Project description:Fear and extinction learning are adaptive processes caused by molecular changes in specific neural circuits. Neurons expressing the corticotropin-releasing hormone gene (Crh) in central amygdala (CeA) are implicated in threat regulation, yet little is known of cell type-specific gene pathways mediating adaptive learning. We translationally profiled the transcriptome of CeA Crh-expressing cells (Crh neurons) after fear conditioning or extinction in mice using translating ribosome affinity purification (TRAP) and RNAseq. Differential gene expression and co-expression network analyses identified diverse networks activated or inhibited by fear vs extinction. Upstream regulator analysis demonstrated that extinction associates with reduced CREB expression, and viral vector-induced increased CREB expression in Crh neurons increased fear expression and inhibited extinction. These findings suggest that CREB, within CeA Crh neurons, may function as a molecular switch that regulates expression of fear and its extinction. Cell-type specific translational analyses may suggest targets useful for understanding and treating stress-related psychiatric illness.
Project description:The interplay between corticotropin-releasing hormone (CRH) and the dopaminergic system has predominantly been studied in addiction and reward, while CRH-dopamine interactions in anxiety are scarcely understood. We describe a new population of CRH-expressing, GABAergic, long-range-projecting neurons in the extended amygdala that innervate the ventral tegmental area and alter anxiety following chronic CRH depletion. These neurons are part of a distinct CRH circuit that acts anxiolytically by positively modulating dopamine release.
Project description:The amygdala is a key limbic area involved in fear responses and pavlovian conditioning with the potential to directly respond to endocrine signals associated with fear or stress. To gain insights into the molecular mechanisms and subregional specificity of fear conditioning, we disrupted type II glucocorticoid receptors (GRs) in the central nucleus of the amygdala (CeA) by delivering lentiviral vectors containing Cre-recombinase into floxed-GR mice. GR deletion in the CeA (CeAGRKO mice) prevented conditioned fear behavior. In contrast, forebrain disruption of GRs excluding the CeA did not. The conditioned fear deficit in CeAGRKO mice was associated with decreases in cFos and corticotropin-releasing hormone (CRH) expression. Moreover, intracerebroventricular delivery of CRH rescued the conditioned fear deficit in CeAGRKO mice. We conclude that fear conditioning involves a neuroendocrine circuit by using GR activation in the CeA for acute CRH induction and long-lasting behavioral modulation.
Project description:RATIONALE:The corticotropin-releasing hormone (CRH) system is a key mediator of stress-induced responses in alcohol-seeking behavior. Recent research has identified the central nucleus of the amygdala (CeA), a brain region involved in the regulation of fear and stress-induced responses that is especially rich in CRH-positive neurons, as a key player in mediating excessive alcohol seeking. However, detailed characterization of the specific influences that local neuronal populations exert in mediating alcohol responses is hampered by current limitations in pharmacological and immunohistochemical tools for targeting CRH receptor subtype 1 (CRHR1). OBJECTIVE:In this study, we investigated the effect of cell- and region-specific overexpression of CRHR1 in the CeA using a novel transgenic tool. METHODS:Co-expression of CRHR1 in calcium-calmodulin-dependent kinase II (?CaMKII) neurons of the amygdala was demonstrated by double immunohistochemistry using a Crhr1-GFP reporter mouse line. A Cre-inducible Crhr1-expressing adeno-associated virus (AAV) was site-specifically injected into the CeA of ?CaMKII-CreERT2 transgenic rats to analyze the role of CRHR1 in ?CaMKII neurons on alcohol self-administration and reinstatement behavior. RESULTS:Forty-eight percent of CRHR1-containing cells showed co-expression of ?CaMKII in the CeA. AAV-mediated gene transfer in ?CaMKII neurons induced a 24-fold increase of Crhr1 mRNA in the CeA which had no effect on locomotor activity, alcohol self-administration, or cue-induced reinstatement. However, rats overexpressing Crhr1 in the CeA increased responding in the stress-induced reinstatement task with yohimbine serving as a pharmacological stressor. CONCLUSION:We demonstrate that CRHR1 overexpression in CeA-?CaMKII neurons is sufficient to mediate increased vulnerability to stress-triggered relapse into alcohol seeking.
