RESUMO
ß-catenin is a multi-functional protein that has an important role in the mature central nervous system; its dysfunction has been implicated in several neuropsychiatric disorders, including depression. Here we show that in mice ß-catenin mediates pro-resilient and anxiolytic effects in the nucleus accumbens, a key brain reward region, an effect mediated by D2-type medium spiny neurons. Using genome-wide ß-catenin enrichment mapping, we identify Dicer1-important in small RNA (for example, microRNA) biogenesis--as a ß-catenin target gene that mediates resilience. Small RNA profiling after excising ß-catenin from nucleus accumbens in the context of chronic stress reveals ß-catenin-dependent microRNA regulation associated with resilience. Together, these findings establish ß-catenin as a critical regulator in the development of behavioural resilience, activating a network that includes Dicer1 and downstream microRNAs. We thus present a foundation for the development of novel therapeutic targets to promote stress resilience.
Assuntos
RNA Helicases DEAD-box/genética , Regulação da Expressão Gênica , MicroRNAs/genética , Resiliência Psicológica , Ribonuclease III/genética , Estresse Fisiológico/genética , beta Catenina/metabolismo , Adaptação Fisiológica/genética , Animais , RNA Helicases DEAD-box/metabolismo , Depressão/fisiopatologia , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Neurônios/metabolismo , Ribonuclease III/metabolismo , Transdução de Sinais , beta Catenina/genéticaRESUMO
Ventral tegmental area (VTA) dopamine neurons in the brain's reward circuit have a crucial role in mediating stress responses, including determining susceptibility versus resilience to social-stress-induced behavioural abnormalities. VTA dopamine neurons show two in vivo patterns of firing: low frequency tonic firing and high frequency phasic firing. Phasic firing of the neurons, which is well known to encode reward signals, is upregulated by repeated social-defeat stress, a highly validated mouse model of depression. Surprisingly, this pathophysiological effect is seen in susceptible mice only, with no apparent change in firing rate in resilient individuals. However, direct evidence--in real time--linking dopamine neuron phasic firing in promoting the susceptible (depression-like) phenotype is lacking. Here we took advantage of the temporal precision and cell-type and projection-pathway specificity of optogenetics to show that enhanced phasic firing of these neurons mediates susceptibility to social-defeat stress in freely behaving mice. We show that optogenetic induction of phasic, but not tonic, firing in VTA dopamine neurons of mice undergoing a subthreshold social-defeat paradigm rapidly induced a susceptible phenotype as measured by social avoidance and decreased sucrose preference. Optogenetic phasic stimulation of these neurons also quickly induced a susceptible phenotype in previously resilient mice that had been subjected to repeated social-defeat stress. Furthermore, we show differences in projection-pathway specificity in promoting stress susceptibility: phasic activation of VTA neurons projecting to the nucleus accumbens (NAc), but not to the medial prefrontal cortex (mPFC), induced susceptibility to social-defeat stress. Conversely, optogenetic inhibition of the VTA-NAc projection induced resilience, whereas inhibition of the VTA-mPFC projection promoted susceptibility. Overall, these studies reveal novel firing-pattern- and neural-circuit-specific mechanisms of depression.
Assuntos
Depressão/fisiopatologia , Neurônios Dopaminérgicos/metabolismo , Mesencéfalo/citologia , Comportamento Social , Estresse Psicológico/fisiopatologia , Animais , Depressão/etiologia , Preferências Alimentares , Masculino , Camundongos , Vias Neurais , Núcleo Accumbens/fisiologia , Optogenética , Fenótipo , Córtex Pré-Frontal/fisiologia , Estresse Psicológico/complicações , Sacarose/administração & dosagem , Fatores de Tempo , Área Tegmentar Ventral/fisiologiaRESUMO
The hippocampus (HPC) is known to play an important role in learning, a process dependent on synaptic plasticity; however, the molecular mechanisms underlying this are poorly understood. ΔFosB is a transcription factor that is induced throughout the brain by chronic exposure to drugs, stress, and variety of other stimuli and regulates synaptic plasticity and behavior in other brain regions, including the nucleus accumbens. We show here that ΔFosB is also induced in HPC CA1 and DG subfields by spatial learning and novel environmental exposure. The goal of the current study was to examine the role of ΔFosB in hippocampal-dependent learning and memory and the structural plasticity of HPC synapses. Using viral-mediated gene transfer to silence ΔFosB transcriptional activity by expressing ΔJunD (a negative modulator of ΔFosB transcriptional function) or to overexpress ΔFosB, we demonstrate that HPC ΔFosB regulates learning and memory. Specifically, ΔJunD expression in HPC impaired learning and memory on a battery of hippocampal-dependent tasks in mice. Similarly, general ΔFosB overexpression also impaired learning. ΔJunD expression in HPC did not affect anxiety or natural reward, but ΔFosB overexpression induced anxiogenic behaviors, suggesting that ΔFosB may mediate attentional gating in addition to learning. Finally, we found that overexpression of ΔFosB increases immature dendritic spines on CA1 pyramidal cells, whereas ΔJunD reduced the number of immature and mature spine types, indicating that ΔFosB may exert its behavioral effects through modulation of HPC synaptic function. Together, these results suggest collectively that ΔFosB plays a significant role in HPC cellular morphology and HPC-dependent learning and memory. SIGNIFICANCE STATEMENT: Consolidation of our explicit memories occurs within the hippocampus, and it is in this brain region that the molecular and cellular processes of learning have been most closely studied. We know that connections between hippocampal neurons are formed, eliminated, enhanced, and weakened during learning, and we know that some stages of this process involve alterations in the transcription of specific genes. However, the specific transcription factors involved in this process are not fully understood. Here, we demonstrate that the transcription factor ΔFosB is induced in the hippocampus by learning, regulates the shape of hippocampal synapses, and is required for memory formation, opening up a host of new possibilities for hippocampal transcriptional regulation.
