RESUMO
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.
Assuntos
Dopamina , Receptores de Dopamina D2 , Consumo de Bebidas Alcoólicas , Animais , Corpo Estriado/metabolismo , Feminino , Macaca mulatta/metabolismo , Masculino , Putamen/metabolismo , Receptores de Dopamina D2/metabolismoRESUMO
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.
Assuntos
Cocaína/administração & dosagem , Comportamento de Procura de Droga/fisiologia , Córtex Pré-Frontal/fisiologia , Receptores de Hormônio Liberador da Corticotropina/biossíntese , Estresse Psicológico/metabolismo , Área Tegmentar Ventral/fisiologia , Animais , Condicionamento Operante/fisiologia , Comportamento de Procura de Droga/efeitos dos fármacos , Masculino , Vias Neurais/química , Vias Neurais/fisiologia , Córtex Pré-Frontal/química , Ratos , Ratos Sprague-Dawley , Receptores de Hormônio Liberador da Corticotropina/genética , Autoadministração , Estresse Psicológico/psicologia , Área Tegmentar Ventral/químicaRESUMO
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.
Assuntos
Regulação da Expressão Gênica/fisiologia , Lactação/fisiologia , Comportamento Materno/fisiologia , Ocitocina/metabolismo , Área Pré-Óptica/metabolismo , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Análise de Variância , Animais , Animais Recém-Nascidos , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Hormônio Liberador da Corticotropina/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Antagonistas de Hormônios/farmacologia , Masculino , Comportamento Materno/psicologia , Aprendizagem em Labirinto/fisiologia , Microdiálise , Área Pré-Óptica/diagnóstico por imagem , Pirimidinas/farmacologia , Pirróis/farmacologia , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Hormônio Liberador da Corticotropina/agonistas , Receptores de Hormônio Liberador da Corticotropina/antagonistas & inibidoresRESUMO
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.
RESUMO
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.
Assuntos
Proteínas de Transporte/genética , Hormônio Liberador da Corticotropina/metabolismo , Sistema Hipotálamo-Hipofisário/metabolismo , Sistema Hipófise-Suprarrenal/metabolismo , Receptores de Hormônio Liberador da Corticotropina/genética , Estresse Psicológico/metabolismo , Animais , Abelhas , Proteínas de Transporte/metabolismo , Copépodes , Peixes , Humanos , Invertebrados , Camundongos , Receptores de Hormônio Liberador da Corticotropina/metabolismoRESUMO
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.
Assuntos
Consumo Excessivo de Bebidas Alcoólicas/metabolismo , Proteínas de Transporte/biossíntese , Córtex Pré-Frontal/metabolismo , Animais , Consumo Excessivo de Bebidas Alcoólicas/genética , Proteínas de Transporte/genética , Hormônio Liberador da Corticotropina/biossíntese , Hormônio Liberador da Corticotropina/genética , Etanol/administração & dosagem , Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Córtex Pré-Frontal/efeitos dos fármacos , Receptores de Hormônio Liberador da Corticotropina/biossíntese , Receptores de Hormônio Liberador da Corticotropina/genéticaRESUMO
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.
Assuntos
Encéfalo/efeitos dos fármacos , Proteínas de Transporte/antagonistas & inibidores , Hormônio Liberador da Corticotropina/farmacologia , Comportamento Materno/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Agressão/efeitos dos fármacos , Agressão/fisiologia , Animais , Ansiedade/metabolismo , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Proteínas de Transporte/metabolismo , Regulação para Baixo/efeitos dos fármacos , Feminino , Lactação/efeitos dos fármacos , Comportamento Materno/fisiologia , Ratos , Receptores de Hormônio Liberador da Corticotropina/metabolismoRESUMO
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.
