Corticotropin-releasing Factor Regulation Research Articles

Corticotropin releasing factor in the nucleus basalis of Meynert impairs attentional performance and reduces levels of glutamate and taurine in male and female rats

Psychiatric disorders that are characterized by impairments in sustained attention, including attention deficit hyperactivity disorder (ADHD), post-traumatic stress disorder (PTSD), and major depression are also sensitive to exacerbation by stress. Sustained attention relies on cholinergic and non-cholinergic projections from the nucleus basalis of Meynert (NBM) in the basal forebrain to the medial prefrontal cortex (mPFC). We have previously shown that central administration of the stress neuropeptide corticotropin releasing factor (CRF) impairs performance on the sustained attention task (SAT) in adult male and female rats. The present study investigated whether this effect was mediated by CRF's action in the NBM. Rats were administered CRF in the NBM and subsequent SAT performance was measured. A high dose of CRF (100 ng) significantly impaired performance on non-signaled events across sex. Because performance on non-signaled events is believed to depend on non-cholinergic (i.e., GABA and glutamate) signaling, high performance liquid chromatography was used to quantify amino acid levels in the NBM and mPFC. We found females have higher levels of glutamate, glutamine, GABA glycine, and alanine in the NBM than males. Importantly, CRF in the NBM led to a local decrease of taurine and several amino acids involved in glutamate synthesis in males and females, changes which may mediate the CRF-induced SAT performance deficit. Together these studies suggest that CRF regulation of amino acids in the NMB contributes to stress-induced attention deficits.

(3α,5α)3-Hydroxypregnan-20-one (3α,5α-THP) Regulation of the HPA Axis in the Context of Different Stressors and Sex.

Corticotropin-releasing factor (CRF) regulates the stress response in the hypothalamus and modulates neurotransmission across the brain through CRF receptors. Acute stress increases hypothalamic CRF and the GABAergic neurosteroid (3α,5α)3-hydroxypregnan-20-one (3α,5α-THP). We previously showed that 3α,5α-THP regulation of CRF is sex and brain region dependent. In this study, we investigated 3α,5α-THP regulation of stress-induced hypothalamic CRF, CRF receptor type 1 (CRFR1), CRF binding protein (CRFBP), pro-opiomelanocortin (POMC), and glucocorticoid receptor (GR) by western blot and circulating corticosterone (CORT) by enzyme-linked immunosorbent assay (ELISA) in male and female Sprague Dawley rats. Tissue was collected after rats were injected with 3α,5α-THP (15 mg/kg, IP) or vehicle 15 min prior to 30 min of restraint stress (RS), or 10 min of forced swim stress (FSS) and 20 min recovery. The initial exposure to a stress stimulus increased circulating CORT levels in both males and females, but 3α,5α-THP attenuated the CORT response only in females after RS. 3α,5α-THP reduced GR levels in male and females, but differently between stressors. 3α,5α-THP decreased the CRF stress response after FSS in males and females, but after RS, only in female rats. 3α,5α-THP reduced the CRFR1, CRFBP, and POMC increases after RS and FSS in males, but in females only after FSS. Our results showed different stress responses following different types of stressors: 3α,5α-THP regulated the HPA axis at different levels, depending on sex.

Enhanced CRFR1-Dependent Regulation of a Ventral Tegmental Area to Prelimbic Cortex Projection Establishes Susceptibility to Stress-Induced Cocaine Seeking.

