms in the etiology of stress-induced psychopathophysiologies. The urocortin neuropeptides are members of the corticotropin-releasing factor family and are associated with the central stress response. In the current study, a triple-knockout (tKO) mouse model lacking all three urocortin genes was generated. Intriguingly, these urocortin tKO mice exhibit increased anxiety-like behaviors 24 h following stress exposure but not under unstressed conditions or immediately following exposure to acute stress. The inability of these mutants to recover properly from the exposure to an acute stress was associated with robust alterations in the expression profile of amygdalar genes and with dysregulated serotonergic function in stress-related neurocircuits. These findings position the urocortins as essential factors in the stress-recovery process and suggest the tKO mouse line as a useful stress-sensitive mouse model.

CRFR1 is expressed on pancreatic beta cells, promotes beta cell proliferation, and potentiates insulin secretion in a glucose-dependent manner.

Corticotropin-releasing factor (CRF), originally characterized as the principal neuroregulator of the hypothalamus-pituitary-adrenal axis, has broad central and peripheral distribution and actions. We demonstrate the presence of CRF receptor type 1 (CRFR1) on primary beta cells and show that activation of pancreatic CRFR1 promotes insulin secretion, thus contributing to the restoration of normoglycemic equilibrium. Stimulation of pancreatic CRFR1 initiates a cAMP response that promotes insulin secretion in vitro and in vivo and leads to the phosphorylation of cAMP response element binding and the induction of the expression of several immediate-early genes. Thus, the insulinotropic actions of pancreatic CRFR1 oppose the activation of CRFR1 on anterior pituitary corticotropes, leading to the release of glucocorticoids that functionally antagonize the actions of insulin. Stimulation of the MIN6 insulinoma line and primary rat islets with CRF also activates the MAPK signaling cascade leading to rapid phosphorylation of Erk1/2 in response to CRFR1-selective ligands, which induce proliferation in primary rat neonatal beta cells. Importantly, CRFR1 stimulates insulin secretion only during conditions of intermediate to high ambient glucose, and the CRFR1-dependent phosphorylation of Erk1/2 is greater with elevated glucose concentrations. This response is reminiscent of the actions of the incretins, which potentiate insulin secretion only during elevated glucose conditions. The presence of CRFR1 on beta cells adds another layer of complexity to the intricate network of paracrine and autocrine factors and their cognate receptors whose coordinated efforts can dictate islet hormone output and regulate beta cell proliferation.

NMR structure of the first extracellular domain of corticotropin-releasing factor receptor 1 (ECD1-CRF-R1) complexed with a high affinity agonist.

The corticotropin-releasing factor (CRF) peptide hormone family members coordinate endocrine, behavioral, autonomic, and metabolic responses to stress and play important roles within the cardiovascular, gastrointestinal, and central nervous systems, among others. The actions of the peptides are mediated by activation of two G-protein-coupled receptors of the B1 family, CRF receptors 1 and 2 (CRF-R1 and CRF-R2α,ß). The recently reported three-dimensional structures of the first extracellular domain (ECD1) of both CRF-R1 and CRF-R2ß (Pioszak, A. A., Parker, N. R., Suino-Powell, K., and Xu, H. E. (2008) J. Biol. Chem. 283, 32900-32912; Grace, C. R., Perrin, M. H., Gulyas, J., Digruccio, M. R., Cantle, J. P., Rivier, J. E., Vale, W. W., and Riek, R. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 4858-4863) complexed with peptide antagonists provided a starting point in understanding the binding between CRF ligands and receptors at a molecular level. We now report the three-dimensional NMR structure of the ECD1 of human CRF-R1 complexed with a high affinity agonist, α-helical cyclic CRF. In the structure of the complex, the C-terminal residues (23-41) of α-helical cyclic CRF bind to the ECD1 of CRF-R1 in a helical conformation mainly along the hydrophobic face of the peptide in a manner similar to that of the antagonists in their corresponding ECD1 complex structures. Unique to this study is the observation that complex formation between an agonist and the ECD1-CRF-R1 promotes the helical conformation of the N terminus of the former, important for receptor activation (Gulyas, J., Rivier, C., Perrin, M., Koerber, S. C., Sutton, S., Corrigan, A., Lahrichi, S. L., Craig, A. G., Vale, W., and Rivier, J. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 10575-10579).

