RESUMO
Escalated aggression can have devastating societal consequences, yet underlying neurobiological mechanisms are poorly understood. Here, we show significantly increased inter-male mouse aggression when neurotransmission is constitutively blocked from either of two subsets of serotonergic, Pet1+ neurons: one identified by dopamine receptor D1(Drd1a)::cre-driven activity perinatally, and the other by Drd2::cre from pre-adolescence onward. Blocking neurotransmission from other Pet1+ neuron subsets of similar size and/or overlapping anatomical domains had no effect on aggression compared with controls, suggesting subtype-specific serotonergic neuron influences on aggression. Using established and novel intersectional genetic tools, we further characterized these subtypes across multiple parameters, showing both overlapping and distinct features in axonal projection targets, gene expression, electrophysiological properties, and effects on non-aggressive behaviors. Notably, Drd2::cre marked 5-HT neurons exhibited D2-dependent inhibitory responses to dopamine in slices, suggesting direct and specific interplay between inhibitory dopaminergic signaling and a serotonergic subpopulation. Thus, we identify specific serotonergic modules that shape aggression.
Assuntos
Agressão/fisiologia , Neurônios Serotoninérgicos/metabolismo , Animais , Axônios/metabolismo , Comportamento Animal , Encéfalo/patologia , Inativação Gênica , Genes Reporter , Integrases/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Fenótipo , Receptores de Dopamina D2/metabolismo , Reprodutibilidade dos Testes , Transmissão SinápticaRESUMO
Newborn neurons enter an extended maturation stage, during which they acquire excitability characteristics crucial for development of presynaptic and postsynaptic connectivity. In contrast to earlier specification programs, little is known about the regulatory mechanisms that control neuronal maturation. The Pet-1 ETS (E26 transformation-specific) factor is continuously expressed in serotonin (5-HT) neurons and initially acts in postmitotic precursors to control acquisition of 5-HT transmitter identity. Using a combination of RNA sequencing, electrophysiology, and conditional targeting approaches, we determined gene expression patterns in maturing flow-sorted 5-HT neurons and the temporal requirements for Pet-1 in shaping these patterns for functional maturation of mouse 5-HT neurons. We report a profound disruption of postmitotic expression trajectories in Pet-1(-/-) neurons, which prevented postnatal maturation of 5-HT neuron passive and active intrinsic membrane properties, G-protein signaling, and synaptic responses to glutamatergic, lysophosphatidic, and adrenergic agonists. Unexpectedly, conditional targeting revealed a postnatal stage-specific switch in Pet-1 targets from 5-HT synthesis genes to transmitter receptor genes required for afferent modulation of 5-HT neuron excitability. Five-HT1a autoreceptor expression depended transiently on Pet-1, thus revealing an early postnatal sensitive period for control of 5-HT excitability genes. Chromatin immunoprecipitation followed by sequencing revealed that Pet-1 regulates 5-HT neuron maturation through direct gene activation and repression. Moreover, Pet-1 directly regulates the 5-HT neuron maturation factor Engrailed 1, which suggests Pet-1 orchestrates maturation through secondary postmitotic regulatory factors. The early postnatal switch in Pet-1 targets uncovers a distinct neonatal stage-specific function for Pet-1, during which it promotes maturation of 5-HT neuron excitability. SIGNIFICANCE STATEMENT: The regulatory mechanisms that control functional maturation of neurons are poorly understood. We show that in addition to inducing brain serotonin (5-HT) synthesis and reuptake, the Pet-1 ETS (E26 transformation-specific) factor subsequently globally coordinates postmitotic expression trajectories of genes necessary for maturation of 5-HT neuron excitability. Further, Pet-1 switches its transcriptional targets as 5-HT neurons mature from 5-HT synthesis genes to G-protein-coupled receptors, which are necessary for afferent synaptic modulation of 5-HT neuron excitability. Our findings uncover gene-specific switching of downstream targets as a previously unrecognized regulatory strategy through which continuously expressed transcription factors control acquisition of neuronal identity at different stages of development.