Assuntos
Proteínas de Transporte/metabolismo , Regulação da Expressão Gênica/fisiologia , Hipófise/metabolismo , Caracteres Sexuais , Estresse Fisiológico/fisiologia , Hormônio Adrenocorticotrópico/sangue , Animais , Proteínas de Transporte/genética , Corticosterona/sangue , Hormônio Liberador da Corticotropina/metabolismo , Feminino , Isótopos de Iodo/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Hipófise/citologia , RNA Mensageiro/metabolismo , Fatores de TempoRESUMO
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.
Assuntos
Memória de Longo Prazo/fisiologia , Memória de Curto Prazo/fisiologia , Reconhecimento Psicológico/fisiologia , Estresse Fisiológico , Animais , Corticosterona/sangue , Masculino , CamundongosRESUMO
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.
Assuntos
Transtorno Depressivo Maior/patologia , Hipocampo/metabolismo , Receptores de Glucocorticoides/metabolismo , Receptores de Mineralocorticoides/metabolismo , Adulto , Idoso , Hipocampo/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Mudanças Depois da Morte , Análise de Regressão , Adulto JovemRESUMO
BACKGROUND: Genetic factors and early-life adversity are critical in the etiology of mood disorders and substance abuse. Because of their role in the transduction of stress responses, glucocorticoid hormones and their receptors could serve as both genetic factors and mediators of environmental influences. We have shown that constitutive overexpression of the glucocorticoid receptor (GR) in forebrain results in increased emotional reactivity and lability in mice. Here, we asked whether there was a critical period for the emergence of this phenotype. METHODS: We generated a mouse line with inducible GR overexpression specifically in forebrain. Anxiety-like behaviors and cocaine-induced sensitization were assessed in adult mice following GR overexpression during different periods in development. The molecular basis of the behavioral phenotype was examined using microarray analyses of dentate gyrus and nucleus accumbens. RESULTS: Transient overexpression of GR during early life led to increased anxiety and cocaine sensitization, paralleling the phenotype of lifelong GR overexpression. This increased emotional reactivity was not observed when GR overexpression was induced after weaning. Glucocorticoid receptor overexpression in early life is sufficient to alter gene expression patterns for the rest of the animal's life, with dentate gyrus being more responsive than nucleus accumbens. The altered transcripts are implicated in GR and axonal guidance signaling in dentate gyrus and dopamine receptor signaling in nucleus accumbens. CONCLUSIONS: Transient overexpression of GR early in life is both necessary and sufficient for inducing transcriptome-wide changes in the brain and producing a lifelong increase in vulnerability to anxiety and drugs of abuse.
Assuntos
Ansiedade/fisiopatologia , Cocaína/farmacologia , Período Crítico Psicológico , Prosencéfalo/metabolismo , Prosencéfalo/fisiopatologia , Receptores de Glucocorticoides/biossíntese , Animais , Ansiedade/genética , Giro Denteado/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Masculino , Camundongos , Camundongos Transgênicos , Núcleo Accumbens/metabolismo , Receptores de Glucocorticoides/genética , Transcriptoma/fisiologiaRESUMO
Leptin acts on leptin receptor (LepRb)-expressing neurons throughout the brain, but the roles for many populations of LepRb neurons in modulating energy balance and behavior remain unclear. We found that the majority of LepRb neurons in the lateral hypothalamic area (LHA) contain neurotensin (Nts). To investigate the physiologic role for leptin action via these LepRb(Nts) neurons, we generated mice null for LepRb specifically in Nts neurons (Nts-LepRbKO mice). Nts-LepRbKO mice demonstrate early-onset obesity, modestly increased feeding, and decreased locomotor activity. Furthermore, consistent with the connection of LepRb(Nts) neurons with local orexin (OX) neurons and the ventral tegmental area (VTA), Nts-LepRbKO mice exhibit altered regulation of OX neurons and the mesolimbic DA system. Thus, LHA LepRb(Nts) neurons mediate physiologic leptin action on OX neurons and the mesolimbic DA system, and contribute importantly to the control of energy balance.
