Effect of Selective Lesions of Nucleus Accumbens µ-Opioid Receptor-Expressing Cells on Heroin Self-Administration in Male and Female Rats: A Study with Novel Oprm1-Cre Knock-in Rats.

The brain µ-opioid receptor (MOR) is critical for the analgesic, rewarding, and addictive effects of opioid drugs. However, in rat models of opioid-related behaviors, the circuit mechanisms of MOR-expressing cells are less known because of a lack of genetic tools to selectively manipulate them. We introduce a CRISPR-based Oprm1-Cre knock-in transgenic rat that provides cell type-specific genetic access to MOR-expressing cells. After performing anatomic and behavioral validation experiments, we used the Oprm1-Cre knock-in rats to study the involvement of NAc MOR-expressing cells in heroin self-administration in male and female rats. Using RNAscope, autoradiography, and FISH chain reaction (HCR-FISH), we found no differences in Oprm1 expression in NAc, dorsal striatum, and dorsal hippocampus, or MOR receptor density (except dorsal striatum) or function between Oprm1-Cre knock-in rats and wildtype littermates. HCR-FISH assay showed that iCre is highly coexpressed with Oprm1 (95%-98%). There were no genotype differences in pain responses, morphine analgesia and tolerance, heroin self-administration, and relapse-related behaviors. We used the Cre-dependent vector AAV1-EF1a-Flex-taCasp3-TEVP to lesion NAc MOR-expressing cells. We found that the lesions decreased acquisition of heroin self-administration in male Oprm1-Cre rats and had a stronger inhibitory effect on the effort to self-administer heroin in female Oprm1-Cre rats. The validation of an Oprm1-Cre knock-in rat enables new strategies for understanding the role of MOR-expressing cells in rat models of opioid addiction, pain-related behaviors, and other opioid-mediated functions. Our initial mechanistic study indicates that lesioning NAc MOR-expressing cells had different effects on heroin self-administration in male and female rats.SIGNIFICANCE STATEMENT The brain µ-opioid receptor (MOR) is critical for the analgesic, rewarding, and addictive effects of opioid drugs. However, in rat models of opioid-related behaviors, the circuit mechanisms of MOR-expressing cells are less known because of a lack of genetic tools to selectively manipulate them. We introduce a CRISPR-based Oprm1-Cre knock-in transgenic rat that provides cell type-specific genetic access to brain MOR-expressing cells. After performing anatomical and behavioral validation experiments, we used the Oprm1-Cre knock-in rats to show that lesioning NAc MOR-expressing cells had different effects on heroin self-administration in males and females. The new Oprm1-Cre rats can be used to study the role of brain MOR-expressing cells in animal models of opioid addiction, pain-related behaviors, and other opioid-mediated functions.

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.

Corticotropin-releasing hormone-binding protein and stress: from invertebrates to humans.

Corticotropin-releasing hormone (CRH) is a key regulator of the stress response. This peptide controls the hypothalamic-pituitary-adrenal (HPA) axis as well as a variety of behavioral and autonomic stress responses via the two CRH receptors, CRH-R1 and CRH-R2. The CRH system also includes an evolutionarily conserved CRH-binding protein (CRH-BP), a secreted glycoprotein that binds CRH with subnanomolar affinity to modulate CRH receptor activity. In this review, we discuss the current literature on CRH-BP and stress across multiple species, from insects to humans. We describe the regulation of CRH-BP in response to stress, as well as genetic mouse models that have been utilized to elucidate the in vivo role(s) of CRH-BP in modulating the stress response. Finally, the role of CRH-BP in the human stress response is examined, including single nucleotide polymorphisms in the human CRHBP gene that are associated with stress-related affective disorders and addiction. Lay summary The stress response is controlled by corticotropin-releasing hormone (CRH), acting via CRH receptors. However, the CRH system also includes a unique CRH-binding protein (CRH-BP) that binds CRH with an affinity greater than the CRH receptors. In this review, we discuss the role of this highly conserved CRH-BP in regulation of the CRH-mediated stress response from invertebrates to humans.

Brain CRF-binding protein modulates aspects of maternal behavior under stressful conditions and supports a hypo-anxious state in lactating rats.