Assuntos
Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Neurônios Serotoninérgicos/fisiologia , Fatores de Transcrição/fisiologia , Transcrição Gênica/fisiologia , Animais , Animais Recém-Nascidos , Feminino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurogênese/fisiologia , Técnicas de Cultura de ÓrgãosRESUMO
BACKGROUND: Differentiation of metazoan cells requires execution of different gene expression programs but recent single-cell transcriptome profiling has revealed considerable variation within cells of seeming identical phenotype. This brings into question the relationship between transcriptome states and cell phenotypes. Additionally, single-cell transcriptomics presents unique analysis challenges that need to be addressed to answer this question. RESULTS: We present high quality deep read-depth single-cell RNA sequencing for 91 cells from five mouse tissues and 18 cells from two rat tissues, along with 30 control samples of bulk RNA diluted to single-cell levels. We find that transcriptomes differ globally across tissues with regard to the number of genes expressed, the average expression patterns, and within-cell-type variation patterns. We develop methods to filter genes for reliable quantification and to calibrate biological variation. All cell types include genes with high variability in expression, in a tissue-specific manner. We also find evidence that single-cell variability of neuronal genes in mice is correlated with that in rats consistent with the hypothesis that levels of variation may be conserved. CONCLUSIONS: Single-cell RNA-sequencing data provide a unique view of transcriptome function; however, careful analysis is required in order to use single-cell RNA-sequencing measurements for this purpose. Technical variation must be considered in single-cell RNA-sequencing studies of expression variation. For a subset of genes, biological variability within each cell type appears to be regulated in order to perform dynamic functions, rather than solely molecular noise.
Assuntos
Variação Genética , Transcriptoma , Animais , Células Cultivadas , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , Camundongos Endogâmicos C57BL , Estabilidade de RNA , Ratos , Ratos Sprague-Dawley , Análise de Sequência de RNA , Análise de Célula ÚnicaRESUMO
The sleep-promoting ventrolateral preoptic nucleus (VLPO) shares reciprocal inhibitory inputs with wake-active neuronal nuclei, including the locus ceruleus. Electrophysiologically, sleep-promoting neurons in the VLPO are directly depolarized by the general anesthetic isoflurane and hyperpolarized by norepinephrine, a wake-promoting neurotransmitter. However, the integration of these competing influences on the VLPO, a sleep- and anesthetic-active structure, has yet to be evaluated in either brain slices in vitro or the intact organism. Single-cell multiplex RT-PCR conducted on both isoflurane-activated, putative sleep-promoting VLPO neurons and neighboring, state-indifferent VLPO neurons in mouse brain slices revealed widespread expression of α2A-, α2B- and α2C-adrenergic receptors in both populations. Indeed, both norepinephrine and the highly selective α2 agonist dexmedetomidine each reversed the VLPO depolarization induced by isoflurane in slices in vitro. When microinjected directly into the VLPO of a mouse lightly anesthetized with isoflurane, dexmedetomidine increased behavioral arousal and reduced the depressant effects of isoflurane on barrel cortex somatosensory-evoked potentials but failed to elicit spectral changes in spontaneous EEG. Based on these observations, we conclude that local modulation of α-adrenergic activity in the VLPO destabilizes, but does not fully antagonize, the anesthetic state, thus priming the brain for anesthetic emergence.
Assuntos
Nível de Alerta/efeitos dos fármacos , Isoflurano/antagonistas & inibidores , Área Pré-Óptica/fisiologia , Receptores Adrenérgicos alfa 2/fisiologia , Agonistas de Receptores Adrenérgicos alfa 2/farmacologia , Anestésicos/farmacologia , Animais , Ondas Encefálicas/efeitos dos fármacos , Dexmedetomidina/administração & dosagem , Dexmedetomidina/farmacologia , Eletroencefalografia , Potenciais Somatossensoriais Evocados/efeitos dos fármacos , Potenciais Somatossensoriais Evocados/fisiologia , Isoflurano/farmacologia , Masculino , Camundongos , Microinjeções , Norepinefrina/farmacologia , Área Pré-Óptica/efeitos dos fármacos , Receptores Adrenérgicos alfa 2/metabolismoRESUMO
Trauma during early life is a major risk factor for the development of anxiety disorders and suggests that the developing brain may be particularly sensitive to perturbation. Increased vulnerability most likely involves altering neural circuits involved in emotional regulation. The role of serotonin in emotional regulation is well established, but little is known about the postnatal development of the raphe where serotonin is made. Using whole-cell patch-clamp recording and immunohistochemistry, we tested whether serotonin circuitry in the dorsal and median raphe was functionally mature during the first 3 postnatal weeks in mice. Serotonin neurons at postnatal day 4 (P4) were hyperexcitable. The increased excitability was due to depolarized resting membrane potential, increased resistance, increased firing rate, lack of 5-HT1A autoreceptor response, and lack of GABA synaptic activity. Over the next 2 weeks, membrane resistance decreased and resting membrane potential hyperpolarized due in part to potassium current activation. The 5-HT1A autoreceptor-mediated inhibition did not develop until P21. The frequency of spontaneous inhibitory and excitatory events increased as neurons extended and refined their dendritic arbor. Serotonin colocalized with vGlut3 at P4 as in adulthood, suggesting enhanced release of glutamate alongside enhanced serotonin release. Because serotonin affects circuit development in other brain regions, altering the developmental trajectory of serotonin neuron excitability and release could have many downstream consequences. We conclude that serotonin neuron structure and function change substantially during the first 3 weeks of life during which external stressors could potentially alter circuit formation.