Assuntos
Hipocampo/metabolismo , Aprendizagem/fisiologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Animais , Aprendizagem da Esquiva , Condicionamento Psicológico/fisiologia , Espinhas Dendríticas/metabolismo , Dependovirus/genética , Meio Ambiente , Comportamento Exploratório/fisiologia , Medo/fisiologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Masculino , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Comportamento EspacialRESUMO
Drugs of abuse modulate the function and activity of the mesolimbic dopamine circuit. To identify novel mediators of drug-induced neuroadaptations in the ventral tegmental area (VTA), we performed RNA sequencing analysis on VTA samples from mice administered repeated saline, morphine, or cocaine injections. One gene that was similarly up-regulated by both drugs was serum- and glucocorticoid-inducible kinase 1 (SGK1). SGK1 activity, as measured by phosphorylation of its substrate N-myc downstream regulated gene (NDRG), was also increased robustly by chronic drug treatment. Increased NDRG phosphorylation was evident 1 but not 24 h after the last drug injection. SGK1 phosphorylation itself was similarly modulated. To determine the role of increased SGK1 activity on drug-related behaviors, we over-expressed constitutively active (CA) SGK1 in the VTA. SGK1-CA expression reduced locomotor sensitization elicited by repeated cocaine, but surprisingly had the opposite effect and promoted locomotor sensitization to morphine, without affecting the initial locomotor responses to either drug. SGK1-CA expression did not significantly affect morphine or cocaine conditioned place preference, although there was a trend toward increased conditioned place preference with both drugs. Further characterizing the role of this kinase in drug-induced changes in VTA may lead to improved understanding of neuroadaptations critical to drug dependence and addiction. We find that repeated, but not acute, morphine or cocaine administration induces an increase in serum- and glucocorticoid-inducible kinase (SGK1) gene expression and activity in the ventral tegmental area (VTA). This increase in SGK1 activity may play a role in drug-dependent behaviors and suggests a novel signaling cascade for potential intervention in drug dependence and addiction.
Assuntos
Cocaína/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas Imediatamente Precoces/biossíntese , Morfina/farmacologia , Proteínas do Tecido Nervoso/biossíntese , Proteínas Serina-Treonina Quinases/biossíntese , Área Tegmentar Ventral/efeitos dos fármacos , Animais , Condicionamento Clássico/efeitos dos fármacos , Condicionamento Clássico/fisiologia , Indução Enzimática/efeitos dos fármacos , Genes Reporter , Vetores Genéticos , Proteínas Imediatamente Precoces/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Proteínas do Tecido Nervoso/genética , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Regulação para Cima/efeitos dos fármacos , Área Tegmentar Ventral/enzimologiaRESUMO
As an approach to elucidating dopamine transporter (DAT) phosphorylation characteristics, we examined in vitro phosphorylation of a recombinant rat DAT N-terminal peptide (NDAT) using purified protein kinases. We found that NDAT becomes phosphorylated at single distinct sites by protein kinase A (Ser-7) and calcium-calmodulin-dependent protein kinase II (Ser-13) and at multiple sites (Ser-4, Ser-7, and Ser-13) by protein kinase C (PKC), implicating these residues as potential sites of DAT phosphorylation by these kinases. Mapping of rat striatal DAT phosphopeptides by two-dimensional thin layer chromatography revealed basal and PKC-stimulated phosphorylation of the same peptide fragments and comigration of PKC-stimulated phosphopeptide fragments with NDAT Ser-7 phosphopeptide markers. We further confirmed by site-directed mutagenesis and mass spectrometry that Ser-7 is a site for PKC-stimulated phosphorylation in heterologously expressed rat and human DATs. Mutation of Ser-7 and nearby residues strongly reduced the affinity of rat DAT for the cocaine analog (-)-2ß-carbomethoxy-3ß-(4-fluorophenyl) tropane (CFT), whereas in rat striatal tissue, conditions that promote DAT phosphorylation caused increased CFT affinity. Ser-7 mutation also affected zinc modulation of CFT binding, with Ala and Asp substitutions inducing opposing effects. These results identify Ser-7 as a major site for basal and PKC-stimulated phosphorylation of native and expressed DAT and suggest that Ser-7 phosphorylation modulates transporter conformational equilibria, shifting the transporter between high and low affinity cocaine binding states.