Reduced corticotropin-releasing factor (CRF) receptor activation in the postpartum period is essential for adequate maternal behavior. One of the factors contributing to this hypo-activity might be the CRF-binding protein (CRF-BP), which likely reduces the availability of free extracellular CRF/urocortin 1. Here, we investigated behavioral effects of acute CRF-BP inhibition using 5µg of CRF(6-33) administered either centrally or locally within different parts of the bed nucleus of the stria terminalis (BNST) in lactating rats. Additionally, we assessed CRF-BP expression in the BNST comparing virgin and lactating rats. Central CRF-BP inhibition increased maternal aggression during maternal defense but did not affect maternal care or anxiety-related behavior. CRF-BP inhibition in the medial-posterior BNST had no effect on maternal care under non-stress conditions but impaired the reinstatement of maternal care following stressor exposure. Furthermore, maternal aggression, particularly threat behavior, and anxiety-related behavior were elevated by CRF-BP inhibition in the medial-posterior BNST. In the anterior-dorsal BNST, CRF-BP inhibition increased only non-maternal behaviors following stress. Finally, CRF-BP expression was higher in the anterior compared to the posterior BNST but was not different between virgin and lactating rats in either region. Our study demonstrates a key role of the CRF-BP, particularly within the BNST, in modulating CRF's impact on maternal behavior. The CRF-BP is important for the reinstatement of maternal care after stress, for modulating threat behavior during an aggressive encounter and for maintaining a hypo-anxious state during lactation. Thus, the CRF-BP likely contributes to the postpartum-associated down-regulation of the CRF system in a brain region-dependent manner.

Binge Drinking Decreases Corticotropin-Releasing Factor-Binding Protein Expression in the Medial Prefrontal Cortex of Mice.

BACKGROUND: Dysregulation of the corticotropin-releasing factor (CRF) system has been observed in rodent models of binge drinking, with a large focus on CRF receptor 1 (CRF-R1). The role of CRF-binding protein (CRF-BP), a key regulator of CRF activity, in binge drinking is less well understood. In humans, single-nucleotide polymorphisms in CRHBP are associated with alcohol use disorder and stress-induced alcohol craving, suggesting a role for CRF-BP in vulnerability to alcohol addiction. METHODS: The role and regulation of CRF-BP in binge drinking were examined in mice exposed to the drinking in the dark (DID) paradigm. Using in situ hybridization, the regulation of CRF-BP, CRF-R1, and CRF mRNA expression was determined in the stress and reward systems of C57BL/6J mice after repeated cycles of DID. To determine the functional role of CRF-BP in binge drinking, CRF-BP knockout (CRF-BP KO) mice were exposed to 6 cycles of DID, during which alcohol consumption was measured and compared to wild-type mice. RESULTS: CRF-BP mRNA expression was significantly decreased in the prelimbic (PL) and infralimbic medial prefrontal cortex (mPFC) of C57BL/6J mice after 3 cycles and in the PL mPFC after 6 cycles of DID. No significant changes in CRF or CRF-R1 mRNA levels were observed in mPFC, ventral tegmental area, bed nucleus of the stria terminalis, or amygdala after 3 cycles of DID. CRF-BP KO mice do not show significant alterations in drinking compared to wild-type mice across 6 cycles of DID. CONCLUSIONS: These results reveal that repeated cycles of binge drinking alter CRF-BP mRNA expression in the mPFC, a region responsible for executive function and regulation of emotion and behavior, including responses to stress. We observed a persistent decrease in CRF-BP mRNA expression in the mPFC after 3 and 6 DID cycles, which may allow for increased CRF signaling at CRF-R1 and contribute to excessive binge-like ethanol consumption.

Conversion of short-term to long-term memory in the novel object recognition paradigm.