Assuntos
Região Hipotalâmica Lateral/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leptina , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Neurotensina/metabolismo , Obesidade/metabolismo , Receptores para Leptina/deficiência , Área Tegmentar Ventral/metabolismo , Animais , Dopamina/metabolismo , Eletrofisiologia , Metabolismo Energético , Expressão Gênica , Técnicas de Silenciamento de Genes , Região Hipotalâmica Lateral/citologia , Região Hipotalâmica Lateral/efeitos dos fármacos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Leptina/metabolismo , Leptina/farmacologia , Masculino , Camundongos , Camundongos Transgênicos , Microtomia , Atividade Motora/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neuropeptídeos/genética , Neurotensina/genética , Obesidade/patologia , Orexinas , Receptores para Leptina/genética , Área Tegmentar Ventral/citologia , Área Tegmentar Ventral/efeitos dos fármacosRESUMO
CRH directs the physiological and behavioral responses to stress. Its activity is mediated by CRH receptors (CRH-R) 1 and 2 and modulated by the CRH-binding protein. Aberrant regulation of this system has been associated with anxiety disorders and major depression, demonstrating the importance of understanding the regulation of CRH activity. An mRNA splice variant of CRH-R2alpha (sCRH-R2alpha) was recently identified that encodes the receptor's ligand-binding extracellular domain but terminates before the transmembrane domains. It was therefore predicted to serve as a secreted decoy receptor, mimicking the ability of CRH-binding protein to sequester free CRH. Although the splice variant contains a premature termination codon, predicting its degradation by nonsense-mediated RNA decay, cycloheximide experiments and polysome profiles demonstrated that sCRH-R2alpha mRNA escaped this regulation and was efficiently translated. However, the resulting protein was unable to serve as a decoy receptor because it failed to traffic for secretion because of an ineffective signal peptide and was ultimately subjected to proteosomal degradation. Several other truncated splice variants of G protein-coupled transmembrane receptors regulate the amount of full-length receptor expression through dimerization and misrouting; however, receptor binding assays and immunofluorescence of cells cotransfected with sCRH-R2alpha and CRH-R2alpha or CRH-R1 indicated that sCRH-R2alpha protein does not alter trafficking or binding of full-length CRH-R. Although sCRH-R2alpha protein does not appear to function as an intracellular or extracellular decoy receptor, the regulated unproductive splicing of CRH-R2alpha pre-mRNA to sCRH-R2alpha may selectively alter the cellular levels of full-length CRH-R2alpha mRNA and hence functional CRH-R2alpha receptor levels.
Assuntos
Receptores de Hormônio Liberador da Corticotropina/genética , Via Secretória/fisiologia , Processamento Alternativo , Animais , Encéfalo/metabolismo , Linhagem Celular , Códon sem Sentido/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Polirribossomos/metabolismo , Transporte Proteico , RNA Mensageiro/metabolismoRESUMO
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.
Assuntos
Adeno-Hipófise/fisiologia , Receptores de Hormônio Liberador da Corticotropina/genética , Animais , Linhagem Celular , Primers do DNA , Feminino , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Adeno-Hipófise/citologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Corticotropin-releasing hormone (CRH) is a key regulator of the mammalian stress response, mediating a wide variety of stress-associated behaviors including stress-induced inhibition of reproductive function. To investigate the potential direct action of CRH on pituitary gonadotrope function, we examined CRH receptor expression and second messenger signaling in alpha T3-1 cells, a murine gonadotrope-like cell line. Reverse transcriptase PCR (RT-PCR) studies demonstrated that alpha T3-1 cells express mRNA for the two CRH receptor subtypes, CRHR1 and CRHR2, with CRHR2alpha as the predominant CRHR2 isoform. Stimulation of the cells with CRH or urocortin (UCN) resulted in rapid, transient increases in the intracellular levels of cAMP that were completely blocked by the addition of alpha-helical CRH 9-41 or astressin, non-selective CRH receptor antagonists. Stimulation of the cells with CRHR2-specific ligands, urocortin 2 (UCN2) or urocortin 3 (UCN3), resulted in rapid increases in intracellular cAMP levels to 50-60% of the levels observed with UCN. Treatment with a selective CRHR2 antagonist, antisauvagine, completely blocked UCN3-mediated increases in cAMP and significantly reduced, but did not completely block UCN-mediated increases in cAMP, demonstrating that both CRHR1 and CRHR2 are functionally active in these gonadotrope-like cells. Finally, UCN treatment significantly increased the transcriptional activity of the glycoprotein hormone alpha-subunit promoter as assessed by alpha-luciferase transfection assays. Together, these results demonstrate the functional signaling of CRH receptors in alpha T3-1 cells, suggesting that CRH may also modulate pituitary gonadotrope function in vivo.