Assuntos
Cocaína/química , Proteínas da Membrana Plasmática de Transporte de Dopamina/química , Serina/química , Animais , Sítios de Ligação , Cromatografia em Camada Fina/métodos , Cocaína/análogos & derivados , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Inibidores da Captação de Dopamina/química , Células HEK293 , Humanos , Cinética , Masculino , Espectrometria de Massas/métodos , Mutagênese Sítio-Dirigida , Mutação , Fosforilação , Ligação Proteica , Conformação Proteica , Ratos , Ratos Sprague-DawleyRESUMO
The molecular mechanism underlying induction by cocaine of ΔFosB, a transcription factor important for addiction, remains unknown. Here, we demonstrate a necessary role for two transcription factors, cAMP response element binding protein (CREB) and serum response factor (SRF), in mediating this induction within the mouse nucleus accumbens (NAc), a key brain reward region. CREB and SRF are both activated in NAc by cocaine and bind to the fosB gene promoter. Using viral-mediated Cre recombinase expression in the NAc of single- or double-floxed mice, we show that deletion of both transcription factors from this brain region completely blocks cocaine induction of ΔFosB in NAc, whereas deletion of either factor alone has no effect. Furthermore, deletion of both SRF and CREB from NAc renders animals less sensitive to the rewarding effects of moderate doses of cocaine when tested in the conditioned place preference (CPP) procedure and also blocks locomotor sensitization to higher doses of cocaine. Deletion of CREB alone has the opposite effect and enhances both cocaine CPP and locomotor sensitization. In contrast to ΔFosB induction by cocaine, ΔFosB induction in NAc by chronic social stress, which we have shown previously requires activation of SRF, is unaffected by the deletion of CREB alone. These surprising findings demonstrate the involvement of distinct transcriptional mechanisms in mediating ΔFosB induction within this same brain region by cocaine versus stress. Our results also establish a complex mode of regulation of ΔFosB induction in response to cocaine, which requires the concerted activities of both SRF and CREB.
Assuntos
Proteína de Ligação a CREB/metabolismo , Cocaína/farmacologia , Inibidores da Captação de Dopamina/farmacologia , Núcleo Accumbens/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Fator de Resposta Sérica/metabolismo , Análise de Variância , Animais , Proteína de Ligação a CREB/deficiência , Imunoprecipitação da Cromatina , Condicionamento Operante/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Atividade Motora/genética , Núcleo Accumbens/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Proteínas Proto-Oncogênicas c-fos/genética , RNA Mensageiro/metabolismo , Fator de Resposta Sérica/deficiência , Estresse Psicológico/genética , Estresse Psicológico/metabolismo , Estresse Psicológico/fisiopatologia , Transdução GenéticaRESUMO
Attention deficit/hyperactivity disorder (ADHD) is the most commonly diagnosed disorder of school-age children. Although genetic and brain-imaging studies suggest a contribution of altered dopamine (DA) signaling in ADHD, evidence of signaling perturbations contributing to risk is largely circumstantial. The presynaptic, cocaine- and amphetamine (AMPH)-sensitive DA transporter (DAT) constrains DA availability at presynaptic and postsynaptic receptors following vesicular release and is targeted by the most commonly prescribed ADHD therapeutics. Using polymorphism discovery approaches with an ADHD cohort, we identified a hDAT (human DAT) coding variant, R615C, located in the distal C terminus of the transporter, a region previously implicated in constitutive and regulated transporter trafficking. Here, we demonstrate that, whereas wild-type DAT proteins traffic in a highly regulated manner, DAT 615C proteins recycle constitutively and demonstrate insensitivity to the endocytic effects of AMPH and PKC (protein kinase C) activation. The disrupted regulation of DAT 615C parallels a redistribution of the transporter variant away from GM1 ganglioside- and flotillin1-enriched membranes, and is accompanied by altered CaMKII (calcium/calmodulin-dependent protein kinase II) and flotillin-1 interactions. Using C-terminal peptides derived from wild-type DAT and the R615C variant, we establish that the DAT 615C C terminus can act dominantly to preclude AMPH regulation of wild-type DAT. Mutagenesis of DAT C-terminal sequences suggests that phosphorylation of T613 may be important in sorting DAT between constitutive and regulated pathways. Together, our studies support a coupling of DAT microdomain localization with transporter regulation and provide evidence of perturbed DAT activity and DA signaling as a risk determinant for ADHD.