It is well-known that stress can significantly impact learning; however, whether this effect facilitates or impairs the resultant memory depends on the characteristics of the stressor. Investigation of these dynamics can be confounded by the role of the stressor in motivating performance in a task. Positing a cohesive model of the effect of stress on learning and memory necessitates elucidating the consequences of stressful stimuli independently from task-specific functions. Therefore, the goal of this study was to examine the effect of manipulating a task-independent stressor (elevated light level) on short-term and long-term memory in the novel object recognition paradigm. Short-term memory was elicited in both low light and high light conditions, but long-term memory specifically required high light conditions during the acquisition phase (familiarization trial) and was independent of the light level during retrieval (test trial). Additionally, long-term memory appeared to be independent of stress-mediated glucocorticoid release, as both low and high light produced similar levels of plasma corticosterone, which further did not correlate with subsequent memory performance. Finally, both short-term and long-term memory showed no savings between repeated experiments suggesting that this novel object recognition paradigm may be useful for longitudinal studies, particularly when investigating treatments to stabilize or enhance weak memories in neurodegenerative diseases or during age-related cognitive decline.

Long-term alcohol consumption alters dorsal striatal dopamine release and regulation by D2 dopamine receptors in rhesus macaques.

The dorsal striatum (DS) is implicated in behavioral and neural processes including action control and reinforcement. Alcohol alters these processes in rodents, and it is believed that the development of alcohol use disorder involves changes in DS dopamine signaling. In nonhuman primates, the DS can be divided into caudate and putamen subregions. As part of a collaborative effort examining the effects of long-term alcohol self-administration in rhesus macaques, we examined DS dopamine signaling using fast-scan cyclic voltammetry. We found that chronic alcohol self-administration resulted in several dopamine system adaptations. Most notably, dopamine release was altered in a sex- and region-dependent manner. Following long-term alcohol consumption, male macaques, regardless of abstinence status, had reduced dopamine release in putamen, while only male macaques in abstinence had reduced dopamine release in caudate. In contrast, female macaques had enhanced dopamine release in the caudate, but not putamen. Dopamine uptake was also enhanced in females, but not males (regardless of abstinence state). We also found that dopamine D2/3 autoreceptor function was reduced in male, but not female, alcohol drinkers relative to control groups. Finally, we found that blockade of nicotinic acetylcholine receptors inhibited evoked dopamine release in nonhuman primates. Altogether, our findings demonstrate that long-term alcohol consumption can sex-dependently alter dopamine release, as well as its feedback control mechanisms in both DS subregions.

Novel expression of type 1 corticotropin-releasing hormone receptor in multiple endocrine cell types in the murine anterior pituitary.

The CRH family of ligands signals via two distinct receptors, CRH-R1 and CRH-R2. Previous studies localized CRH-R1 and CRH-R2 to a subset of anterior pituitary corticotropes and gonadotropes, respectively. However, numerous studies have indicated that stress and CRH activity can alter the secretion of multiple anterior pituitary hormones, suggesting a broader expression of the CRH receptors in pituitary. To examine this hypothesis, the in vivo expression of CRH-R1 and CRH-R2 mRNA was further characterized in adult mouse pituitary. Quantitative RT-PCR analysis demonstrated that CRH-R1 mRNA is greater than 100-fold more abundant than CRH-R2 mRNA in male and female mouse pituitaries. Dual in situ hybridization analysis identified cell-specific CRH-R1 expression in the anterior pituitary. At least half of the CRH-R1-positive cells expressed proopiomelanocortin-mRNA (50% in females; 70% in males). In females, a significant percentage of the cells expressing CRH-R1 also expressed transcript for prolactin (40%), LHbeta (10%), or TSH (3%), all novel sites of CRH-R1 expression. Similarly in males, a percentage of CRH-R1-positive cells expressed prolactin (12%), LHbeta (13%), and TSH (5%). RT-PCR studies with immortalized murine anterior pituitary cell lines showed CRH-R1 and/or CRH-R2 expression in corticotropes (AtT-20 cells), gonadotropes (alphaT3-1 and LbetaT2 cells), and thyrotropes (alphaTSH cells). Whereas CRH-R1 expression in corticotropes is well established, the presence of CRH-R1 mRNA in a subset of lactotropes, gonadotropes, and thyrotropes establishes these cell types as novel sites of murine CRH-R1 expression and highlights the pituitary as an important site of interaction between the hypothalamus-pituitary-adrenal and multiple endocrine axes.

Overexpressing the glucocorticoid receptor in forebrain causes an aging-like neuroendocrine phenotype and mild cognitive dysfunction.