Urocortin2 inhibits tumor growth via effects on vascularization and cell proliferation.

The corticotropin-releasing factor (CRF) receptor CRFR2 is expressed widely in peripheral tissues and in the vasculature, although its functional roles in those tissues have only recently begun to be elucidated. Previously we found that genetic deletion of CRFR2 resulted in profound postnatal hypervascularization in mice, characterized by both an increase in total vessel number and a dramatic increase in vessel diameter. These data strongly suggested that ligands for CRFR2 act to limit tissue vascularity, perhaps as a counterbalance to factors that promote neovascularization. Urocortin 2 (Ucn2) is a specific ligand for the CRFR2. We hypothesized that activation of CRFR2 by Ucn2 might thus suppress tumor vascularization and consequently limit tumor growth. Here, we show that viral-mediated expression of Ucn2 strikingly inhibits the growth and vascularization of Lewis Lung Carcinoma Cell (LLCC) tumors in vivo. Further, we found that this effect on tumor growth inhibition was independent of whether exposure to Ucn2 occurred before or after establishment of measurable tumors. In vitro, Ucn2 directly inhibited the proliferation of LLCC, suggesting that the tumor-suppressing effects of CRFR2 activation involve a dual mechanism of both a direct inhibition of tumor cell cycling and the suppression of tumor vascularization. These results establish that Ucn2 inhibits tumor growth, suggesting a potential therapeutic role for CRFR2 ligands in clinical malignancies.

Photo-cross-linkers incorporated into G-protein-coupled receptors in mammalian cells: a ligand comparison.

Capturing the right ligand at the right spot: a well-balanced system for non-natural amino acid mutagenesis allows the ligand binding sites of a class II G-protein coupled receptor to be mapped and distinct binding domains to be identified for different ligands in the native environment of mammalian cells.

Common and divergent structural features of a series of corticotropin releasing factor-related peptides.

Members of the corticoliberin family include the corticotropin releasing factors (CRFs), sauvagine, the urotensins, and urocortin 1 (Ucn1), which bind to both the CRF receptors CRF-R1 and CRF-R2, and the urocortins 2 (Ucn2) and 3 (Ucn3), which are selective agonists of CRF-R2. Structure activity relationship studies led to several potent and long-acting analogues with selective binding to either one of the receptors. NMR structures of six ligands of this family (the antagonists astressin B and astressin2-B, the agonists stressin1, and the natural ligands human Ucn1, Ucn2, and Ucn3) were determined in DMSO. These six peptides show differences in binding affinities, receptor-selectivity, and NMR structure. Overall, their backbones are alpha-helical, with a small kink or a turn around residues 25-27, resulting in a helix-loop-helix motif. The C-terminal helices are of amphipathic nature, whereas the N-terminal helices vary in their amphipathicity. The C-terminal helices thereby assume a conformation very similar to that of astressin bound to the ECD1 of CRF-R2 recently reported by our group.1 On the basis of an analysis of the observed 3D structures and relative potencies of [Ala]-substituted analogues, it is proposed that both helices could play a crucial role in receptor binding and selectivity. In conclusion, the C-terminal helices may interact along their hydrophobic faces with the ECD1, whereas the entire N-terminal helical surface may be involved in receptor activation. On the basis of the common and divergent features observed in the 3D structures of these ligands, multiple binding models are proposed that may explain their plurality of actions.

Delayed satiety-like actions and altered feeding microstructure by a selective type 2 corticotropin-releasing factor agonist in rats: intra-hypothalamic urocortin 3 administration reduces food intake by prolonging the post-meal interval.