Assuntos
Transtorno do Deficit de Atenção com Hiperatividade/genética , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Microdomínios da Membrana/genética , Polimorfismo de Nucleotídeo Único/genética , Adolescente , Anfetamina/farmacologia , Análise de Variância , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Benzilaminas/farmacologia , Biotinilação , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Linhagem Celular Transformada , Criança , Pré-Escolar , Toxina da Cólera/metabolismo , Estudos de Coortes , Dopamina/metabolismo , Dopamina/farmacologia , Inibidores da Captação de Dopamina/farmacologia , Relação Dose-Resposta a Droga , Eletroquímica , Feminino , Humanos , Imunoprecipitação , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Microdomínios da Membrana/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Sulfonamidas/farmacologia , Transfecção/métodos , Trítio/metabolismoRESUMO
Intestinal epithelial barrier function is compromised in inflammatory bowel disease and barrier dysfunction contributes to disease progression. Extracellular nucleotides/nucleosides generated in gut inflammation may regulate barrier function through actions on diverse cell types. Enteric glia modulate extracellular purinergic signaling and exert pathophysiological effects on mucosal permeability. These glia may regulate inflammation with paracrine responses, theoretically mediated via adenosine 2B receptor (A2BR) signaling. As the cell-specific roles of A2BRs in models of colitis and barrier dysfunction are unclear, we studied glial A2BRs in acute dextran sodium sulfate (DSS) colitis. We performed and validated conditional ablation of glial A2BRs in Sox10CreERT2+/-;Adora2bf/f mice. Overt intestinal disease activity indices in DSS-colitis were comparable between Sox10CreERT2+/-;Adora2bf/f mice and littermate controls. However, ablating glial A2BRs protected against barrier dysfunction following acute DSS-colitis. These benefits were associated with the normalization of tight junction protein expression and localization including claudin-1, claudin-8, and occludin. Glial A2BR signaling increased levels of proinflammatory mediators in the colon and cell-intrinsic regulation of genes including Csf3, Cxcl1, Cxcl10, and Il6. Our studies show that glial A2BR signaling exacerbates immune responses during DSS-colitis and that this adenosinergic cell-specific mechanism contributes to persistent gut epithelial barrier dysfunction.
Assuntos
Colite , Mucosa Intestinal , Adenosina/metabolismo , Animais , Colite/induzido quimicamente , Colite/genética , Colite/metabolismo , Colo/metabolismo , Sulfato de Dextrana , Modelos Animais de Doenças , Inflamação/metabolismo , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neuroglia/metabolismoRESUMO
Opioid drugs are highly valued as potent analgesics; however, there are significant risks associated with long-term use because of their abuse liability. Opioids cause changes in ventral tegmental area (VTA) gene expression and cell activity that have been linked to addiction-related behaviors in rodent models. Here, we focus on VTA dopamine (DA) neurons and review the cellular, structural, and synaptic plasticity changes induced by acute and chronic opioid exposure. We also discuss many avenues for future research including determination of whether opioid neuroadaptations are specific for subpopulations of VTA DA neurons. A better understanding of the molecular adaptations within the cells and circuits that drive opioid abuse is crucial for the development of better treatments for substance use disorders and to create novel, safer pain-relieving therapeutics.