The ability of stress to trigger cocaine seeking in humans and rodents is variable and is determined by the amount and pattern of prior drug use. This study examined the role of a corticotropin releasing factor (CRF)-regulated dopaminergic projection from the ventral tegmental area (VTA) to the prelimbic cortex in shock-induced cocaine seeking and its recruitment under self-administration conditions that establish relapse vulnerability. Male rats with a history of daily long-access (LgA; 14 × 6 h/d) but not short-access (ShA; 14 × 2 h/d) self-administration showed robust shock-induced cocaine seeking. This was associated with a heightened shock-induced prelimbic cortex Fos response and activation of cholera toxin b retro-labeled VTA neurons that project to the prelimbic cortex. Chemogenetic inhibition of this pathway using a dual virus intersectional hM4Di DREADD (designer receptor exclusively activated by designer drug) based approach prevented shock-induced cocaine seeking. Both shock-induced reinstatement and the prelimbic cortex Fos response were prevented by bilateral intra-VTA injections of the CRF receptor 1 (CRFR1) antagonist, antalarmin. Moreover, pharmacological disconnection of the CRF-regulated dopaminergic projection to the prelimbic cortex by injection of antalarmin into the VTA in one hemisphere and the D1 receptor antagonist, SCH23390, into the prelimbic cortex of the contralateral hemisphere prevented shock-induced cocaine seeking. Finally, LgA, but not ShA, cocaine self-administration resulted in increased VTA CRFR1 mRNA levels as measured using in situ hybridization. Altogether, these findings suggest that excessive cocaine use may establish susceptibility to stress-induced relapse by recruiting CRF regulation of a stressor-responsive mesocortical dopaminergic pathway.SIGNIFICANCE STATEMENT Understanding the neural pathways and mechanisms through which stress triggers relapse to cocaine use is critical for the development of more effective treatment approaches. Prior work has demonstrated a critical role for the neuropeptide corticotropin releasing factor (CRF) in stress-induced cocaine seeking. Here we provide evidence that stress-induced reinstatement in a rat model of relapse is mediated by a CRF-regulated dopaminergic projection from the ventral tegmental area (VTA) that activates dopamine D1 receptors in the prelimbic cortex. Moreover, we report that this pathway may be recruited as a result of daily cocaine self-administration under conditions of extended drug access/heightened drug intake, likely as a result of increased CRFR1 expression in the VTA, thereby promoting susceptibility to stress-induced cocaine seeking.

Amygdala-mediated mechanisms regulate visceral hypersensitivity in adult females following early life stress: importance of the glucocorticoid receptor and corticotropin-releasing factor.

Alterations in amygdala activity are apparent in women who report a history of early life stress (ELS) and those diagnosed with chronic pain disorders. Chronic stress in adulthood induces visceral hypersensitivity by alterations in glucocorticoid receptor (GR) and corticotropin-releasing factor (CRF) expression within the central amygdala (CeA). Here, we hypothesized that unpredictable ELS, previously shown to induce visceral hypersensitivity in adult female rats, alters GR and CRF expression in the CeA. After neonatal ELS, visceral sensitivity and GR and CRF gene expression were quantified in adult female rats. After unpredictable ELS, adult female rats exhibited visceral hypersensitivity and increased expression of GR and CRF in the CeA. After predictable ELS, adult female rats demonstrated normosensitive behavioral pain responses and upregulation of GR but not CRF in the CeA. After the ELS paradigms, visceral sensitivity and gene expression within the CeA were unaffected in adult male rats. The role of GR and CRF in modulating visceral sensitivity in adult female rats after ELS was investigated using oligodeoxynucleotide sequences targeted to the CeA for knockdown of GR or CRF. Knockdown of GR increased visceral sensitivity in all rats but revealed an exaggerated visceral hypersensitivity in females with a history of predictable or unpredictable ELS compared with that of controls. Knockdown of CRF expression or antagonism of CRF1R in the CeA attenuated visceral hypersensitivity after unpredictable ELS. This study highlights a shift in GR and CRF regulation within the CeA after ELS that underlies the development of visceral hypersensitivity in adulthood.

Stimulation of corticotropin-releasing factor gene expression by FosB in rat hypothalamic 4B cells

The Fos- and Jun family proteins are immediate-early gene products, and the Fos/Jun heterodimer, activator protein-1 (AP-1), may be involved in the regulation of corticotropin-releasing factor (CRF) gene expression. FosB is a member of the Fos family proteins that is expressed in the paraventricular nucleus of the hypothalamus upon stress exposure, but it has not been clear whether FosB participates in the regulation of CRF gene expression. This study aimed to explore the effect of the FosB and cJun proteins on CRF gene expression in rat hypothalamic 4B cells. The levels of FosB mRNA and cJun mRNA increased following treatment with forskolin, phorbol-12-myristate-13-acetate (PMA), or A23187 in the hypothalamic cells. Overexpression of FosB or cJun potently increased CRF mRNA levels. Furthermore, downregulation of FosB or cJun suppressed the CRF gene expression induced by forskolin, PMA, or A23187. In addition, the basal CRF mRNA levels were partially reduced by cJun downregulation. These findings suggest that FosB, together with cJun, may mediate CRF gene expression in the hypothalamic cells.