Repeated stress enhances vulnerability to neural dysfunction that is cumulative over the course of the lifespan. This dysfunction contributes to cognitive deficits observed during aging. In addition, aging is associated with dysregulation of the limbic-hypothalamic-pituitary-adrenal (LHPA) axis, leading to a delayed termination of the stress response. This delay, in turn, increases exposure to glucocorticoids and exacerbates the likelihood of neural damage. Here we asked whether similar effects could emerge at an early age as a result of genetic variations in the level or function of the brain glucocorticoid receptor (GR). We investigated the effect of forebrain-specific overexpression of GR on LHPA axis activity. Transgenic mice with GR overexpression in forebrain (GRov) display normal basal circulating adrenocorticotropic hormone and corticosterone levels. However, young GRov mice exhibit a number of LHPA alterations, including a blunted initial response to acute restraint stress followed by a delayed turn-off of the stress response. This deficit in negative feedback is paradoxical in the face of elevated GR levels, resembles the stress response in aged animals, and continues to worsen as GRov mice age. The neuroendocrine dysregulation in young GRov mice is coupled with a mild cognitive deficit, also consistent with the accelerated aging hypothesis. The molecular basis of this phenotype was examined using microarray analysis of the hippocampus, which revealed a broad downregulation of glutamate receptor signaling in GRov mice. Thus, even in the absence of chronic stress, elevation of GR gene expression can lead to an increased allostatic load and result in an "aging-like" phenotype in young animals.

Maternal stress and the MPOA: Activation of CRF receptor 1 impairs maternal behavior and triggers local oxytocin release in lactating rats.

Maternal behavior and anxiety are potently modulated by the brain corticotropin-releasing factor (CRF) system postpartum. Downregulation of CRF in limbic brain regions is essential for appropriate maternal behavior and an adaptive anxiety response. Here, we focus our attention on arguably the most important brain region for maternal behavior, the hypothalamic medial preoptic area (MPOA). Within the MPOA, mRNA for CRF receptor subtype 1 (protein: CRFR1, gene: Crhr1) was more abundantly expressed than for subtype 2 (protein: CRFR2, gene: Crhr2), however expression of Crhr1, Crhr2 and CRF-binding protein (protein: CRFBP, gene: Crhbp) mRNA was similar between virgin and lactating rats. Subtype-specific activation of CRFR, predominantly CRFR1, in the MPOA decreased arched back nursing and total nursing under non-stress conditions. Following acute stressor exposure, only CRFR1 inhibition rescued the stress-induced reduction in arched back nursing while CRFR1 activation prolonged the decline in nursing. Furthermore, inhibition of CRFR1 strongly increased maternal aggression in the maternal defense test. CRFR1 activation had anxiogenic actions and reduced locomotion on the elevated plus-maze, however neither CRFR1 nor R2 manipulation affected maternal motivation. In addition, activation of CRFR1, either centrally or locally in the MPOA, increased local oxytocin release. Finally, inhibition of CRFBP (a potent regulator of CRFR activity) in the MPOA did not affect any of the maternal parameters investigated. In conclusion, activity of CRFR in the MPOA, particularly of subtype 1, needs to be dampened during lactation to ensure appropriate maternal behavior. Furthermore, oxytocin release in the MPOA may provide a regulatory mechanism to counteract the negative impact of CRFR activation on maternal behavior.

Mouse models for psychiatric disorders.

Genes involved in psychiatric disorders are difficult to identify, and those that have been proposed so far remain ambiguous. As it is unrealistic to expect the development of, say, a 'schizophrenic' or 'autistic' mouse, mice are unlikely to have the same role in gene identification in psychiatry as circling mice did in the discovery of human deafness genes. However, many psychiatric disorders are associated with intermediate phenotypes that can be modeled and studied in mice, including physiological or anatomical brain changes and behavioral traits. Mouse models help to evaluate the effect of a human candidate gene mutation on an intermediate trait, and to identify new candidate genes. Once a gene or pathway has been identified, mice are also used to study the interplay of different genes in that system.

Cell Type-Specific Expression of Corticotropin-Releasing Hormone-Binding Protein in GABAergic Interneurons in the Prefrontal Cortex.

