RESUMO
Increasing evidence suggests that the neurobiological processes that govern learning and memory can be different in males and females, but many of the specific mechanisms underlying these sex differences have not been fully defined. Here we investigated potential sex differences in endocannabinoid (eCB) modulation of Pavlovian fear conditioning and extinction, examining multiple defensive behaviors, including shock responsivity, conditioned freezing, and conditioned darting. We found that while systemic administration of drugs acting on eCB receptors did not influence the occurrence of darting, females that were classified as Darters responded differently to the drug administration than those classified as Non-darters. Most notably, CB1R antagonist AM251 produced an increase in cue-elicited freezing and context generalization selectively in female Non-darters that persisted across extinction and extinction retrieval tests but was prevented by co-administration of TRPV1R antagonist Capsazepine. To identify a potential synaptic mechanism for these sex differences, we next employed biochemical and neuroanatomical tracing techniques to quantify anandamide (AEA), TRPV1R, and perisomatic CB1R expression, focusing on the ventral hippocampus (vHip) given its known role in mediating contextual fear generalization. These assays identified sex-specific effects of both fear conditioning-elicited AEA release and vHip-BLA circuit structure. Together, our data support a model in which sexual dimorphism in vHip-BLA circuitry promotes a female-specific dependence on CB1Rs for context processing that is sensitive to TRPV1-mediated disruption when CB1Rs are blocked.
Assuntos
Medo , Aprendizagem , Feminino , Ratos , Animais , Masculino , Hipocampo , Condicionamento ClássicoRESUMO
Increasing evidence suggests that the neurobiological processes that govern learning and memory can be different in males and females, and here we asked specifically whether the endocannabinoid (eCB) system could modulate Pavlovian fear conditioning in a sex-dependent manner. Systemic (i.p.) injection of CB1R antagonist AM251 in adult male and female Sprague Dawley rats prior to auditory cued fear conditioning produced a female-specific increase in freezing that persisted across extinction and extinction retrieval tests but was prevented by co-administration of TRPV1R antagonist Capsazepine. Notably, AM251 also produced robust freezing in a novel context prior to auditory cue presentation the day following drug administration, but not the day of, suggesting that CB1R blockade elicited contextual fear generalization in females. To identify a potential synaptic mechanism for these sex differences, we next used liquid chromatography/tandem mass spectrometry, Western Blot, and confocal-assisted immunofluorescence techniques to quantify anandamide (AEA), TRPV1R, and perisomatic CB1R expression, respectively, focusing on the ventral hippocampus (vHip). Fear conditioning elicited increased vHip AEA levels in females only, and in both sexes, CB1R expression around vHip efferents targeting the basolateral amygdala (BLA) was twice that at neighboring vHip neurons. Finally, quantification of the vHip-BLA projections themselves revealed that females have over twice the number of neurons in this pathway that males do. Together, our data support a model in which sexual dimorphism in vHip-BLA circuitry promotes a female-specific dependence on CB1Rs for context processing that is sensitive to TRPV1-mediated disruption when CB1Rs are blocked.
RESUMO
Differential expression of myelin-related genes and changes in myelin thickness have been demonstrated in mice after chronic psychosocial stress, a risk factor for anxiety disorders. To determine whether and how stress affects structural remodeling of nodes of Ranvier, another form of myelin plasticity, we developed a 3D reconstruction analysis of node morphology in C57BL/6NCrl and DBA/2NCrl mice. We identified strain-dependent effects of chronic social defeat stress on node morphology in the medial prefrontal cortex (mPFC) gray matter, including shortening of paranodes in C57BL/6NCrl stress-resilient and shortening of node gaps in DBA/2NCrl stress-susceptible mice compared to controls. Neuronal activity has been associated with changes in myelin thickness. To investigate whether neuronal activation is a mechanism influencing also node of Ranvier morphology, we used DREADDs to repeatedly activate the ventral hippocampus-to-mPFC pathway. We found reduced anxiety-like behavior and shortened paranodes specifically in stimulated, but not in the nearby non-stimulated axons. Altogether, our data demonstrate (1) nodal remodeling of the mPFC gray matter axons after chronic stress and (2) axon-specific regulation of paranodes in response to repeated neuronal activity in an anxiety-associated pathway. Nodal remodeling may thus contribute to aberrant circuit function associated with anxiety disorders.