Assuntos
Analgésicos Opioides/farmacologia , Plasticidade Neuronal/efeitos dos fármacos , Área Tegmentar Ventral/efeitos dos fármacos , Analgésicos Opioides/administração & dosagem , Animais , Neurônios Dopaminérgicos/efeitos dos fármacos , HumanosRESUMO
Despite the high prevalence of major depressive disorder (MDD), understanding of the biological underpinnings remains limited. Rodent models suggest that changes in activity and output of dopamine (DA) neurons in the ventral tegmental area (VTA) are important for depressive-like phenotypes. Additionally, brain inflammatory processes are thought to contribute to MDD pathology and inflammation in the VTA has been linked to changes in VTA DA neuronal activity. Thus, we sought to determine whether there is increased inflammatory signaling in the VTA following forms of chronic stress that induce depressive-like symptoms. First, we subjected male mice to either physical or vicarious chronic social defeat stress (CSDS), paradigms known to induce long-term depressive-like behavior and changes in VTA signaling. Second, we subjected male and female mice to subchronic variable stress (SCVS), a paradigm that induces depressive-like behavior only in female mice. We then isolated mRNA from the VTA and assessed proinflammatory gene regulation via RT-PCR. Our results show that physical, but not vicarious, CSDS increases interleukin 1ß (IL-1ß) mRNA expression and this inversely correlates with social interaction score. In contrast, IL-1ß expression was unchanged in male or female mice following SCVS. No significant increases in VTA ionized calcium binding adapter molecule 1 (Iba1) and glial fibrillary acidic protein (GFAP) immunochemistry were detected following CSDS that would be indicative of a robust inflammatory response. In conclusion, we show that chronic stressors distinctively alter expression of proinflammatory genes in the VTA and changes may depend on the severity and time-course of the stress exposure.
Assuntos
Transtorno Depressivo Maior , Área Tegmentar Ventral , Animais , Modelos Animais de Doenças , Neurônios Dopaminérgicos , Feminino , Masculino , Camundongos , Estresse PsicológicoRESUMO
Drugs of abuse regulate the activity of the mesolimbic dopamine (DA) system, and drug-induced changes in ventral tegmental area (VTA) cellular activity and gene regulation are linked to behavioral outputs associated with addiction. Previous work from our lab determined that VTA serum- and glucocorticoid-inducible kinase 1 (SGK1) transcription and catalytic activity were increased by repeated cocaine administration; however, it was unknown if these biochemical changes contributed to cocaine-elicited behaviors. Using transgenic and viral-mediated manipulations, we investigated the role of VTA SGK1 catalytic activity in regulating cocaine conditioned place preference and self-administration. We showed intra-VTA infusion of a catalytically inactive SGK1 mutant (K127Q) significantly decreased cocaine conditioned place preference (CPP). Further, we found that K127Q expression in VTA DA neurons significantly decreased cocaine CPP, while this same manipulation in VTA GABA neurons had no effect. However, blunted VTA DA SGK1 catalytic activity did not alter cocaine self-administration. Altogether, these studies identify the specific VTA cells critical for SGK1-mediated effects on cocaine CPP but not self-administration.
Assuntos
Cocaína , Área Tegmentar Ventral , Cocaína/farmacologia , Condicionamento Clássico , Neurônios Dopaminérgicos , GlucocorticoidesRESUMO
Although chronic cocaine-induced changes in dendritic spines on nucleus accumbens (NAc) neurons have been correlated with behavioral sensitization, the molecular pathways governing these structural changes, and their resulting behavioral effects, are poorly understood. The transcription factor, nuclear factor kappa B (NFkappaB), is rapidly activated by diverse stimuli and regulates expression of many genes known to maintain cell structure. Therefore, we evaluated the role of NFkappaB in regulating cocaine-induced dendritic spine changes on medium spiny neurons of the NAc and the rewarding effects of cocaine. We show that chronic cocaine induces NFkappaB-dependent transcription in the NAc of NFkappaB-Lac transgenic mice. This induction of NFkappaB activity is accompanied by increased expression of several NFkappaB genes, the promoters of which show chromatin modifications after chronic cocaine exposure consistent with their transcriptional activation. To study the functional significance of this induction, we used viral-mediated gene transfer to express either a constitutively active or dominant-negative mutant of Inhibitor of kappa B kinase (IKKca or IKKdn), which normally activates NFkappaB signaling, in the NAc. We found that activation of NFkappaB by IKKca increases the number of dendritic spines on NAc neurons, whereas inhibition of NFkappaB by IKKdn decreases basal dendritic spine number and blocks the increase in dendritic spines after chronic cocaine. Moreover, inhibition of NFkappaB blocks the rewarding effects of cocaine and the ability of previous cocaine exposure to increase an animal's preference for cocaine. Together, these studies establish a direct role for NFkappaB pathways in the NAc to regulate structural and behavioral plasticity to cocaine.