Corticotropin-releasing hormone-binding protein (CRH-BP) is a secreted glycoprotein that binds CRH with very high affinity to modulate CRH receptor activity. CRH-BP is widely expressed throughout the brain, with particularly high expression in regions such as the amygdala, hippocampus, ventral tegmental area and prefrontal cortex (PFC). Recent studies suggest a role for CRH-BP in stress-related psychiatric disorders and addiction, with the PFC being a potential site of interest. However, the molecular phenotype of CRH-BP-expressing cells in this region has not been well-characterized. In the current study, we sought to determine the cell type-specific expression of CRH-BP in the PFC to begin to define the neural circuits in which this key regulator is acting. To characterize the expression of CRH-BP in excitatory and/or inhibitory neurons, we utilized dual in situ hybridization to examine the cellular colocalization of CRH-BP mRNA with vesicular glutamate transporter (VGLUT) or glutamic acid decarboxylase (GAD) mRNA in different subregions of the PFC. We show that CRH-BP is expressed predominantly in GABAergic interneurons of the PFC, as revealed by the high degree of colocalization (>85%) between CRH-BP and GAD. To further characterize the expression of CRH-BP in this heterogenous group of inhibitory neurons, we examined the colocalization of CRH-BP with various molecular markers of GABAergic interneurons, including parvalbumin (PV), somatostatin (SST), vasoactive intestinal peptide (VIP) and cholecystokinin (CCK). We demonstrate that CRH-BP is colocalized predominantly with SST in the PFC, with lower levels of colocalization in PV- and CCK-expressing neurons. Our results provide a more comprehensive characterization of the cell type-specific expression of CRH-BP and begin to define its potential role within circuits of the PFC. These results will serve as the basis for future in vivo studies to manipulate CRH-BP in a cell type-specific manner to better understand its role in stress-related psychiatric disorders, including anxiety, depression and addiction.

Corticotropin-releasing factor is cytoprotective in Xenopus tadpole tail: coordination of ligand, receptor, and binding protein in tail muscle cell survival.

Upon metamorphosis, amphibian tadpoles lose their tails through programmed cell death induced by thyroid hormone (T3). Before transformation, the tail functions as an essential locomotory organ. The binding protein for the stress neuropeptide corticotropin-releasing factor (CRF; CRF-BP) is strongly up-regulated in the tail of Xenopus tadpoles during spontaneous or T3-induced metamorphosis. This finding led us to investigate physiological roles for CRF and CRF-BP in tadpole tail. We found CRF, CRF-BP, and functional CRF1 receptor in tail and CRF and functional CRF1 receptors, but not CRF-BP, in the tail muscle-derived cell line XLT-15. CRF, acting via the CRF1 receptor, slowed spontaneous tail regression in explant culture and caused a reduction in caspase 3/7 activity. CRF increased, but stable CRF-BP overexpression decreased, [3H]thymidine incorporation in XLT-15 cells. Overexpression of CRF-BP in vivo accelerated the loss of tail muscle cells during spontaneous metamorphosis. Lastly, exposure of tail explants to hypoxia increased CRF and urocortin 1 but strongly decreased CRF-BP mRNA expression. We show that CRF is expressed in tadpole tail, is up-regulated by environmental stressors, and is cytoprotective. The inhibitory binding protein for CRF is regulated by hormones or by environmental stressors and can modulate CRF bioactivity.

CRH-BP: the regulation and function of a phylogenetically conserved binding protein.

Corticotropin Releasing Hormone-Binding Protein (CRH-BP), a 37 kDa secreted glycoprotein, binds both CRH and urocortin with high affinity and is structurally unrelated to the CRH receptors. CRH-BP orthologues have been identified in multiple invertebrate and vertebrate species. It is strongly conserved throughout evolution, suggesting the maintenance of a structural conformation necessary for biological activity. CRH-BP is an important modulator of CRH activity; it inhibits CRH-induced ACTH secretion from pituitary corticotropes and may exert similar actions at central sites of CRH release. While the function of CRH-BP is thought to be primarily inhibitory, recent studies indicate that novel functional roles may exist in both the brain and pituitary. Regulation of CRH-BP expression by stress and metabolic factors are consistent with in vivo models of altered CRH-BP expression. Positive regulation of pituitary CRH-BP by reproductive hormones suggests that additional interactions between the stress and reproductive axes may exist. While recent research has focused on the evolutionary conservation, expanded sites of expression, regulation and in vivo function of CRH-BP, a more complete understanding of the central and peripheral functions of CRH-BP and its mechanisms of action will help elucidate its potential role in the etiology or treatment of disorders of CRH dysregulation.