Regulation of corticotropin-releasing factor and urocortin 2/3 mRNA by leptin in hypothalamic N39 cells

Corticotropin-releasing factor (CRF) activates the pituitary-adrenal axis during stress, and shows anorectic effects via CRF type 1 receptors in the hypothalamus. Both urocortin (Ucn) 2 and Ucn3 also act as anorectic neuropeptides via CRF type 2 receptors. Leptin, a product of the obesity gene secreted mainly from adipose tissue, reduces food intake and increases energy expenditure. A possible interaction between leptin and CRF/Ucns has been suggested, as leptin can regulate expression and activation of CRF and Ucns in the hypothalamus. This study aimed to explore the possible function of leptin in the hypothalamus, and its effects in regulating CRF and Ucns. The study identified mRNA expression of the leptin receptor (Ob-R) and its subtypes, CRF, and Ucn2/3 in mouse hypothalamic N39 cells. Leptin stimulated signal transducer and activators of transcription type 3 (STAT3) phosphorylation, directly increased the mRNA levels of both CRF and Ucn2/3 in hypothalamic cells, and increased Ob-Rb mRNA levels. A Janus kinase inhibitor inhibited the leptin-mediated increase in STAT3 phosphorylation, and then the increases in CRF and Ucn2/3 mRNA levels. Leptin may contribute to a stress response or anorectic effect via the regulation of CRF and Ucn2/3 in the hypothalamus.

Binge Ethanol‐Drinking Potentiates Corticotropin Releasing Factor R1 Receptor Activity in the Ventral Tegmental Area

Corticotropin releasing factor (CRF) and urocortin play an important role in many stress responses and also can regulate ethanol (EtOH) intake. Adaptations in CRF signaling in the central amygdala promote EtOH consumption after long-term EtOH intake in dependent animals and also after brief periods of binge EtOH intake. Thus, even brief episodes of EtOH consumption can alter the function of the CRF system, allowing CRF to regulate EtOH intake. Here, we examined whether brief binge EtOH consumption leads to CRF receptor adaptations within the ventral tegmental area (VTA), a structure involved in signaling rewarding and aversive events and important in the development and expression of drug and alcohol addiction. We utilized a mouse model of binge drinking known as drinking in the dark (DID), where C57BL/6J mice drink approximately 6 g/kg in 4 hours and achieve blood EtOH concentrations of approximately 100 mg/dl, which is equivalent to binge drinking in humans. We used ex vivo whole-cell recordings from putative VTA dopamine (DA) neurons to examine CRF regulation of NMDA receptor (NMDAR) currents. We also examined the impact of CRF receptor antagonist injection in the VTA on binge EtOH intake. Ex vivo whole-cell recordings from putative VTA DA neurons showed enhanced CRF-mediated potentiation of NMDAR currents in juvenile mice that consumed EtOH in the DID procedure. CRF-induced potentiation of NMDAR currents in EtOH-drinking mice was blocked by administration of CP-154,526 (3 μM), a selective CRF1 receptor antagonist. Furthermore, intra-VTA infusion of CP-154,526 (1 μg) significantly reduced binge EtOH consumption in adult mice. These results were not due to alterations of VTA NMDAR number or function, suggesting that binge drinking may enhance signaling through VTA CRF1 receptors onto NMDARs. Altered CRF1 receptor-mediated signaling in the VTA promotes binge-like EtOH consumption in mice, which supports the idea that CRF1 receptors may therefore be a promising pharmacological target for reducing binge drinking in humans.