Assuntos
Transtornos de Ansiedade , Ansiedade , Camundongos , Animais , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Ansiedade/metabolismo , Transtornos de Ansiedade/metabolismo , Estresse Psicológico/metabolismo , Córtex Pré-Frontal/metabolismoRESUMO
Pavlovian fear conditioning is a prevalent tool in the study of aversive learning, which is a key component of stress-related psychiatric disorders. Adult rats can exhibit various threat-related behaviors, including freezing, motor responses, and ultrasonic vocalizations (USVs). While these responses can all signal aversion, we know little about how they relate to one another. Here we characterize USVs emitted by male and female rats during cued fear acquisition and extinction, and assess the relationship between different threat-related behaviors. We found that males consistently emitted >22 kHz calls (referred to here as "alarm calls") than females, and that alarm call frequency in males, but not females, related to the intensity of the shock stimulus. Interestingly, 25% of males and 45% of females did not emit any alarm calls at all. Males that did make alarm calls had significantly higher levels of freezing than males who did not, while no differences in freezing were observed between female Alarm callers and Non-alarm callers. Alarm call emission was also affected by the predictability of the shock; when unpaired from a tone cue, both males and females started emitting alarm calls significantly later. During extinction learning and retrieval sessions, males were again more likely than females to emit alarm calls, which followed an extinction-like reduction in frequency. Collectively these data suggest sex dependence in how behavioral readouts relate to innate and conditioned threat responses. Importantly, we suggest that the same behaviors can signal sex-dependent features of aversion.
Assuntos
Caracteres Sexuais , Vocalização Animal , Ratos , Masculino , Feminino , Animais , Vocalização Animal/fisiologia , Ultrassom , Medo/fisiologia , Condicionamento ClássicoRESUMO
Pavlovian fear conditioning is a widely used behavioral paradigm for studying associative learning in rodents. Despite early recognition that subjects may engage in a variety of both conditioned and unconditioned responses, the last several decades have seen the field narrow its focus to measure freezing as the sole indicator of conditioned fear. We previously reported that female rats were more likely than males to engage in darting, an escape-like conditioned response that is associated with heightened shock reactivity. To determine how experimental parameters contribute to the frequency of darting in both males and females, we manipulated factors such as chamber size, shock intensity, and number of trials. To better capture fear-related behavioral repertoires in our animals, we developed ScaredyRat, an open-source custom Python tool that analyzes Noldus Ethovision-generated raw data files to identify darters and quantify both conditioned and unconditioned responses. We found that, like freezing, conditioned darting occurrences scale with experimental alterations. While most darting occurs in females, we found that with an extended training protocol, darting can emerge in males as well. Collectively, our data suggest that darting reflects a behavioral switch in conditioned responding that is a product of an individual animal's sex, shock reactivity, and experimental parameters, underscoring the need for careful consideration of sex as a biological variable in classic learning paradigms.