Assuntos
Cocaína/administração & dosagem , NF-kappa B/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Recompensa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Animais , Células Cultivadas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/ultraestrutura , Núcleo Accumbens/citologia , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/fisiologia , Células PC12 , RatosRESUMO
The chronic social defeat stress model has been instrumental in shaping our understanding of neurobiology relevant to affect-related illnesses, including major depressive disorder. However, the classic chronic social defeat stress procedure is limited by its exclusive application to adult male rodents. We have recently developed a novel vicarious social defeat stress procedure wherein one mouse witnesses the physical defeat bout of a conspecific from the safety of an adjacent compartment. This witness mouse develops a similar behavioral phenotype to that of the mouse that physically experiences social defeat stress, modeling multiple aspects of major depressive disorder. Importantly, this new procedure allows researchers to perform vicarious social defeat stress in males or females and in juvenile mice, which typically are excluded from classic social defeat experiments. Here we discuss several recent advances made using this procedure and how its application provides a new preclinical approach to study the neurobiology of psychological stress-induced phenotypes.
Assuntos
Comportamento Animal , Transtorno Depressivo Maior , Afeto , Animais , Feminino , Masculino , Camundongos , Comportamento Social , Estresse PsicológicoRESUMO
The hippocampus has a well-known role in mediating learning and memory, and its function can be directly regulated by both stress and glucocorticoid receptor activation. Hippocampal contributions to learning are thought to be dependent on changes in the plasticity of synapses within specific subregions, and these functional changes are accompanied by morphological changes in the number and shape of dendritic spines, the physical correlates of these glutamatergic synapses. Serum- and glucocorticoid-inducible kinase 1 (SGK1) regulates dendritic spine morphology in the prefrontal cortex, and modulation of SGK1 expression in mouse hippocampus regulates learning. However, the role of SGK1 in dendritic spine morphology within the CA1 and dentate gyrus regions of the hippocampus are unknown. Thus, herpes simplex viral vectors expressing GFP and various SGK1 constructs, including wild type SGK1, a catalytically inactive version of SGK1 (K127Q), and a phospho-defective version of SGK1 (S78A), were infused into the hippocampus of adult mice and confocal fluorescent microscopy was used to visualize dendritic spines. We show that increasing expression of SGK1 in the dentate gyrus increased the total number of spines, driven primarily by an increase in mushroom spines, while decreasing SGK1 activity (K127Q) in the CA1 region increased the total number of dendritic spines, driven by a significant increase in mushroom and stubby spines. The differential effects of SGK1 in these regions may be mediated by the interactions of SGK1 with multiple pathways required for spine formation and stability. As the formation of mature synapses is a crucial component of learning and memory, this indicates that SGK1 is a potential target in the pathway underlying stress-associated changes in cognition and memory.
Assuntos
Espinhas Dendríticas/metabolismo , Hipocampo/metabolismo , Proteínas Imediatamente Precoces/biossíntese , Proteínas Serina-Treonina Quinases/biossíntese , Animais , Espinhas Dendríticas/química , Ativação Enzimática/fisiologia , Vetores Genéticos/administração & dosagem , Vetores Genéticos/análise , Proteínas de Fluorescência Verde/administração & dosagem , Proteínas de Fluorescência Verde/análise , Hipocampo/química , Proteínas Imediatamente Precoces/análise , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Serina-Treonina Quinases/análiseRESUMO
Drugs of abuse cause significant neuroadaptations within the ventral tegmental area (VTA), with alterations in gene expression tied to changes in reward behavior. Serum- and glucocorticoid-inducible kinase 1 (SGK1) transcription, catalytic activity, and phosphorylation are upregulated in the VTA by chronic cocaine or morphine treatment, positioning SGK1 as a critical mediator of reward behavior. Using transgenic mouse models, we investigated the effect of SGK1 knockout in the VTA and in dopamine (DA) neurons to evaluate the necessity of protein expression for natural and drug reward behaviors. SGK1 knockdown in the VTA did not impact reward behaviors. Given VTA cellular heterogeneity, we also investigated a DA neuron-specific SGK1 knockout (KO). DA SGK1 KO significantly decreased body weight of adult mice as well as increased general locomotor activity; however, reward behaviors were similarly unaltered. Given that SGK1 mutants virally overexpressed in the VTA are capable of altering drug-associated behavior, our current results suggest that changes in SGK1 protein signaling may be distinct from expression. This work yields novel information on the impact of SGK1 deletion, critical for understanding the role of SGK1 signaling in the central nervous system and evaluating SGK1 as a potential therapeutic target for treatment of substance use disorders.