Sex differences in the effects of chronic stress and food restriction on body weight gain and brain expression of CRF and relaxin‐3 in rats

This study investigated sex-specific effects of repeated stress and food restriction on food intake, body weight, corticosterone plasma levels and expression of corticotropin-releasing factor (CRF) in the hypothalamus and relaxin-3 in the nucleus incertus (NI). The CRF and relaxin-3 expression is affected by stress, and these neuropeptides produce opposite effects on feeding (anorexigenic and orexigenic, respectively), but sex-specific regulation of CRF and relaxin-3 by chronic stress is not fully understood. Male and female rats were fed ad libitum chow (AC) or ad libitum chow and intermittent palatable liquid Ensure without food restriction (ACE), or combined with repeated food restriction (60% chow, 2 days per week; RCE). Half of the rats were submitted to 1-h restraint stress once a week. In total, seven weekly cycles were applied. The body weight of the RCE stressed male rats significantly decreased, whereas the body weight of the RCE stressed female rats significantly increased compared with the respective control groups. The stressed female RCE rats considerably overate chow during recovery from stress and food restriction. The RCE female rats showed elevated plasma corticosterone levels and low expression of CRF mRNA in the paraventricular hypothalamic nucleus but not in the medial preoptic area. The NI expression of relaxin-3 mRNA was significantly higher in the stressed RCE female rats compared with other groups. An increase in the expression of orexigenic relaxin-3 and misbalanced hypothalamic-pituitary-adrenal axis activity may contribute to the overeating and increased body weight seen in chronically stressed and repeatedly food-restricted female rats.

Corticotropin-releasing factor in the mouse central nucleus of the amygdala: Ultrastructural distribution in NMDA-NR1 receptor subunit expressing neurons as well as projection neurons to the bed nucleus of the stria terminalis

Corticotropin-releasing factor (CRF) and glutamate are critical signaling molecules in the central nucleus of the amygdala (CeA). Central amygdala CRF, acting via the CRF type 1 receptor (CRF-R1), plays an integral role in stress responses and emotional learning, processes that are generally known to involve functional NMDA-type glutamate receptors. There is also evidence that CRF expressing CeA projection neurons to the bed nucleus of the stria terminalis (BNST) play an important role in stress related behaviors. Despite the potentially significant interactions between CRF and NMDA receptors in the CeA, the synaptic organization of these systems is largely unknown. Using dual labeling high resolution immunocytochemical electron microscopy, it was found that individual somata and dendrites displayed immunoreactivity for CRF and the NMDA-NR1 (NR1) subunit in the mouse CeA. In addition, CRF-containing axon terminals contacted postsynaptic targets in the CeA, some of which also expressed NR1. Neuronal profiles expressing the CRF type 1 receptor (CRF-R1), identified by the expression of green fluorescent protein (GFP) in bacterial artificial chromosome (BAC) transgenic mice, also contained NR1, and GFP immunoreactive terminals formed synapses with NR1 containing dendrites. Although CRF and GFP were only occasionally co-expressed in individual somata and dendritic profiles, contacts between labeled axon terminals and dendrites were frequently observed. A combination of tract tracing and immunocytochemistry revealed that a population of CeA CRF neurons projected to the BNST. It was also found that CRF, or GFP expressing terminals directly contacted CeA-BNST projection neurons. These results indicate that the NMDA receptor is positioned for the postsynaptic regulation of CRF expressing CeA neurons and the modulation of signals conveyed by CRF inputs. Interactions between CRF and NMDA receptor mediated signaling in CeA neurons, including those projecting to the BNST, may provide the synaptic basis for integrating the experience of stress and relevant environmental stimuli with behaviors that may be of particular relevance to stress-related learning and the emergence of psychiatric disorders, including drug addiction.