Assuntos
Condicionamento Clássico , Medo , Animais , Condicionamento Clássico/fisiologia , Medo/fisiologia , Feminino , Humanos , Masculino , RatosRESUMO
Stressful life events are major environmental risk factors for anxiety disorders, although not all individuals exposed to stress develop clinical anxiety. The molecular mechanisms underlying the influence of environmental effects on anxiety are largely unknown. To identify biological pathways mediating stress-related anxiety and resilience to it, we used the chronic social defeat stress (CSDS) paradigm in male mice of two inbred strains, C57BL/6NCrl (B6) and DBA/2NCrl (D2), that differ in their susceptibility to stress. Using a multi-omics approach, we identified differential mRNA, miRNA and protein expression changes in the bed nucleus of the stria terminalis (BNST) and blood cells after chronic stress. Integrative gene set enrichment analysis revealed enrichment of mitochondrial-related genes in the BNST and blood of stressed mice. To translate these results to human anxiety, we investigated blood gene expression changes associated with exposure-induced panic attacks. Remarkably, we found reduced expression of mitochondrial-related genes in D2 stress-susceptible mice and in exposure-induced panic attacks in humans, but increased expression of these genes in B6 stress-susceptible mice. Moreover, stress-susceptible vs. stress-resilient B6 mice displayed more mitochondrial cross-sections in the post-synaptic compartment after CSDS. Our findings demonstrate mitochondrial-related alterations in gene expression as an evolutionarily conserved response in stress-related behaviors and validate the use of cross-species approaches in investigating the biological mechanisms underlying anxiety disorders.
Assuntos
Ansiedade/genética , Ansiedade/metabolismo , Estresse Psicológico/metabolismo , Animais , Comportamento Animal/fisiologia , Modelos Animais de Doenças , Genômica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , MicroRNAs/genética , Mitocôndrias , Proteômica , RNA Mensageiro/genética , Núcleos Septais/metabolismo , Estresse Psicológico/fisiopatologia , Transcriptoma/genéticaRESUMO
Importance: Anxiety and stress-related disorders are among the most common mental disorders. Although family and twin studies indicate that both genetic and environmental factors play an important role underlying their etiology, the genetic underpinnings of anxiety and stress-related disorders are poorly understood. Objectives: To estimate the single-nucleotide polymorphism-based heritability of anxiety and stress-related disorders; to identify novel genetic risk variants, genes, or biological pathways; to test for pleiotropic associations with other psychiatric traits; and to evaluate the association of psychiatric comorbidities with genetic findings. Design, Setting, Participants: This genome-wide association study included individuals with various anxiety and stress-related diagnoses and controls derived from the population-based Lundbeck Foundation Initiative for Integrative Psychiatric Research (iPSYCH) study. Lifetime diagnoses of anxiety and stress-related disorders were obtained through the national Danish registers. Genes of interest were further evaluated in mice exposed to chronic social defeat. The study was conducted between June 2016 and November 2018. Main Outcomes and Measures: Diagnoses of a relatively broad diagnostic spectrum of anxiety and stress-related disorders. Results: The study sample included 12â¯655 individuals with various anxiety and stress-related diagnoses and 19â¯225 controls. Overall, 17â¯740 study participants (55.6%) were women. A total of 7308 participants (22.9%) were born between 1981-1985, 8840 (27.7%) between 1986-1990, 8157 (25.6%) between 1991-1995, 5918 (18.6%) between 1996-2000, and 1657 (5.2%) between 2001-2005. Standard association analysis revealed variants in PDE4B to be associated with anxiety and stress-related disorder (rs7528604; P = 5.39 × 10-11; odds ratio = 0.89; 95% CI, 0.86-0.92). A framework of sensitivity analyses adjusting for mental comorbidity supported this result showing consistent association of PDE4B variants with anxiety and stress-related disorder across analytical scenarios. In mouse models, alterations in Pde4b expression were observed in those mice displaying anxiety-like behavior after exposure to chronic stress in the prefrontal cortex (P = .002; t = -3.33) and the hippocampus (P = .001; t = -3.72). We also found a single-nucleotide polymorphism heritability of 28% (standard error = 0.027) and that the genetic signature of anxiety and stress-related overlapped with psychiatric traits, educational outcomes, obesity-related phenotypes, smoking, and reproductive success. Conclusions and Relevance: This study highlights anxiety and stress-related disorders as complex heritable phenotypes with intriguing genetic correlations not only with psychiatric traits, but also with educational outcomes and multiple obesity-related phenotypes. Furthermore, we highlight the candidate gene PDE4B as a robust risk locus pointing to the potential of PDE4B inhibitors in treatment of these disorders.