Assuntos
Meio Ambiente , Potenciais Pós-Sinápticos Excitadores/fisiologia , Núcleos da Rafe/citologia , Núcleos da Rafe/crescimento & desenvolvimento , Neurônios Serotoninérgicos/fisiologia , Fatores Etários , Animais , Animais Recém-Nascidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bicuculina/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Antagonistas de Receptores de GABA-A/farmacologia , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Técnicas In Vitro , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Quinoxalinas/farmacologia , Neurônios Serotoninérgicos/efeitos dos fármacos , Serotonina/análogos & derivados , Serotonina/farmacologia , Agonistas do Receptor de Serotonina/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
It has been well established that modulating serotonin (5-HT) levels in humans and animals affects perception and response to social threats, however the circuit mechanisms that control 5-HT output during social interaction are not well understood. A better understanding of these systems could provide groundwork for more precise and efficient therapeutic interventions. Here we examined the organization and plasticity of microcircuits implicated in top-down control of 5-HT neurons in the dorsal raphe nucleus (DRN) by excitatory inputs from the ventromedial prefrontal cortex (vmPFC) and their role in social approach-avoidance decisions. We did this in the context of a social defeat model that induces a long lasting form of social aversion that is reversible by antidepressants. We first used viral tracing and Cre-dependent genetic identification of vmPFC glutamatergic synapses in the DRN to determine their topographic distribution in relation to 5-HT and GABAergic subregions and found that excitatory vmPFC projections primarily localized to GABA-rich areas of the DRN. We then used optogenetics in combination with cFos mapping and slice electrophysiology to establish the functional effects of repeatedly driving vmPFC inputs in DRN. We provide the first direct evidence that vmPFC axons drive synaptic activity and immediate early gene expression in genetically identified DRN GABA neurons through an AMPA receptor-dependent mechanism. In contrast, we did not detect vmPFC-driven synaptic activity in 5-HT neurons and cFos induction in 5-HT neurons was limited. Finally we show that optogenetically increasing or decreasing excitatory vmPFC input to the DRN during sensory exposure to an aggressor's cues enhances or diminishes avoidance bias, respectively. These results clarify the functional organization of vmPFC-DRN pathways and identify GABAergic neurons as a key cellular element filtering top-down vmPFC influences on affect-regulating 5-HT output.
RESUMO
BACKGROUND: Cortical deep brain stimulation (DBS) is a promising therapeutic option for treatment-refractory depression, but its mode of action remains enigmatic. Serotonin (5-HT) systems are engaged indirectly by ventromedial prefrontal cortex (vmPFC) DBS. Resulting neuroplastic changes in 5-HT systems could thus coincide with the long-term therapeutic activity of vmPFC DBS. METHODS: We tested this hypothesis by evaluating the antidepressant-like activity of vmPFC DBS in the chronic social defeat stress (CSDS) model of depression (n = 8-13 mice/group). Circuit-wide activation induced by vmPFC DBS was mapped with c-Fos immunolabeling. The effects of chronic vmPFC DBS on the physiology and morphology of genetically identified 5-HT cells from the dorsal raphe nucleus (DRN) were examined with whole-cell recording, somatodendritic three-dimensional reconstructions and morphometric analyses of presynaptic boutons along 5-HT axons. RESULTS: Acute DBS drove c-Fos expression locally in the vmPFC and in several distal monosynaptically connected regions, including the DRN. Chronic DBS reversed CSDS-induced social avoidance, restored the disrupted balance of excitatory/inhibitory inputs onto 5-HT neurons, and reversed 5-HT hypoexcitability observed after CSDS. Furthermore, vmPFC DBS reversed CSDS-induced arborization of 5-HT dendrites in the DRN and increased the size and density of 5-HT presynaptic terminals in the dentate gyrus and vmPFC. CONCLUSIONS: We validate a new preclinical paradigm to examine cellular mechanisms underlying the antidepressant-like activity of vmPFC DBS and identify dramatic circuit-mediated cellular adaptations that coincide with this treatment. These neuroplastic changes of 5-HT neurons might contribute to the progressive mood improvements reported in patients treated with chronic courses of cortical DBS.