Action of glucagon-like peptide 1 and glucose levels on corticotropin-releasing factor and vasopressin gene expression in rat hypothalamic 4B cells

Corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) are the two major regulatory peptides in the hypothalamic–pituitary–adrenal (HPA) axis. Glucagon-like peptide-1 (GLP-1), an important regulator of metabolism or energy homeostasis, is implicated in the regulation of the HPA axis in response to stress and may act directly on CRF and AVP neurons. To elucidate the direct regulation of CRF and AVP genes by GLP-1 in the hypothalamus, we examined the effect of GLP-1 in hypothalamic 4B cells, which show the characteristics of hypothalamic paraventricular nucleus neurons. The mRNA of GLP-1 receptor was detected in 4B cells by RT-PCR. GLP-1 significantly stimulated both CRF and AVP mRNA levels. Cells were transfected with CRF or AVP promoter to examine the activity of each promoter. GLP-1 directly stimulated the activities of both CRF and AVP promoters in hypothalamic 4B cells. Basal promoter activities of both CRF and AVP were increased in higher glucose medium. In addition, CRF and AVP promoter activities were increased by GLP-1 in standard or low glucose medium but not in higher glucose medium. An equimolar concentration of metabolically inactive l-glucose failed to mimic the effect of d-glucose, indicating that the event was caused by changes in glucose levels and not by hyperosmolality. Together, these data suggest that GLP-1 would contribute to stress responses through activation of CRF and AVP genes in the hypothalamic cells. Hyperglycemia may be one of the stressors enhancing the syntheses of CRF and AVP in the hypothalamus.

Increased vulnerability of the brain norepinephrine system of females to corticotropin-releasing factor overexpression

Stress-related psychiatric disorders are more prevalent in women than men. Because hypersecretion of the stress neuromediator, corticotropin-releasing factor (CRF) has been implicated in these disorders, sex differences in CRF sensitivity could underlie this disparity. Hyperarousal is a core symptom that is shared by stress-related disorders and this has been attributed to CRF regulation of the locus coeruleus (LC)-norepinephrine arousal system. We recently identified sex differences in CRF1 receptor (CRF1) signaling and trafficking that render LC neurons of female rats more sensitive to CRF and potentially less able to adapt to excess CRF compared to male rats. The present study used a genetic model of CRF overexpression to test the hypothesis that females would be more vulnerable to LC dysregulation by conditions of excess CRF. In both male and female CRF overexpressing (CRF-OE) mice, the LC was more densely innervated by CRF compared to wild type controls. Despite the equally dense CRF innervation of the LC in male and female CRF-OE mice, LC discharge rates recorded in slices in vitro were selectively elevated in female CRF-OE mice. Immunoelectron microscopy revealed that this sex difference resulted from differential CRF1 trafficking. In male CRF-OE mice, CRF1 immunolabeling was prominent in the cytoplasm of LC neurons, indicative of internalization, a process that would protect cells from excessive CRF. However, in female CRF-OE mice, CRF1 labeling was more prominent on the plasma membrane, suggesting that the compensatory response of internalization was compromised. Together, the findings suggest that the LC-norepinephrine system of females will be particularly affected by conditions resulting in elevated CRF because of differences in receptor trafficking. As excessive LC activation has been implicated in the arousal components of stress-related psychiatric disorders, this may be a cellular mechanism that contributes to the increased incidence of these disorders in females.

Sex Differences in Molecular and Cellular Substrates of Stress

Women are twice as likely as men to suffer from stress-related psychiatric disorders, like unipolar depression and post-traumatic stress disorder. Although the underlying neural mechanisms are not well characterized, the pivotal role of stress in the onset and severity of these diseases has led to the idea that sex differences in stress responses account for this sex bias. Corticotropin-releasing factor (CRF) orchestrates stress responses by acting both as a neurohormone to initiate the hypothalamic-pituitary-adrenal (HPA) axis and as a neuromodulator in the brain. One target of CRF modulation is the locus coeruleus (LC)-norepinephrine system, which coordinates arousal components of the stress response. Hypersecretion of CRF and dysregulation of targets downstream from CRF, such as the HPA axis and LC-norepinephrine system, are characteristic features of many stress-related psychiatric diseases, suggesting a causal role for CRF and its targets in the development of these disorders. This review will describe sex differences in CRF and the LC-norepinephrine system that can increase stress sensitivity in females, making them vulnerable to stress-related disorders. Evidence for gonadal hormone regulation of hypothalamic CRF is discussed as an effect that can lead to increased HPA axis activity in females. Sex differences in the structure of LC neurons that create the potential for hyperarousal in response to emotional stimuli are described. Finally, sex differences at the molecular level of the CRF(1) receptor that make the LC-norepinephrine system more reactive in females are reviewed. The implications of these sex differences for the treatment of stress-related psychiatric disorders also will be discussed.