Gonadotropin-releasing hormone (GnRH) positively regulates corticotropin-releasing hormone-binding protein expression via multiple intracellular signaling pathways and a multipartite GnRH response element in alphaT3-1 cells.

CRH-binding protein (CRH-BP) binds CRH with high affinity and inhibits CRH-mediated ACTH release from anterior pituitary corticotrope-like cells in vitro. In female mouse pituitary, CRH-BP is localized not only in corticotropes, but is also expressed in gonadotropes and lactotropes. To investigate the functional significance of gonadotrope CRH-BP, we examined the molecular mechanisms underlying GnRH-regulated CRH-BP expression in alphaT3-1 gonadotrope-like cells. CRH-BP is endogenously expressed in alphaT3-1 cells, and quantitative real-time RT-PCR and ribonuclease protection assays demonstrate that GnRH induces a 3.7-fold increase in CRH-BP mRNA levels. GnRH also induces intracellular CRH-BP (2.0-fold) and secreted CRH-BP (5.3-fold) levels, as measured by [125I]CRH:CRH-BP chemical cross-linking. Transient transfection assays using CRH-BP promoter-luciferase constructs indicate that GnRH regulation involves protein kinase C-, ERK- and calcium-dependent signaling pathways and is mediated via a multipartite GnRH response element that includes activator protein 1 and cAMP response element (CRE) sites. The CRE site significantly contributes to GnRH responsiveness, independent of protein kinase A, representing a unique form of multipartite GnRH regulation in alphaT3-1 cells. Furthermore, EMSAs indicate that alphaT3-1 nuclear proteins specifically bind at activator protein 1 and CRE sites. These data demonstrate novel regulation of pituitary CRH-BP, highlighting the importance of the pituitary gonadotrope as a potential interface between the stress and reproductive axes.

Estrogen receptor (ER)-mediated transcriptional regulation of the human corticotropin-releasing hormone-binding protein promoter: differential effects of ERalpha and ERbeta.

CRH-binding protein (CRH-BP) regulates activation of the hypothalamic-pituitary-adrenal (HPA) axis by binding and inhibiting CRH. We investigated for the first time transcriptional regulation of the human CRH-BP promoter using transient transfections. Estrogen receptors (ERs) contributed to ligand-independent constitutive activation of the promoter, whereas in the presence of estradiol ERalpha induced and ERbeta repressed promoter activity in a dose-dependent manner. TNFalpha inhibited promoter induction by ERalpha in the absence and presence of estradiol. Three ERE half-sites in the CRH-BP promoter bound ERalpha and ERbeta in an EMSA, and disruption of ERE half-sites by site-directed mutagenesis abolished ligand-independent induction by ERalpha and ERbeta and promoter enhancement by estradiol-activated ERalpha. Repression by estradiol/ERbeta was unaffected by disruption of ERE half-sites, activating protein 1, cAMP response element, GATA, or nuclear factor kappaB sites, and reversed to promoter induction by estrogen antagonists, tamoxifen and ICI 182,780, suggesting corepressor involvement. In hypothalamic GT1-7 cells, Western blotting demonstrated rapid induction of endogenous CRH-BP expression by estradiol-bound ER, which was inhibited by TNFalpha. We propose a model in which ERs maintain basal CRH-BP expression in pituitary and neurosecretory cells, whereas in the presence of ERalpha estrogen enhances CRH-BP transcription, causing down-regulation of the HPA axis, and nuclear factor kappaB-activating cytokines activate the HPA axis by inhibiting ERalpha.

Pituitary CRH-binding protein and stress in female mice.