Assuntos
Adaptação Fisiológica , Estimulação Encefálica Profunda , Depressão/terapia , Núcleo Dorsal da Rafe/fisiopatologia , Córtex Pré-Frontal/fisiopatologia , Neurônios Serotoninérgicos/fisiologia , Animais , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Núcleo Dorsal da Rafe/metabolismo , Potenciais Pós-Sinápticos Inibidores , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Córtex Pré-Frontal/metabolismoRESUMO
STUDY OBJECTIVES: Delayed hypercapnic arousals may occur in obstructive sleep apnea. The impaired arousal response is expected to promote more pronounced oxyhemoglobin desaturations. We hypothesized that long-term sleep fragmentation (SF) results in injury to or dysfunction of wake-active neurons that manifests, in part, as a delayed hypercapnic arousal response. DESIGN: Adult male mice were implanted for behavioral state recordings and randomly assigned to 4 weeks of either orbital platform SF (SF4wk, 30 events/h) or control conditions (Ct4wk) prior to behavioral, histological, and locus coeruleus (LC) whole cell electrophysiological evaluations. MEASUREMENTS AND RESULTS: SF was successfully achieved across the 4 week study, as evidenced by a persistently increased arousal index, P < 0.01 and shortened sleep bouts, P < 0.05, while total sleep/wake times and plasma corticosterone levels were unaffected. A multiple sleep latency test performed at the onset of the dark period showed a reduced latency to sleep in SF4wk mice (P < 0.05). The hypercapnic arousal latency was increased, Ct4wk 64 ± 5 sec vs. SF4wk 154 ± 6 sec, P < 0.001, and remained elevated after a 2 week recovery (101 ± 4 sec, P < 0.001). C-fos activation in noradrenergic, orexinergic, histaminergic, and cholinergic wake-active neurons was reduced in response to hypercapnia (P < 0.05-0.001). Catecholaminergic and orexinergic projections into the cingulate cortex were also reduced in SF4wk (P < 0.01). In addition, SF4wk resulted in impaired LC neuron excitability (P < 0.01). CONCLUSIONS: Four weeks of sleep fragmentation (SF4wk) impairs arousal responses to hypercapnia, reduces wake neuron projections and locus coeruleus neuronal excitability, supporting the concepts that some effects of sleep fragmentation may contribute to impaired arousal responses in sleep apnea, which may not reverse immediately with therapy.
Assuntos
Nível de Alerta/fisiologia , Hipercapnia/fisiopatologia , Neurônios/fisiologia , Apneia Obstrutiva do Sono/fisiopatologia , Privação do Sono/patologia , Privação do Sono/fisiopatologia , Vigília/fisiologia , Animais , Axônios/fisiologia , Doença Crônica , Corticosterona/sangue , Eletroencefalografia , Hipercapnia/sangue , Hipercapnia/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Locus Cerúleo/citologia , Locus Cerúleo/patologia , Locus Cerúleo/fisiopatologia , Masculino , Camundongos , Neuropeptídeos/metabolismo , Orexinas , Polissonografia , Córtex Pré-Frontal/patologia , Córtex Pré-Frontal/fisiopatologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Sono/fisiologia , Apneia Obstrutiva do Sono/sangue , Apneia Obstrutiva do Sono/patologia , Privação do Sono/sangue , Fatores de TempoRESUMO
The serotonin 1A receptor (5-HT1A) has a major role in modulating the effects of serotonin on mood and behavior. Previous studies have shown that knockout of 5-HT1A selectively in the raphe leads to higher levels of anxiety during adulthood. However, it remains unclear whether this phenotype is due to variation in receptor levels specifically during development or throughout life. To test the hypothesis that developmental sensitivity may underlie the effects of 5-HT1A on anxiety, we used an inducible transgenic system to selectively suppress 5-HT1A levels in serotonergic raphe neurons from post-natal days (P) 14 to P30, with a maximal reduction of 40% at P21 and return to regular levels by P30. This developmental decrease in receptor levels has long-lasting consequences, increasing anxiety and decreasing social investigation in adulthood. In addition, post-natal knockdown of autoreceptors leads to long-term increases in the excitability of serotonergic neurons, which may represent a mechanism underlying the effects of post-natal receptor variation on behavior later in life. Finally, we also examined the interplay between receptor variation and juvenile exposure to stress (applied from P14 to P21). Similar to receptor knockdown, juvenile exposure to stress led to increased anxiety phenotypes but did not exacerbate 5-HT1A knockdown-mediated anxiety levels. This work indicates that the effects of 5-HT1A autoreceptors on anxiety and social behaviors are developmentally mediated and suggests that natural variations in the expression of 5-HT1A may act during development to influence individual anxiety levels and contribute to susceptibility to anxiety disorders.