Project description:Corticotropin-releasing hormone (CRH) is a critical neuropeptide modulating the mammalian stress response. It is involved in many functional activities within various brain regions, among which there is a subset of CRH neurons occupying a considerable proportion of the cortical GABAergic interneurons. Here, we utilized rabies virus-based monosynaptic retrograde tracing system to map the whole-brain afferent presynaptic partners of the CRH neurons in the anterior cingulate cortex (ACC). We find that the ACC CRH neurons integrate information from the cortex, thalamus, hippocampal formation, amygdala, and also several other midbrain and hindbrain nuclei. Furthermore, our results reveal that ACC CRH neurons receive direct inputs from two neuromodulatory systems, the basal forebrain cholinergic neurons and raphe serotoninergic neurons. These findings together expand our knowledge about the connectivity of the cortical GABAergic neurons and also provide a basis for further investigation of the circuit function of cortical CRH neurons.
Project description:BACKGROUND:Anhedonia, the diminished ability to experience pleasure, is an important dimensional entity linked to depression, schizophrenia, and other emotional disorders, but its origins and mechanisms are poorly understood. We have previously identified anhedonia, manifest as decreased sucrose preference and social play, in adolescent male rats that experienced chronic early-life adversity/stress (CES). Here we probed the molecular, cellular, and circuit processes underlying CES-induced anhedonia and tested them mechanistically. METHODS:We examined functional brain circuits and neuronal populations activated by social play in adolescent CES and control rats. Structural connectivity between stress- and reward-related networks was probed using high-resolution diffusion tensor imaging, and cellular/regional activation was probed using c-Fos. We employed viral-genetic approaches to reduce corticotropin-releasing hormone (Crh) expression in the central nucleus of the amygdala in anhedonic rats, and tested for anhedonia reversal in the same animals. RESULTS:Sucrose preference was reduced in adolescent CES rats. Social play, generally considered an independent measure of pleasure, activated brain regions involved in reward circuitry in both control and CES groups. In CES rats, social play activated Crh-expressing neurons in the central nucleus of the amygdala, typically involved in anxiety/fear, indicating aberrant functional connectivity of pleasure/reward and fear circuits. Diffusion tensor imaging tractography revealed increased structural connectivity of the amygdala to the medial prefrontal cortex in CES rats. Crh-short hairpin RNA, but not control short hairpin RNA, given into the central nucleus of the amygdala reversed CES-induced anhedonia without influencing other emotional measures. CONCLUSIONS:These findings robustly demonstrate aberrant interactions of stress and reward networks after early-life adversity and suggest mechanistic roles for Crh-expressing amygdala neurons in emotional deficits portending major neuropsychiatric disorders.
Project description:Corticotropin-releasing hormone receptor 1 (CRH-R1) in the amygdala and the stria terminalis plays an important role in the activation of central stress circuits. Genetic factors may contribute to the hyperresponsiveness of these circuits in irritable bowel syndrome (IBS).To determine if CRH-R1 SNPs are associated with: (1) a diagnosis of IBS, (2) gastrointestinal (GI) symptoms, and (3) acoustic startle response (ASR) to threat, which is mediated by the amygdala via CRH.Three CRH-R1 SNPS (rs110402, rs242924, and rs7209436) were genotyped using salivary DNA from IBS and healthy control subjects (HCs). Eye blink ASR was obtained during safe (no shock), anticipation (abdominal shock may soon occur) and threat (abdominal shock likely) conditions in a subset of subjects. Associations between each SNP with IBS status, clinical traits and ASR were measured.235 IBS patients (mean age 37.5 yrs, 74% F) and 264 HCs (mean age 32.1 yrs, 70% F) were studied. Of these, 57 IBS and 41 HCs underwent the ASR protocol. The presence of IBS was associated with the major allele for all three CRH-R1 SNPs (p=0.009-0.025). Within IBS, the major allele for all three SNPs (p=0.017-0.065) was associated with GI symptom anxiety scores. Within subjects with at least one copy of the major allele for the CRH-R1 SNPs, IBS had significantly lower ASR compared to HCs during threat conditions (p=0.001-0.002). Within IBS, CRH-R1 SNPs were associated with a graded increase in ASR to threat (p=0.007-0.008).These findings support that CRH-R1 contributes to the dysregulated stress responsiveness in IBS.