Assuntos
Hormônio Liberador da Corticotropina/biossíntese , AMP Cíclico/biossíntese , Gonadotrofos/metabolismo , Receptores de Hormônio Liberador da Corticotropina/genética , Transdução de Sinais , Animais , Linhagem Celular , Expressão Gênica , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Isoformas de Proteínas , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
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.
Assuntos
Envelhecimento/fisiologia , Transtornos Cognitivos , Sistemas Neurossecretores/metabolismo , Prosencéfalo/metabolismo , Receptores de Glucocorticoides/metabolismo , Hormônio Adrenocorticotrópico/sangue , Análise de Variância , Animais , Comportamento Animal , Transtornos Cognitivos/genética , Transtornos Cognitivos/patologia , Transtornos Cognitivos/fisiopatologia , Corticosterona/sangue , Dexametasona , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Análise em Microsséries/métodos , Sistemas Neurossecretores/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Receptores de Glucocorticoides/genética , Receptores de Glutamato/genética , Receptores de Glutamato/metabolismo , Restrição Física/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Estresse Psicológico/sangue , Estresse Psicológico/etiologiaRESUMO
Although numerous stress-related molecules have been implicated in vulnerability to psychiatric illness, especially major depression and anxiety disorders, the role of the brain mineralocorticoid receptor (MR) in stress, depression, and affective function is not well defined. MR is a steroid hormone receptor that detects circulating glucocorticoids with high affinity and has been primarily implicated in controlling their basal level and circadian rhythm. To specifically address the role of MR in hypothalamic-pituitary-adrenal axis activity and anxiety-related behaviors, we generated transgenic mice with increased levels of MR in the forebrain (MRov mice) by using the forebrain-specific calcium/calmodulin-dependent protein kinase II alpha promoter to direct expression of MR cDNA. A mild but chronic elevation in forebrain MR results in decreased anxiety-like behavior in both male and female transgenic mice. Female MRov mice also exhibit a moderate suppression of the corticosterone response to restraint stress. Increased forebrain MR expression alters the expression of two genes associated with stress and anxiety, leading to a decrease in the hippocampal glucocorticoid receptor (GR) and an increase in serotonin receptor 5HT-1a, consistent with the decreased anxiety phenotype. These data suggest that the functions of forebrain MR may overlap with GR in hypothalamic-pituitary-adrenal axis regulation, but they dissociate significantly from GR in the modulation of affective responses, with GR overexpression increasing anxiety-like behavior and MR overexpression dampening it. These findings point to the importance of the MR:GR ratio in the control of emotional reactivity.
Assuntos
Ansiedade/metabolismo , Regulação da Expressão Gênica , Prosencéfalo/metabolismo , Receptores de Mineralocorticoides/biossíntese , Animais , Comportamento Animal , Feminino , Hipocampo/metabolismo , Masculino , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas , Receptor 5-HT1A de Serotonina/metabolismo , Receptores de Glucocorticoides/metabolismo , Receptores de Mineralocorticoides/genética , Estresse FisiológicoRESUMO
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.