Assuntos
Comportamento Animal/efeitos dos fármacos , Cocaína/farmacologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Proteínas Imediatamente Precoces/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Recompensa , Área Tegmentar Ventral/efeitos dos fármacos , Animais , Inibidores da Captação de Dopamina/farmacologia , Neurônios Dopaminérgicos/patologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais , Área Tegmentar Ventral/patologiaRESUMO
Dopamine (DA) signaling at synapses is tightly coordinated through opposing mechanisms of vesicular fusion-mediated DA release and transporter-mediated DA clearance. Altered brain DA signaling is suspected to underlie multiple brain disorders, including schizophrenia, Parkinson's disease, bipolar disorder, and attention-deficit hyperactivity disorder (ADHD). We identified a pedigree containing two male children diagnosed with ADHD who share a rare human DA transporter (DAT; SLC6A3) coding variant, Ala559Val. Among >1000 control and affected subjects, the Val559 variant has only been isolated once previously, in a female subject with bipolar disorder. Although hDAT Ala559Val supports normal DAT protein and cell surface expression, as well as normal DA uptake, the variant exhibits anomalous DA efflux from DA-loaded cells. We also demonstrate that hDAT Ala599Val exhibits increased sensitivity to intracellular Na(+), but not intracellular DA, and displays exaggerated DA efflux at depolarized potentials. Remarkably, the two most common ADHD medications, amphetamine and methylphenidate, both block hDAT Ala559Val-mediated DA efflux, whereas these drugs have opposite actions at wild-type hDAT. Our findings reveal that DA efflux, typically associated with amphetamine-like psychostimulants, can be produced through a heritable change in hDAT structure. Because multiple gene products are known to coordinate to support amphetamine-mediated DA efflux, the properties of hDAT Ala559Val may have broader significance in identifying a new mechanism through which DA signaling disorders arise. Additionally, they suggest that block of inappropriate neurotransmitter efflux may be an unsuspected mechanism supporting the therapeutic actions of existing transporter-directed medications.
Assuntos
Proteínas da Membrana Plasmática de Transporte de Dopamina/fisiologia , Dopamina/metabolismo , Mutação/genética , Alanina/genética , Anfetamina/farmacologia , Análise de Variância , Biotinilação/métodos , Linhagem Celular Transformada , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Inibidores da Captação de Dopamina/farmacologia , Humanos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Metilfenidato/farmacologia , Técnicas de Patch-Clamp , Transporte Proteico/efeitos dos fármacos , Sódio/metabolismo , Transfecção , Valina/genéticaRESUMO
Drugs of abuse produce widespread effects on the structure and function of neurons throughout the brain's reward circuitry, and these changes are believed to underlie the long-lasting behavioral phenotypes that characterize addiction. Although the intracellular mechanisms regulating the structural plasticity of neurons are not fully understood, accumulating evidence suggests an essential role for neurotrophic factor signaling in the neuronal remodeling which occurs after chronic drug administration. Brain-derived neurotrophic factor (BDNF), a growth factor enriched in brain and highly regulated by several drugs of abuse, regulates the phosphatidylinositol 3'-kinase (PI3K), mitogen-activated protein kinase (MAPK), phospholipase Cgamma (PLCgamma), and nuclear factor kappa B (NFkappaB) signaling pathways, which influence a range of cellular functions including neuronal survival, growth, differentiation, and structure. This review discusses recent advances in our understanding of how BDNF and its signaling pathways regulate structural and behavioral plasticity in the context of drug addiction.
Assuntos
Fatores de Crescimento Neural/metabolismo , Plasticidade Neuronal/fisiologia , Neurônios/fisiologia , Transdução de Sinais/fisiologia , Transtornos Relacionados ao Uso de Substâncias , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/fisiologia , Transtornos Relacionados ao Uso de Substâncias/metabolismo , Transtornos Relacionados ao Uso de Substâncias/patologia , Transtornos Relacionados ao Uso de Substâncias/fisiopatologiaRESUMO
Chronic opiate exposure induces neuroadaptations in the mesocorticolimbic system including ventral tegmental area (VTA) dopamine (DA) neurons, whose soma size is decreased following opiate exposure. Yet it is now well documented that VTA DA neurons are heterogeneous, with notable differences between VTA DA neurons based on their projection target. Therefore, we sought to determine whether chronic morphine induced similar changes in the morphology of VTA DA neurons that project to the nucleus accumbens (NAc) and prefrontal cortex (PFC). We utilized Cre-dependent retrograde viral vectors in DA Cre driver lines to label VTA DA neurons that projected to NAc and PFC and assessed neuronal soma size. Consistent with previous data, the soma size of VTA DA neurons that projected to the NAc medial shell was decreased following morphine exposure. However, soma size of VTA DA neurons that projected to the NAc core was unaltered by morphine. Interestingly, morphology of PFC-projecting VTA DA neurons was also altered by morphine, but in this case soma size was increased compared to sham controls. Differences in basal soma size were also noted, suggesting stable differences in projection-specific morphology in addition to drug-induced changes. Together, these data suggest morphine-induced changes in VTA DA morphology occur within distinct VTA DA populations and that study of opiate-induced structural plasticity of individual VTA DA subcircuits may be critical for understanding addiction-related behavior.