Antagonizing Corticotropin-Releasing Factor in the Central Nucleus of the Amygdala Attenuates Fear-induced Reductions in Sleep but not Freezing

Contextual fear is followed by significant reductions in rapid eye movement sleep (REM) that are regulated by the central nucleus of the amygdala (CNA). Corticotropin-releasing factor (CRF) plays a major role in regulating the stress response as well as arousal, and CRF in CNA is implicated in stress-related behavior. To test the hypothesis that CRF regulation of CNA is involved in fear-induced alterations in REM, we determined the effects of microinjections into CNA of the CRF1 antagonist, antalarmin (ANT) on fear-induced reductions in REM. We also evaluated c-Fos activation in the hypothalamic paraventricular nucleus (PVN), locus coeruleus (LC), and dorsal raphe nucleus (DRN) to determine whether activation of these regions was consistent with their roles in regulating stress and in the control of REM. On separate days, rats were subjected to baseline and 2 shock training sessions (S1 and S2). Five days later, the rats received bilateral microinjections of ANT (4.8 mM) or vehicle (VEH) prior to exposure to the fearful context. Sleep was recorded for 20 h in each condition. Freezing was assessed during S1, S2, and context. Separate groups of rats received identical training and microinjections or handling control (HC) only, but were sacrificed 2 h after context exposure to assess c-Fos expression. NA. NA. NA. Compared to baseline, S1 and S2 significantly reduced REM. Exposure to the fearful context reduced REM in VEH treated rats, whereas REM in ANT treated rats did not differ from baseline. ANT did not significantly alter freezing. Fear-induced c-Fos expression was decreased in PVN and LC after ANT compared to VEH. The results demonstrate that CRF receptors in CNA are involved in fear-induced reductions in REM and neural activation (as indicated by c-Fos) in stress and REM regulatory regions, but not in fear-induced freezing.

Stress and glucocorticoids regulated corticotropin releasing factor in rat prefrontal cortex

Corticotropin releasing factor (CRF) is considered as the central driving force in the stress response and plays a key role in the pathogenesis of depression. CRF neurons have been identified to locate in most regions of the prefrontal cortex (PFC), a brain region that is highly associated with the control of emotion and cognition. However, little is known on the regulation of CRF in this region. In this study, we aimed to identify the regulatory effect of acute restraint stress and glucocorticoid on PFC CRF and characterize the possible function of CRF in the PFC. We found that acute restraint stress increased and glucocorticoid decreased PFC CRF mRNA expression. The expression of glucocorticoid receptor (GR) was found to colocalize with CRF neurons in the PFC. In addition, recruitment of GR by the CRF promoter was observed in vivo. Specific attention was paid to the effect of CRF on CRF receptor 1 (CRFR1) expression in primary PFC cultures. The results showed that CRF increased CRFR1 expression through the MEK-ERK1/2 pathway. In summary, this study may contribute to the better understanding of CRF functions in the PFC.

Ghrelin stimulates corticotropin-releasing factor and vasopressin gene expression in rat hypothalamic 4B cells

Corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) play a central role in regulating the stress response. In response to stress, CRF and AVP neurons in the hypothalamic paraventricular nucleus secrete the peptides to stimulate the release of adrenocorticotropic hormone from the anterior pituitary. Ghrelin, an endogenous ligand of the growth hormone-releasing peptide receptors (GHSR), has been shown to stimulate the release of CRF and AVP by rat hypothalamic explants. However, little is known about the ability of the ghrelin signaling pathways to activate the CRF and AVP genes in the hypothalamus. In the present study, we examined the direct effect of ghrelin on CRF and AVP gene expression in hypothalamic 4B cells, which show the characteristics of the hypothalamic parvocellular paraventricular nucleus neurons. Cells were transfected with CRF or AVP promoter to examine the activity of each promoter. Ghrelin stimulated the promoter activities and mRNA levels for both CRF and AVP. The involvement of a protein kinase pathway was examined using inhibitors. Protein kinase A and phospholipase C pathways were shown to be involved in ghrelin-induced increases in both CRF and AVP promoter activities. GHSR type 1a (GHSR1a) mRNA levels were also increased by ghrelin, and these ghrelin-induced levels were suppressed by a GHSR1a antagonist. Thus, ghrelin-dependent pathways are involved in the regulation of CRF and AVP gene expression in the hypothalamus: ghrelin, an orexigenic hormone, stimulates CRF, an anorexigenic/anxiogenic factor in the hypothalamus, resulting in hypothalamic–pituitary–adrenal axis activation to stimulate the release of glucocorticoids.