Assuntos
Transtornos de Ansiedade/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Estudo de Associação Genômica Ampla , Sistema de Registros , Transtornos Relacionados a Trauma e Fatores de Estresse/genética , Adolescente , Adulto , Animais , Comportamento Animal/fisiologia , Comorbidade , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Dinamarca , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Hipocampo/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Polimorfismo de Nucleotídeo Único , Córtex Pré-Frontal/metabolismo , Derrota Social , Adulto JovemRESUMO
Anxiety disorders often manifest in genetically susceptible individuals after psychosocial stress, but the mechanisms underlying these gene-environment interactions are largely unknown. We used the chronic social defeat stress (CSDS) mouse model to study resilience and susceptibility to chronic psychosocial stress. We identified a strong genetic background effect in CSDS-induced social avoidance (SA) using four inbred mouse strains: 69% of C57BL/6NCrl (B6), 23% of BALB/cAnNCrl, 19% of 129S2/SvPasCrl, and 5% of DBA/2NCrl (D2) mice were stress resilient. Furthermore, different inbred mouse strains responded differently to stress, suggesting they use distinct coping strategies. To identify biological pathways affected by CSDS, we used RNA-sequencing (RNA-seq) of three brain regions of two strains, B6 and D2: medial prefrontal cortex (mPFC), ventral hippocampus (vHPC), and bed nucleus of the stria terminalis (BNST). We discovered overrepresentation of oligodendrocyte (OLG)-related genes in the differentially expressed gene population. Because OLGs myelinate axons, we measured myelin thickness and found significant region and strain-specific differences. For example, in resilient D2 mice, mPFC axons had thinner myelin than controls, whereas susceptible B6 mice had thinner myelin than controls in the vHPC. Neither myelin-related gene expression in several other regions nor corpus callosum thickness differed between stressed and control animals. Our unbiased gene expression experiment suggests that myelin plasticity is a substantial response to chronic psychosocial stress, varies across brain regions, and is genetically controlled. Identification of genetic regulators of the myelin response will provide mechanistic insight into the molecular basis of stress-related diseases, such as anxiety disorders, a critical step in developing targeted therapy.
Assuntos
Transtornos de Ansiedade/metabolismo , Comportamento Animal/fisiologia , Expressão Gênica/fisiologia , Hipocampo/metabolismo , Bainha de Mielina/metabolismo , Oligodendroglia/metabolismo , Córtex Pré-Frontal/metabolismo , Resiliência Psicológica , Núcleos Septais/metabolismo , Estresse Psicológico/metabolismo , Animais , Transtornos de Ansiedade/etiologia , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Microscopia Eletrônica de Transmissão , Análise de Sequência de RNA , Estresse Psicológico/complicaçõesRESUMO
Chronic psychosocial stress is a well-established risk factor for neuropsychiatric diseases. Abnormalities in brain activity have been demonstrated in patients with stress-related disorders. Global brain activation patterns during chronic stress exposure are less well understood but may have strong modifying effects on specific brain circuits and thereby influence development of stress-related pathologies. We determined neural activation induced by chronic social defeat stress, a mouse model of psychosocial stress. To assess chronic activation with an unbiased brain-wide focus we used manganese-enhanced magnetic resonance imaging (MEMRI) and immunohistochemical staining of ∆FOSB, a transcription factor induced by repeated neural activity. One week after 10-day social defeat we observed significantly more activation in several brain regions known to regulate depressive and anxiety-like behaviour, including the prefrontal cortex, bed nucleus of stria terminalis, ventral hippocampus and periaqueductal grey in stressed compared to control mice. We further established that the correlation of ∆FOSB positive cells between specific brain regions was altered following chronic social defeat. Chronic activation of these neural circuits may relate to persistent brain activity changes occurring during chronic psychosocial stress exposure, with potential relevance for the development of anxiety and depression in humans.