Project description:Chronic or prolonged exposure to stress ranks among the most important socioenvironmental factors contributing to the development of neuropsychiatric diseases, a process generally associated with loss of inhibitory tone in amygdala. Recent studies have identified distinct neuronal circuits within the basolateral amygdala (BLA) engaged in different emotional processes. However, the potential circuit involved in stress-induced dysregulation of inhibitory tones in BLA remains elusive. Here, a transgenic mouse model expressing yellow fluorescent protein under control of the Thy1 promoter was used to differentiate subpopulations of projection neurons (PNs) within the BLA. We observed that the tonic inhibition in amygdala neurons expressing and not expressing Thy1 (Thy1+/-) was oppositely regulated by chronic social defeat stress (CSDS). In unstressed control mice, the tonic inhibitory currents were significantly stronger in Thy1- PNs than their Thy1+ counterparts. CSDS markedly reduced the currents in Thy1- projection neurons (PNs), but increased that in Thy1+ ones. By contrast, CSDS failed to affect both the phasic A-type ?-aminobutyric acid receptor (GABAAR) currents and GABABR currents in these two PN populations. Moreover, chronic corticosterone administration was sufficient to mimic the effect of CSDS on the tonic inhibition of Thy1+ and Thy1- PNs. As a consequence, the suppression of tonic GABAAR currents on the excitability of Thy1- PNs was weakened by CSDS, but enhanced in Thy1+ PNs. The differential regulation of chronic stress on the tonic inhibition in Thy1+ and Thy1- neurons may orchestrate cell-specific adaptation of amygdala neurons to chronic stress.
Project description:Rett syndrome (RTT), a postnatal neurodevelopmental disorder, is caused by mutations in the methyl-CpG-binding protein 2 (MECP2) gene. Children with RTT display cognitive and motor abnormalities as well as autistic features. We studied mice bearing a truncated Mecp2 allele (Mecp2(308/Y) mice) and found evidence of increased anxiety-like behavior and an abnormal stress response as evidenced by elevated serum corticosterone levels. We found increased corticotropin-releasing hormone (Crh) gene expression in the paraventricular nucleus of the hypothalamus, the central amygdala, and the bed nucleus of the stria terminalis. Finally, we discovered that MeCP2 binds the Crh promoter, which is enriched for methylated CpG dinucleotides. In contrast, the MeCP2(308) protein was not detected at the Crh promoter. This study identifies Crh as a target of MeCP2 and implicates Crh overexpression in the development of specific features of the Mecp2(308/Y) mouse, thereby providing opportunities for clinical investigation and therapeutic intervention in RTT.
Project description:Memory formation is crucial for the survival of animals. Here, we study the effect of different crh-1 [Caenorhabditis elegans homolog of mammalian cAMP response element binding protein 1 (CREB1)] isoforms on the ability of C. elegans to form long-term memory (LTM). Null mutants in creb1/crh-1 are defective in LTM formation across phyla. We show that a specific isoform of CREB1/CRH-1, CRH-1e, is primarily responsible for memory related functions of the transcription factor in C. elegans Silencing of CRH-1e-expressing neurons during training for LTM formation abolishes the LTM of the animal. Further, CRH-1e expression in RIM neurons is sufficient to rescue LTM defects of creb1/crh-1-null mutants. We go on to show that apart from being LTM defective, creb1/crh-1-null animals show defects in innate chemotaxis behavior. We further characterize the amino acids K247 and K266 as responsible for the LTM related functions of CREB1/CRH-1 while being dispensable for its innate chemotaxis behavior. These findings provide insight into the spatial and temporal workings of a crucial transcription factor that can be further exploited to find CREB1 targets involved in the process of memory formation.SIGNIFICANCE STATEMENT This study elucidates the role of a specific isoform of CREB1/CRH-1, CRH-1e, in Caenorhabditis elegans memory formation and chemosensation. Removal of this single isoform of creb1/crh-1 shows defects in long-term memory formation in the animal and expression of CREB1/CRH-1e in a single pair of neurons is sufficient to rescue the memory defects seen in the mutant animals. We further show that two specific amino acids of CRH-1 are required for the process of memory formation in the animal.