The CRH-binding protein (CRH-BP) binds CRH with very high affinity and inhibits CRH-mediated ACTH release from anterior pituitary cells in vitro, suggesting that the CRH-BP functions as a negative regulator of CRH activity. Our previous studies have demonstrated sexually dimorphic expression of CRH-BP in the murine pituitary. Basal CRH-BP expression is higher in the female pituitary, where CRH-BP mRNA is detected in multiple anterior pituitary cell types. In this study, we examined stress-induced changes in CRH-BP mRNA and protein expression in mouse pituitary and assessed the in vivo role of CRH-BP in modulating the stress response. Pituitary CRH-BP mRNA was greater than 200-fold more abundant in females than males, and restraint stress increased pituitary CRH-BP mRNA by 11.8-fold in females and 3.2-fold in males as assessed by qRT-PCR. In females, restraint stress increased CRH-BP mRNA levels not only in POMC-expressing cells, but also in PRL-expressing cells. The increase in female pituitary CRH-BP mRNA following stress resulted in significant increases in CRH-BP protein 4-6h after a 30-minute restraint stress as detected by [(125)I]-CRH:CRH-BP cross-linking analyses. Based on this temporal profile, the physiological role of CRH-BP was assessed using a stressor of longer duration. In lipopolysaccharide (LPS) stress studies, female CRH-BP-deficient mice showed elevated levels of stress-induced corticosterone release as compared to wild-type littermates. These studies demonstrate a role for the pituitary CRH-BP in attenuating the HPA response to stress in female mice.

Sexually dimorphic expression of corticotropin-releasing hormone-binding protein in the mouse pituitary.

In the pituitary, CRH-binding protein (CRH-BP) neutralizes the ACTH-releasing activity of CRH. Because sexual dimorphisms exist at multiple levels of the hypothalamic-pituitary-adrenal axis, these studies examined expression of pituitary CRH-BP in the male and female mouse pituitary. Ribonuclease protection assays and (125)I-CRH cross-linking assays demonstrate greater expression of pituitary CRH-BP in female than male mice. Normalized CRH-BP mRNA levels in female mice are 2.58 times greater at proestrus than diestrus. Ovariectomy reduces pituitary CRH-BP mRNA levels to 11% of sham-ovariectomy control levels, and estradiol benzoate treatment restores CRH-BP mRNA to control levels. These data suggest that estrogen positively regulates pituitary CRH-BP. Dual in situ hybridization analysis reveals that CRH-BP expression increases significantly in proopiomelanocortin-expressing cells at proestrus, compared with metestrus (P = 0.003), suggesting that CRH-BP expression is estrogen regulated in corticotropes. Further studies reveal that approximately 80% of the CRH-BP transcripts in the proestrus mouse pituitary localize to prolactin-expressing cells, a novel site for CRH-BP expression. CRH-BP mRNA levels increase significantly at proestrus, compared with metestrus in prolactin-positive cells (P < 0.0001). This robust, estrogen-regulated expression of CRH-BP in lactotropes in female mice suggests that the pituitary is an important site for interactions between the hypothalamic-pituitary-adrenal axis and other endocrine systems.

Glucocorticoid and mineralocorticoid receptor expression in the human hippocampus in major depressive disorder.

Approximately 50% of mood disorder patients exhibit hypercortisolism. Cortisol normally exerts its functions in the CNS via binding to mineralocorticoid receptors (MR) and glucocorticoid receptors (GR). Both MR and GR are highly expressed in human hippocampus and several studies have suggested that alterations in the levels of MR or GR within this region may contribute to the dysregulation in major depressive disorder (MDD). Studies have also shown functional heterogeneity across the hippocampus, with posterior hippocampus preferentially involved in cognitive processes and anterior hippocampus involved in stress, emotion and affect. We therefore hypothesize that GR and MR expression in hippocampus of control and MDD patients may vary not only with disease, but also with regional specificity along the anterior/posterior axis. Student's t-test analysis showed decreased expression of MR in the MDD group compared to controls in the anterior, but not the posterior hippocampus, with no significant changes in GR. Linear regression analysis showed a marked difference in MR:GR correlation between suicide and non-suicide patients in the posterior hippocampus. Our findings are consistent with previous reports of hippocampal corticosteroid receptor dysregulation in mood disorders, but extend those findings by analysis across the anterior/posterior axis of the hippocampus. A decrease in MR in the anterior but not posterior hippocampus of MDD patients emphasizes the important functional role of the anterior hippocampus in neuroendocrine regulation in humans.