Brain corticotropin-releasing factor/urocortin (CRF/Ucn) systems are hypothesized to control feeding, with central administration of 'type 2' urocortins producing delayed anorexia. The present study sought to identify the receptor subtype, brain site, and behavioral mode of action through which Ucn 3 reduces nocturnal food intake in rats. Non-food-deprived male Wistar rats (n=176) were administered Ucn 3 into the lateral (LV) or fourth ventricle, or into the ventromedial or paraventricular nuclei of the hypothalamus (VMN, PVN) or the medial amygdala (MeA), regions in which Ucn 3 is expressed in proximity to CRF(2) receptors. LV Ucn 3 suppressed ingestion during the third-fourth post-injection hours. LV Ucn 3 anorexia was reversed by cotreatment with astressin(2)-B, a selective CRF(2) antagonist and not observed following equimole subcutaneous or fourth ventricle administration. Bilateral intra-VMN and intra-PVN infusion, more potently than LV infusion, reduced the quantity (57-73%) and duration of ingestion (32-68%) during the third-fourth post-infusion hours. LV, intra-PVN and intra-VMN infusion of Ucn 3 slowed the eating rate and reduced intake by prolonging the post-meal interval. Intra-VMN Ucn 3 reduced feeding bout size, and intra-PVN Ucn 3 reduced the regularity of eating from pellet to pellet. Ucn 3 effects were behaviorally specific, because minimal effective anorectic Ucn 3 doses did not alter drinking rate or promote a conditioned taste aversion, and site-specific, because intra-MeA Ucn 3 produced a nibbling pattern of more, but smaller meals without altering total intake. The results implicate the VMN and PVN of the hypothalamus as sites for Ucn 3-CRF(2) control of food intake.

Chronic cocaine administration switches corticotropin-releasing factor2 receptor-mediated depression to facilitation of glutamatergic transmission in the lateral septum.

Corticotropin-releasing factor (CRF) and urocortin (Ucn I) are endogenous members among a family of CRF-related peptides that activate two different and synaptically localized G-protein-coupled receptors, CRF1 and CRF2. These peptides and their receptors have been implicated in stress responses and stress with cocaine abuse. In this study, we observed significant alterations in excitatory transmission and CRF-related peptide regulation of excitatory transmission in the lateral septum mediolateral nucleus (LSMLN) after chronic cocaine administration. In brain slice recordings from the LSMLN of control (saline-treated) rats, glutamatergic synaptic transmission was facilitated by activation of CRF1 receptors with CRF but was depressed after activation of CRF2 receptors with Ucn I. After acute withdrawal from a chronic cocaine administration regimen, CRF1 activation remained facilitatory, but CRF2 activation facilitated rather than depressed LSMLN EPSCs. These alterations in CRF2 effects occurred through both presynaptic and postsynaptic mechanisms. In saline-treated rats, CRF1 and CRF2 coupled predominantly to protein kinase A signaling pathways, whereas after cocaine withdrawal, protein kinase C activity was more prominent and likely contributed to the CRF2-mediated presynaptic facilitation. Neither CRF nor Ucn I altered monosynaptic GABA(A)-mediated IPSCs before or after chronic cocaine administration, suggesting that loss of GABAA-mediated inhibition could not account for the facilitation. This switch in polarity of Ucn I-mediated neuromodulation, from a negative to positive regulation of excitatory glutamatergic transmission after chronic cocaine administration, could generate an imbalance in the brain reward circuitry associated with the LSMLN.

Cocaine- and amphetamine-regulated transcript is localized in pituitary lactotropes and is regulated during lactation.

Cocaine- and amphetamine-regulated transcript (CART) is a highly expressed peptide implicated in the regulation of feeding, reward and reinforcement, and stress-related behaviors. CART has been localized to discrete cell populations in the brain, gut, adrenal gland, and pancreas. In contrast, CART-producing cell types in the pituitary gland remain ill defined. In the present study, double-label immunohistochemistry, employing a high-affinity antiserum we generated against CART-(62-102), was used to identify CART-producing cells in the pituitary gland. In the anterior pituitary, the majority of CART immunoreactivity (-ir) was localized in lactotropes; minor populations of CART-ir cells were identified as somatotropes and corticotropes. In the posterior pituitary, CART-ir extensively colocalized with oxytocin-containing fibers; in contrast, only a few vasopressin fibers contained CART-ir. As expected, CART colocalized with oxytocin in magnocellular neurons of the supraoptic nucleus. The effects of bromocriptine, a potent dopamine receptor agonist, were examined to determine whether CART mRNA expression and protein release are regulated in a similar fashion as prolactin. Similar to prolactin, CART mRNA expression and protein release were significantly decreased after bromocriptine treatment of dispersed rat anterior pituitary cells in culture. To explore the putative physiological role of pituitary CART, we compared levels of CART mRNA expression in lactating and nonlactating female rats. CART mRNA levels were significantly increased in the anterior pituitary and supraoptic nucleus of lactating rats. Furthermore, levels of CART in the systemic circulation were significantly elevated at the onset of lactation, peaked on d 10 of lactation and returned to baseline values 10 d after pups were weaned. The current study describes the cellular localization and regulation of CART expression and protein release from the rat pituitary gland. These findings suggest a putative role for CART in lactation.