Assuntos
Transtornos de Ansiedade/genética , Transtornos de Ansiedade/metabolismo , Autorreceptores/deficiência , Receptor 5-HT1A de Serotonina/deficiência , Comportamento Social , Fatores Etários , Animais , Transtornos de Ansiedade/psicologia , Autorreceptores/genética , Predisposição Genética para Doença , Humanos , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Receptor 5-HT1A de Serotonina/genéticaRESUMO
BACKGROUND: Women are twice as likely as men to suffer from stress-related affective disorders. Corticotropin-releasing factor (CRF) is an important link between stress and mood, in part through its signaling in the serotonergic dorsal raphe (DR). Development of CRF receptor-1 (CRFr1) antagonists has been a focus of numerous clinical trials but has not yet been proven efficacious. We hypothesized that sex differences in CRFr1 modulation of DR circuits might be key determinants in predicting therapeutic responses and affective disorder vulnerability. METHODS: Male and female mice received DR infusions of the CRFr1 antagonist, NBI 35965, or CRF and were evaluated for stress responsivity. Sex differences in indices of neural activation (cFos) and colocalization of CRFr1 throughout the DR were examined. Whole-cell patch-clamp electrophysiology assessed sex differences in serotonin neuron membrane characteristics and responsivity to CRF. RESULTS: Males showed robust behavioral and hypothalamic-pituitary-adrenal axis responses to DR infusion of NBI 35965 and CRF, whereas females were minimally responsive. Sex differences were also found for both CRF-induced DR cFos and CRFr1 co-localization throughout the DR. Electrophysiologically, female serotonergic neurons showed blunted membrane excitability and divergent inhibitory postsynaptic current responses to CRF application. CONCLUSIONS: These studies demonstrate convincing sex differences in CRFr1 activity in the DR, where blunted female responses to NBI 35965 and CRF suggest unique stress modulation of the DR. These sex differences might underlie affective disorder vulnerability and differential sensitivity to pharmacologic treatments developed to target the CRF system, thereby contributing to a current lack of CRFr1 antagonist efficacy in clinical trials.
Assuntos
Núcleo Dorsal da Rafe/efeitos dos fármacos , Receptores de Hormônio Liberador da Corticotropina/antagonistas & inibidores , Estresse Psicológico/metabolismo , Acenaftenos/farmacologia , Animais , Corticosterona/sangue , Hormônio Liberador da Corticotropina/farmacologia , Núcleo Dorsal da Rafe/metabolismo , Feminino , Hormônios/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Receptores de Hormônio Liberador da Corticotropina/agonistas , Fatores SexuaisRESUMO
Despite the recognized importance of the dorsal raphe (DR) serotonergic (5-HT) nuclei in the pathophysiology of depression and anxiety, the molecular components/putative drug targets expressed by these neurons are poorly characterized. Utilizing the promoter of an ETS domain transcription factor that is a stable marker of 5-HT neurons (Pet-1) to drive 5-HT neuronal expression of YFP, we identified 5-HT neurons in live acute slices. We isolated RNA from single 5-HT neurons in the ventromedial and lateral wings of the DR and performed single-cell RNA-Seq analysis identifying >500 G-protein coupled receptors (GPCRs) including receptors for classical transmitters, lipid signals, and peptides as well as dozens of orphan-GPCRs. Using these data to inform our selection of receptors to assess, we found that oxytocin and lysophosphatidic acid 1 receptors are translated and active in costimulating, with the α1-adrenergic receptor, the firing of DR 5-HT neurons, while the effects of histamine are inhibitory and exerted at H3 histamine receptors. The inhibitory histamine response provides evidence for tonic in vivo histamine inhibition of 5-HT neurons. This study illustrates that unbiased single-cell transcriptomics coupled with functional analyses provides novel insights into how neurons and neuronal systems are regulated.