Individual Differences in Reactivity to Social Stress Predict Susceptibility and Resilience to a Depressive Phenotype: Role of Corticotropin-Releasing Factor

Previous social stress exposure is a common risk factor for affective disorders. However, factors that determine vulnerability or resiliency to social stress-induced psychopathologies remain unclear. Using a rodent model of social stress, the present study was designed to identify putative neurobiological substrates that contribute to social stress-induced psychopathology and factors that influence or predict vulnerability. The resident-intruder model of defeat was used as a social stressor in adult male Sprague Dawley rats. The average latency to assume a subordinate posture (signaling defeat) over seven daily defeat exposures was calculated and examined with respect to endpoints of hypothalamic-pituitary-adrenal activity, components of the corticotropin-releasing factor (CRF) system, and behaviors that are relevant to human depression. In the present studies, a bimodal distribution emerged in an otherwise homogeneous population of Sprague Dawley rats such that 42% of rats exhibited short defeat latencies (<300 sec), whereas 58% of rats resisted defeat and exhibited longer latencies (>300 sec). These two phenotypes were associated with distinct endocrine and behavioral profiles as well as differences in components of the CRF system. Notably, the short-latency subpopulation exhibited hypothalamic-pituitary-adrenal dysregulation and behavior similar to that observed in melancholic depression. Examination of components of the CRF system suggested that proactive behavior in resisting defeat exhibited by long-latency rats was associated with decreased efficacy of CRF. Together, these data suggest that inherent differences in stress reactivity, perhaps as a result of differences in CRF regulation, may predict long-term consequences of social stress and vulnerability to depressive-like symptoms.

Corticotropin-releasing factor in the dorsal raphe nucleus: Linking stress coping and addiction

Addiction and stress are linked at multiple levels. Drug abuse is often initiated as a maladaptive mechanism for coping with stress. It is maintained in part by negative reinforcement to prevent the aversive consequences of stress associated with abstinence. Finally, stress is a major factor leading to relapse in subjects in which drug seeking behavior has extinguished. These associations imply overlapping or converging neural circuits and substrates that underlie the processes of addiction and the expression of the stress response. Here we discuss the major brain serotonin (5-HT) system, the dorsal raphe nucleus (DRN)-5-HT system as a point of convergence that links these processes and how the stress-related neuropeptide, corticotropin-releasing factor (CRF) directs this by a bimodal regulation of DRN neuronal activity. The review begins by describing a structural basis for CRF regulation of the DRN-5-HT system. This is followed by a review of the effects of CRF and stress on DRN function based on electrophysiological and microdialysis studies. The concept that multiple CRF receptor subtypes in the DRN facilitate distinct coping behaviors is reviewed with recent evidence for a unique cellular mechanism by which stress history can determine the type of coping behavior. Finally, work on CRF regulation of the DRN-5-HT system is integrated with literature on the role of 5-HT–dopamine interactions in addiction.

Regulation and role of suppressor of cytokine signaling-3 in hypothalamic 4B cells