The three-dimensional structure of the N-terminal domain of corticotropin-releasing factor receptors: sushi domains and the B1 family of G protein-coupled receptors.

The corticotropin-releasing factor (CRF) receptors, CRF-R1 and CRF-R2, belong to the B1 subfamily of G protein-coupled Receptors (GPCRs), including receptors for secretin, growth hormone-releasing hormone (GHRH), vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating polypeptide (PACAP), calcitonin, parathyroid hormone (PTH), glucagon, and glucagon-like peptide-1 (GLP-1). The peptide ligand family comprises CRF, Ucn 1, 2, and 3. CRF plays the major role in integrating the response to stress. Additionally, the ligands exhibit many effects on muscle, pancreas, heart, and the GI, reproductive, and immune systems. CRF-R1 has higher affinity for CRF than does CRF-R2 while both receptors bind Ucn 1 equally. CRF-R2 shows specificity for Ucns 2 and 3. A major binding domain of the CRFRs is the N terminus/first extracellular domain (ECD1). Soluble proteins corresponding to the ECD1s of each receptor bind CRF ligands with nanomolar affinities. Our three-dimensional (3D) nuclear magnetic resonance (NMR) structure of a soluble protein corresponding to the ECD1 of CRF-R2beta (1) identified its structural fold as a Sushi domain/short consensus repeat (SCR), stabilized by three disulfide bridges, two tryptophan residues, and an internal salt bridge (Asp65-Arg101). Disruption of the bridge by D65A mutation abrogates ligand recognition and results in loss of the well-defined disulfide pattern and Sushi domain structure. NMR analysis of the ECD1 in complex with astressin identified key amino acids involved in ligand recognition. Mutation of some of these residues in the full-length receptor reduces its affinity for CRF ligands. A structure-based sequence comparison shows conservation of key amino acids in all the B1 subfamily receptors, suggesting a corresponding conservation of a Sushi domain structural fold of their ECD1s.

Antagonists of activin signaling: mechanisms and potential biological applications.

Activins are members of the transforming growth factor-beta (TGF-beta) superfamily that control many physiological processes such as cell proliferation and differentiation, immune responses, wound repair and various endocrine activities. Activins elicit these diverse biological responses by signaling via type I and type II receptor serine kinases. Recent studies have revealed details of the roles of inhibin, betaglycan, follistatin and its related protein follistatin-related gene (FLRG), Cripto and BAMBI in antagonizing activin action, and exogenous antagonists against the activin type I (SB-431542 and SB-505124) and type II (activin-M108A) receptors have been developed. Understanding how activin signaling is controlled extracellularly is the first step in providing treatment for wound healing and for disorders such as cachexia and cancer, which result from a deregulated activin pathway.

The role of the hypothalamic-pituitary-adrenal axis in neuroendocrine responses to stress.

Animals respond to stress by activating a wide array of behavioral and physiological responses that are collectively referred to as the stress response. Corticotropin-releasing factor (CRF) plays a central role in the stress response by regulating the hypothalamic-pituitary-adrenal (HPA) axis. In response to stress, CRF initiates a cascade of events that culminate in the release of glucocorticoids from the adrenal cortex. As a result of the great number of physiological and behavioral effects exerted by glucocorticoids, several mechanisms have evolved to control HPA axis activation and integrate the stress response. Glucocorticoid feedback inhibition plays a prominent role in regulating the magnitude and duration of glucocorticoid release. In addition to glucocorticoid feedback, the HPA axis is regulated at the level of the hypothalamus by a diverse group of afferent projections from limbic, midbrain, and brain stem nuclei. The stress response is also mediated in part by brain stem noradrenergic neurons, sympathetic andrenomedullary circuits, and parasympathetic systems. In summary, the aim of this review is to discuss the role of the HPA axis in the integration of adaptive responses to stress. We also identify and briefly describe the major neuronal and endocrine systems that contribute to the regulation of the HPA axis and the maintenance of homeostasis in the face of aversive stimuli.