Assuntos
Neurônios Serotoninérgicos/metabolismo , Animais , Eletrofisiologia , Técnicas In Vitro , Masculino , Camundongos , Receptores Acoplados a Proteínas G/metabolismo , Serotonina/metabolismoRESUMO
Serotonin (5-HT) modulates neural responses to socioaffective cues and can bias approach or avoidance behavioral decisions, yet the cellular mechanisms underlying its contribution to the regulation of social experiences remain poorly understood. We hypothesized that GABAergic neurons in the dorsal raphe nucleus (DRN) may participate in socioaffective regulation by controlling serotonergic tone during social interaction. We tested this hypothesis using whole-cell recording techniques in genetically identified DRN GABA and 5-HT neurons in mice exposed to social defeat, a model that induces long-lasting avoidance behaviors in a subset of mice responsive to serotonergic antidepressants. Our results revealed that social defeat engaged DRN GABA neurons and drove GABAergic sensitization that strengthened inhibition of 5-HT neurons in mice that were susceptible, but not resilient to social defeat. Furthermore, optogenetic silencing of DRN GABA neurons disinhibited neighboring 5-HT neurons and prevented the acquisition of social avoidance in mice exposed to a social threat, but did not affect a previously acquired avoidance phenotype. We provide the first characterization of GABA neurons in the DRN that monosynaptically inhibit 5-HT neurons and reveal their key role in neuroplastic processes underlying the development of social avoidance.
Assuntos
Agressão , Aprendizagem da Esquiva , Neurônios GABAérgicos/fisiologia , Núcleos da Rafe/citologia , Neurônios Serotoninérgicos/fisiologia , Animais , Potenciais Pós-Sinápticos Excitadores , Potenciais Pós-Sinápticos Inibidores , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Optogenética , Fenótipo , Núcleos da Rafe/fisiologiaRESUMO
Anxiety disorders are among the most prevalent psychiatric disorders, yet much is unknown about the underlying mechanisms. The dorsal raphe (DR) is at the crux of the anxiety-inducing effects of uncontrollable stress, a key component of models of anxiety. Though DR serotonin (5-HT) neurons play a prominent role, anxiety-associated changes in the physiology of 5-HT neurons remain poorly understood. A 5-day social defeat model of anxiety produced a multifaceted, anxious phenotype in intruder mice that included increased avoidance behavior in the open field test, increased stress-evoked grooming, and increased bladder and heart weights when compared to control mice. Intruders were further compared to controls using electrophysiology recordings conducted in midbrain slices wherein recordings targeted 5-HT neurons of the ventromedial (vmDR) and lateral wing (lwDR) subfields of the DR. Though defining membrane characteristics of 5-HT neurons were unchanged, γ-aminobutyric-acid-mediated (GABAergic) synaptic regulation of 5-HT neurons was altered in a topographically specific way. In the vmDR of intruders, there was a decrease in the frequency and amplitude of GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs). However, in the lwDR, there was an increase in the strength of inhibitory signals due to slower sIPSC kinetics. Synaptic changes were selective for GABAergic input, as glutamatergic synaptic input was unchanged in intruders. The distinct inhibitory regulation of DR subfields provides a mechanism for increased 5-HT output in vmDR target regions and decreased 5-HT output in lwDR target regions, divergent responses to uncontrollable stress that have been reported in the literature but were previously poorly understood.
Assuntos
Transtornos de Ansiedade/etiologia , Núcleos da Rafe/fisiopatologia , Neurônios Serotoninérgicos/fisiologia , Estresse Psicológico/fisiopatologia , Animais , Transtornos de Ansiedade/fisiopatologia , Potenciais Pós-Sinápticos Inibidores/fisiologia , Masculino , Camundongos , Ratos Sprague-Dawley , Receptores de GABA/fisiologia , Ácido gama-Aminobutírico/fisiologiaRESUMO
BACKGROUND: Despite seventeen decades of continuous clinical use, the neuronal mechanisms through which volatile anesthetics act to produce unconsciousness remain obscure. One emerging possibility is that anesthetics exert their hypnotic effects by hijacking endogenous arousal circuits. A key sleep-promoting component of this circuitry is the ventrolateral preoptic nucleus (VLPO), a hypothalamic region containing both state-independent neurons and neurons that preferentially fire during natural sleep. RESULTS: Using c-Fos immunohistochemistry as a biomarker for antecedent neuronal activity, we show that isoflurane and halothane increase the number of active neurons in the VLPO, but only when mice are sedated or unconscious. Destroying VLPO neurons produces an acute resistance to isoflurane-induced hypnosis. Electrophysiological studies prove that the neurons depolarized by isoflurane belong to the subpopulation of VLPO neurons responsible for promoting natural sleep, whereas neighboring non-sleep-active VLPO neurons are unaffected by isoflurane. Finally, we show that this anesthetic-induced depolarization is not solely due to a presynaptic inhibition of wake-active neurons as previously hypothesized but rather is due to a direct postsynaptic effect on VLPO neurons themselves arising from the closing of a background potassium conductance. CONCLUSIONS: Cumulatively, this work demonstrates that anesthetics are capable of directly activating endogenous sleep-promoting networks and that such actions contribute to their hypnotic properties.