Corticotropin-releasing factor (CRF) plays a central role in regulating stress responses. In the hypothalamic paraventricular nucleus (PVN), CRF, produced in response to stress, stimulates the release of ACTH from the anterior pituitary. ACTH then stimulates the release of glucocorticoids from the adrenal glands; circulating glucocorticoids are critical for recovery from stress conditions. Cytokines are also implicated in the regulation of CRF expression. Among them, interleukin (IL)-6 plays a role in the regulation of CRF. Factors other than glucocorticoids are likely to be involved in limiting the stimulation of CRF during stress. Suppressor of cytokine signaling (SOCS)-3 acts as a potent negative regulator of cytokine signaling. Little is known about the ability of the inhibitory signaling pathways to limit activation of the CRF gene in parvocellular PVN neurons. Hypothalamic 4B cells are useful for exploring the mechanisms, because these cells show characteristics of the parvocellular neurons of the PVN. In the present study, we examined whether SOCS-3 is regulated by IL-6 and cAMP in hypothalamic 4B cells. We also explored the involvement of SOCS-3 in the regulation of CRF gene expression. SOCS-3 was found to be regulated by IL-6 and via the cAMP/protein kinase A pathway in the hypothalamic cells. SOCS-3 knockdown increased IL-6- or forskolin-induced CRF gene transcription and mRNA levels. Therefore, SOCS-3, induced by a cAMP stimulant and IL-6, would be involved in the negative regulation of CRF gene expression in hypothalamic cells.

Fluoxetine inhibits corticotropin-releasing factor (CRF)-induced behavioural responses in rats

Corticotropin-releasing factor (CRF) is a potent neuromodulator of stress-related behaviour but the neural mechanisms underlying these effects are not clear. Studies were designed to test the hypothesis that CRF-induced behavioural arousal involves interactions with brainstem serotonergic systems. To examine interactions between CRF and serotonergic systems in the regulation of behaviour, CRF (1 μg, intracerebroventricular (i.c.v.)) or vehicle was infused in the presence or absence of the selective serotonin re-uptake inhibitor fluoxetine (0, 0.1, 1 or 10 mg/kg, intravenous (i.v.)). Fluoxetine was used at these doses because it is known to decrease serotonin cell firing rates while increasing extracellular serotonin concentrations in select forebrain regions. We then measured behavioural, neurochemical and endocrine responses. CRF increased locomotion and spontaneous non-ambulatory motor activity (SNAMA) in the home cages. Fluoxetine decreased tissue 5-hydroxyindoleacetic acid concentrations, a measure of serotonin metabolism, in specific limbic brain regions of CRF-treated rats (nucleus accumbens shell region, entorhinal cortex, central nucleus of the amygdala). Furthermore, fluoxetine inhibited CRF-induced SNAMA. CRF and fluoxetine independently increased plasma corticosterone concentrations, but the responses had distinct temporal profiles. Overall, these data are consistent with the hypothesis that CRF-induced facilitation of behavioural activity is dependent on brainstem serotonergic systems. Therefore, fluoxetine may attenuate or alleviate some behavioural responses to stress by interfering with CRF-induced responses.

CRF1 and CRF2 Receptors are Required for Potentiated Startle to Contextual but not Discrete Cues

Corticotropin-releasing factor (CRF) peptides and their receptors have crucial roles in behavioral and endocrine responses to stress. Dysregulation of CRF signaling has been linked to post-traumatic stress disorder, which is associated with increased startle reactivity in response to threat. Thus, understanding the mechanisms underlying CRF regulation of startle may identify pathways involved in this disorder. Here, we tested the hypothesis that both CRF1 and CRF2 receptors contribute to fear-induced increases in startle. Startle responses of wild type (WT) and mice with null mutations (knockout, KO) for CRF1 or CRF2 receptor genes were measured immediately after footshock (shock sensitization) or in the presence of cues previously associated with footshock (ie fear-potentiated startle, FPS). WT mice exhibited robust increases in startle immediately after footshock, which was dependent upon contextual cues. This effect was completely absent in CRF1 KO mice, and significantly attenuated in CRF2 KO mice. In contrast, CRF1 and CRF2 KO mice exhibited normal potentiation of startle by discrete conditioned cues. Blockade of both receptors via CRF1 receptor antagonist treatment in CRF2 KO mice also had no effect on FPS. These results support an additive model of CRF1 and CRF2 receptor activation effects on potentiated startle. These data also indicate that both CRF receptor subtypes contribute to contextual fear but are not required for discrete cued fear effects on startle reactivity. Thus, we suggest that either CRF1 or CRF2 could contribute to the increased startle observed in anxiety disorders with CRF system abnormalities.