Assuntos
Adaptação Fisiológica/efeitos dos fármacos , Neurônios Dopaminérgicos/patologia , Morfina/farmacologia , Rede Nervosa/patologia , Área Tegmentar Ventral/patologia , Animais , Biomarcadores/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Feminino , Masculino , Camundongos , Proteínas do Tecido Nervoso/metabolismoRESUMO
BACKGROUND: Our previous study revealed that adult female rats respond differently to trauma than adult males, recapitulating sex differences in symptoms of post-traumatic stress disorder (PTSD) exhibited by women and men. Here, we asked two questions: does the female phenotype depend on (1) social housing condition and/or (2) circulating gonadal hormones? METHODS: For the first study, the effects of single prolonged stress (SPS) were compared for females singly or pair-housed. For the second study, adult male and female rats were gonadectomized or sham-gonadectomized 2 weeks prior to exposure to SPS, with half the gonadectomized rats given testosterone. In addition to the typical measures of the trauma response in rats, acoustic startle response (ASR), and the dexamethasone suppression test (DST), we also used two other measures typically used to assess depressive-like responses, social interaction and sucrose preference. Glucocorticoid receptor (GR) expression in the hypothalamus was also examined. RESULTS: We now report that the distinct trauma response of female rats is not influenced by social housing condition. Moreover, sex differences in the response to SPS based on ASR and DST, replicated in the current study, are independent of adult gonadal hormones. Regardless of hormonal status, traumatized males show a hyper-responsive phenotype whereas traumatized females do not. Moreover, testosterone treatment in adulthood did not masculinize the response to trauma in females. Notably, both sucrose preference and social interaction tests revealed an effect of trauma in females but not in males, with the effects of SPS on sucrose preference dependent on ovarian hormones. Effects of SPS on GR expression in the hypothalamus also depended on gonadal hormones in females. CONCLUSIONS: We propose that the trauma response for female rats is depressive in nature, recapitulating the female bias in PTSD for internalizing symptoms and major depression in contrast to the externalizing symptoms of males. Presumed core markers of PTSD (enhanced ASR and negative feedback control of corticosterone) are apparently relevant only to males and are independent of adult gonadal hormones. Such sex differences in trauma responding are likely determined earlier in life. We conclude that males and females show fundamentally different responses to trauma that do not simply reflect differences in resilience.
Assuntos
Hormônios Gonadais/fisiologia , Caracteres Sexuais , Estresse Psicológico , Anedonia , Animais , Encéfalo/metabolismo , Dexametasona/administração & dosagem , Éter/administração & dosagem , Feminino , Relações Interpessoais , Masculino , Ratos Sprague-Dawley , Receptores de Glucocorticoides/metabolismo , Reflexo de Sobressalto , Restrição Física , Estresse Fisiológico , NataçãoRESUMO
BACKGROUND: Post-traumatic stress disorder (PTSD) affects men and women differently. Not only are women twice as likely as men to develop PTSD, they experience different symptoms and comorbidities associated with PTSD. Yet the dearth of preclinical research on females leaves a notable gap in understanding the underlying neuropathology of this sex difference. METHODS: Using two standard measures of PTSD-like responses in rats, the acoustic startle response (ASR) and dexamethasone suppression test (DST), we tested the effects of traumatic stress in adult male and female rats using two rodent models of PTSD, single prolonged stress and predator exposure. We then examined the neural correlates underlying these responses with cFos and glucocorticoid receptor immunohistochemistry in brain regions implicated in the traumatic stress response. RESULTS: We now report that adult male and female rats across two models of PTSD show consistent sex-specific responses that recapitulate fundamental differences of PTSD in men and women. Trauma-exposed males showed the well-established hyper-responsive phenotype of enhanced ASR and exaggerated negative feedback control of the hypothalamic-pituitary-adrenal axis, while the same traumatic event had little effect on these same measures in females. Dramatic sex differences in how trauma affected cFos and glucocorticoid receptor expression in the brain lend further support to the idea that the trauma response of male and female rats is fundamentally different. CONCLUSIONS: Two standard measures, ASR and DST, might suggest that females are resilient to the effects of traumatic stress, but other measures make it clear that females are not resilient, but simply respond differently to trauma. The next important question to answer is why. We conclude that males and females show fundamentally different responses to trauma that do not simply reflect differences in resilience. The divergent effects of trauma in the brains of males and females begin to shed light on the neurobiological underpinnings of these sex differences, paving the way for improved diagnostics and therapeutics that effectively treat both men and women.