Assuntos
Anestésicos/farmacologia , Halotano/farmacologia , Hipnose Anestésica , Isoflurano/farmacologia , Neurônios/efeitos dos fármacos , Área Pré-Óptica/efeitos dos fármacos , Anestésicos/administração & dosagem , Animais , Hipnóticos e Sedativos , Camundongos , Neurônios/fisiologia , Potássio/metabolismo , Área Pré-Óptica/fisiologia , Proteínas Proto-Oncogênicas c-fos/química , Sono/efeitos dos fármacos , Sono/fisiologia , InconsciênciaRESUMO
Genetic variations in certain components of the glucocorticoid receptor (GR) chaperone complex have been associated with the development of stress-related affective disorders and individual variability in therapeutic responses to antidepressants. Mechanisms that link GR chaperoning and stress susceptibility are not well understood. Here, we show that the effects of glucocorticoid hormones on socioaffective behaviors are critically regulated via reversible acetylation of Hsp90, a key component of the GR chaperone complex. We provide pharmacological and genetic evidence indicating that the cytoplasmic lysine deacetylase HDAC6 controls Hsp90 acetylation in the brain, and thereby modulates Hsp90-GR protein-protein interactions, as well as hormone- and stress-induced GR translocation, with a critical impact on GR downstream signaling and behavior. Pet1-Cre-driven deletion of HDAC6 in serotonin neurons, the densest HDAC6-expressing cell group in the mouse brain, dramatically reduced acute anxiogenic effects of the glucocorticoid hormone corticosterone in the open-field, elevated plus maze, and social interaction tests. Serotonin-selective depletion of HDAC6 also blocked the expression of social avoidance in mice exposed to chronic social defeat and concurrently prevented the electrophysiological and morphological changes induced, in serotonin neurons, by this murine model of traumatic stress. Together, these results identify HDAC6 inhibition as a potential new strategy for proresilience and antidepressant interventions through regulation of the Hsp90-GR heterocomplex and focal prevention of GR signaling in serotonin pathways. Our data thus uncover an alternate mechanism by which pan-HDAC inhibitors may regulate stress-related behaviors independently of their action on histones.
Assuntos
Comportamento Animal/fisiologia , Histona Desacetilases/fisiologia , Núcleos da Rafe/fisiologia , Receptores de Glucocorticoides/fisiologia , Resiliência Psicológica , Neurônios Serotoninérgicos/fisiologia , Estresse Psicológico/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/fisiologia , Células Cultivadas , Corticosterona/antagonistas & inibidores , Corticosterona/farmacologia , Dexametasona/farmacologia , Modelos Animais de Doenças , Deleção de Genes , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP90/metabolismo , Desacetilase 6 de Histona , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Imipramina/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Chaperonas Moleculares/metabolismo , Núcleos da Rafe/efeitos dos fármacos , Núcleos da Rafe/metabolismo , Receptores de Glucocorticoides/metabolismo , Neurônios Serotoninérgicos/citologia , Neurônios Serotoninérgicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Estresse Psicológico/fisiopatologiaRESUMO
Characterization of glutamatergic input to dorsal raphe (DR) serotonin (5-HT) neurons is crucial for understanding how the glutamate and 5-HT systems interact in psychiatric disorders. Markers of glutamatergic terminals, vGlut1, 2 and 3, reflect inputs from specific forebrain and midbrain regions. Punctate staining of vGlut2 was homogeneous throughout the mouse DR whereas vGlut1 and vGlut3 puncta were less dense in the lateral wing (lwDR) compared with the ventromedial (vmDR) subregion. The distribution of glutamate terminals was consistent with the lower miniature excitatory postsynaptic current frequency found in the lwDR; however, it was not predictive of glutamatergic synaptic input with local activity intact, as spontaneous excitatory postsynaptic current (sEPSC) frequency was higher in the lwDR. We examined the morphology of recorded cells to determine if variations in dendrite structure contributed to differences in synaptic input. Although lwDR neurons had longer, more complex dendrites than vmDR neurons, glutamatergic input was not correlated with dendrite length in the lwDR, suggesting that dendrite length did not contribute to subregional differences in sEPSC frequency. Overall, glutamatergic input in the DR was the result of selective innervation of subpopulations of 5-HT neurons and was rooted in the topography of DR neurons and the activity of glutamate neurons located within the midbrain slice. Increased glutamatergic input to lwDR cells potentially synergizes with previously reported increased intrinsic excitability of lwDR cells to increase 5-HT output in lwDR target regions. Because the vmDR and lwDR are involved in unique circuits, subregional differences in glutamate modulation may result in diverse effects on 5-HT output in stress-related psychopathology.