Increased depression-like behaviors in corticotropin-releasing factor receptor-2-deficient mice: sexually dichotomous responses.

Depressive disorders affect nearly 19 million American adults, making depression and the susceptibility for developing depression a critical focus of mental health research today. Females are twice as likely to develop depression as males. Stress is a known risk factor for developing depression, and recent hypotheses suggest an involvement of an overactive stress axis. As mediators of the stress response, corticotropin-releasing factor (CRF) and its receptors (CRFR1 and CRFR2) have been implicated in the propensity for developing stress-related mood disorders. Mice deficient in CRFR2 display increased anxiety-like behaviors and a hypersensitive stress response. As a possible animal model of depression, these mice were tested for depression-like behaviors in the forced swim test. Comparisons were made between wild-type and mutant animals, as well as between sexes. Male and female CRFR2-mutant mice showed increased immobility as an indicator of depression compared with wild-type mice of the same sex. In addition, mutant and wild-type female mice demonstrated increased immobile time compared with males of the same genotype. Treatment of CRFR2-deficient mice with the CRFR1 antagonist antalarmin decreased immobile time and increased swim time in both sexes. We found a significant effect of sex for both time spent immobile and swimming after antalarmin treatment. Because differences in behaviors in the forced swim test are good indicators of serotonergic and catecholaminergic involvement, our results may reveal an interaction of CRF pathways with other known antidepressant systems and may also support an involvement of CRF receptors in the development of depression such that elevated CRFR1 activity, in the absence of CRFR2, increases depression-like behaviors.

Urocortin III-immunoreactive projections in rat brain: partial overlap with sites of type 2 corticotrophin-releasing factor receptor expression.

Urocortin (Ucn) III, or stresscopin, is a new member of the corticotropin-releasing factor (CRF) peptide family identified in mouse and human. Pharmacological studies showed that Ucn III is a high-affinity ligand for the type 2 CRF receptor (CRF-R2). To further understand physiological functions the peptide may serve in the brain, the distribution of Ucn III neurons and fibers was examined by in situ hybridization and immunohistochemistry in the rat brain. Ucn III-positive neurons were found predominately within the hypothalamus and medial amygdala. In the hypothalamus, Ucn III neurons were observed in the median preoptic nucleus and in the rostral perifornical area lateral to the paraventricular nucleus. The Ucn III fibers were distributed mainly in the hypothalamus and limbic structures. Hypothalamic regions that were innervated prominently by Ucn III fibers included the ventromedial nucleus, medial preoptic nucleus, and ventral premammillary nucleus. Outside the hypothalamus, the densest projections were found in the intermediate part of the lateral septum, posterior division of the bed nucleus stria terminalis, and the medial nucleus of the amygdala. Several major Ucn III terminal fields identified in the present study, including the lateral septum and the ventromedial hypothalamus, are known to express high levels of CRF-R2. Thus, these anatomical data strongly support the notion that Ucn III is an endogenous ligand for CRF-R2 in these areas. These results also suggest that Ucn III is positioned to play a role in mediating physiological functions, including food intake and neuroendocrine regulation.

Selective activation of corticotropin-releasing factor-2 receptors on neurochemically identified neurons in the rat dorsal raphe nucleus reveals dual actions.

The dorsal raphe (DR)-serotonin (5-HT) system has been implicated in stress-related psychiatric disorders. Stress may impact on this system through corticotropin-releasing factor (CRF), which densely innervates the DR. CRF binds to CRF-R1 and CRF-R2 receptors in the DR and has complex and opposing effects depending on the dose used and the endpoint examined. To clarify the impact of CRF on the DR-5-HT system, the effects of selectively activating CRF-R2 receptors (the predominant subtype) on extracellular DR neuronal activity were examined in halothane-anesthetized rats. Because the DR is neurochemically heterogeneous, when possible, neurons were labeled with neurobiotin for subsequent neurochemical classification as 5-HT or non-5-HT. Relatively low doses of urocortin II (UII) (0.1-10 ng) injected into the DR inhibited most (79%; n = 34) neurons, whereas a higher dose (30 ng) inhibited 28% and activated 41% (n = 29). An analysis of effects on neurochemically identified neurons revealed that 5-HT neurons were inhibited by 0.1-10 ng of UII and activated by 30 ng of UII. Activation of 5-HT neurons by 30 ng of UII likely resulted from disinhibition because the majority of non-5-HT neurons were inhibited by this dose. Antisauvagine-30, but not antalarmin, antagonized UII, implicating CRF-R2 receptors in the effects. The results suggest that activation of CRF-R2 on DR-5-HT neurons inhibits neuronal activity, whereas activation of CRF-R2 receptors on non-5-HT neurons may indirectly excite DR-5-HT neurons through disinhibition. Importantly, the tone of the DR-5-HT system can be regulated in a dynamic manner through CRF-R2 activation, being either decreased or increased depending on the level of endogenous or exogenous ligand.

Corticotropin-releasing factor receptors CRF1 and CRF2 exert both additive and opposing influences on defensive startle behavior.

The corticotropin-releasing factor (CRF) receptors (CRF1 and CRF2) are crucial mediators of physiological and behavioral responses to stress. In animals, CRF1 appears to primarily mediate CRF-induced anxiety-like responses, but the role of CRF2 during stress is still unclear. Here we report the effects of CRF1 and CRF2 on the magnitude and plasticity of defensive startle responses in mice. Startle plasticity is measured by inhibition of startle by sensory stimuli, i.e., prepulse inhibition (PPI), and is disrupted in patients with panic or posttraumatic stress disorders in which CRF neurotransmission may be overactive. Pharmacological blockade of CRF1 reversed both CRF-induced increases in startle and CRF-induced deficits in PPI. CRF2 blockade attenuated high-dose but not low-dose CRF-induced increases in startle and reduced PPI. Conversely, activation of CRF2 enhanced PPI. CRF had no effect on startle and increased PPI in CRF1 knock-out mice. These data indicate that CRF receptors act in concert to increase the magnitude of defensive startle yet in opposition to regulate the flexibility of startle. These data support a new model of respective CRF receptor roles in stress-related behavior such that, although both receptors enhance the magnitude of defensive responses, CRF1 receptors contravene, whereas CRF2 receptors enhance, the impact of sensory information on defensive behavior. We hypothesize that excessive CRF1 activation combined with reduced CRF2 signaling may contribute to information processing deficits seen in panic and posttraumatic stress disorder patients and support CRF1-specific pharmacotherapy.

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 mother's 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 dam's 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 pup's 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.

Corticotropin-releasing factor and Urocortin I modulate excitatory glutamatergic synaptic transmission.

Corticotropin-releasing factor (CRF)-related peptides serve as hormones and neuromodulators of the stress response and play a role in affective disorders. These peptides are known to alter complex behaviors and neuronal properties, but their receptor-mediated effects at CNS synapses are not well described. Here we show that excitatory glutamatergic transmission is modulated by two endogenous CRF-related peptide ligands, corticotropin-releasing factor [CRF rat/human (r/h)] and Urocortin I (Ucn I), within the central nucleus of the amygdala (CeA) and the lateral septum mediolateral nucleus (LSMLN). These limbic nuclei are reciprocally innervated, are involved in stress and affective disorders, and have high densities of the CRF receptors CRF1 and CRF2. Activation of these receptors exerts diametrically opposed actions on glutamatergic transmission in these nuclei. In the CeA, CRF(r/h) depressed excitatory glutamatergic transmission through a CRF1-mediated postsynaptic action, whereas Ucn I facilitated synaptic responses through presynaptic and postsynaptic CRF2-mediated mechanisms. Conversely, in the LSMLN, CRF caused a CRF1-mediated facilitation of glutamatergic transmission via postsynaptic mechanisms, whereas Ucn I depressed EPSCs by postsynaptic and presynaptic CRF2-mediated actions. Furthermore, antagonists of these receptors also affected glutamatergic neurotransmission, indicating that endogenous ligands tonically modulated synoptic activity at these synapses. These data show that CRF receptors in CeA and LSMLN synapses exert and maintain a significant synaptic tone and thereby regulate excitatory glutamatergic transmission. The results also suggest that CRF receptors may provide novel targets in affective disorders and stress.