Assuntos
Ácido Glutâmico/metabolismo , Núcleos da Rafe/citologia , Neurônios Serotoninérgicos/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Animais , Potenciais Pós-Sinápticos Excitadores/fisiologia , Masculino , Camundongos , Técnicas de Patch-Clamp , Núcleos da Rafe/metabolismo , Neurônios Serotoninérgicos/citologia , Serotonina/metabolismo , Sinapses/metabolismo , Sinapses/ultraestrutura , Transmissão Sináptica/fisiologia , Proteína Vesicular 1 de Transporte de Glutamato/metabolismo , Proteína Vesicular 2 de Transporte de Glutamato/metabolismoRESUMO
The median (MR) and dorsal raphe (DR) nuclei contain the majority of the 5-hydroxytryptamine (5-HT, serotonin) neurons that project to limbic forebrain regions, are important in regulating homeostatic functions and are implicated in the etiology and treatment of mood disorders and schizophrenia. The primary synaptic inputs within and to the raphe are glutamatergic and GABAergic. The DR is divided into three subfields, i.e., ventromedial (vmDR), lateral wings (lwDR) and dorsomedial (dmDR). Our previous work shows that cell characteristics of 5-HT neurons and the magnitude of the 5-HT(1A) and 5-HT(1B) receptor-mediated responses in the vmDR and MR are not the same. We extend these observations to examine the electrophysiological properties across all four raphe subfields in both 5-HT and non-5-HT neurons. The neurochemical topography of glutamatergic and GABAergic cell bodies and nerve terminals were identified using immunohistochemistry and the morphology of the 5-HT neurons was measured. Although 5-HT neurons possessed similar physiological properties, important differences existed between subfields. Non-5-HT neurons were indistinguishable from 5-HT neurons. GABA neurons were distributed throughout the raphe, usually in areas devoid of 5-HT neurons. Although GABAergic synaptic innervation was dense throughout the raphe (immunohistochemical analysis of the GABA transporters GAT1 and GAT3), their distributions differed. Glutamate neurons, as defined by vGlut3 anti-bodies, were intermixed and co-localized with 5-HT neurons within all raphe subfields. Finally, the dendritic arbor of the 5-HT neurons was distinct between subfields. Previous studies regard 5-HT neurons as a homogenous population. Our data support a model of the raphe as an area composed of functionally distinct subpopulations of 5-HT and non-5-HT neurons, in part delineated by subfield. Understanding the interaction of the cell properties of the neurons in concert with their morphology, local distribution of GABA and glutamate neurons and their synaptic input, reveals a more complicated and heterogeneous raphe. These results provide an important foundation for understanding how specific subfields modulate behavior and for defining which aspects of the circuitry are altered during the etiology of psychological disorders.
Assuntos
Química Encefálica , Neurônios/fisiologia , Neurônios/ultraestrutura , Núcleos da Rafe/fisiologia , Núcleos da Rafe/ultraestrutura , Receptor 5-HT1A de Serotonina/metabolismo , Receptor 5-HT1B de Serotonina/metabolismo , Transmissão Sináptica , Animais , Dendritos/fisiologia , Dendritos/ultraestrutura , Impedância Elétrica , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Transportador de Glucose Tipo 3/metabolismo , Glutamato Descarboxilase/metabolismo , Imuno-Histoquímica , Masculino , Potenciais da Membrana , Técnicas de Patch-Clamp , Núcleos da Rafe/química , Ratos , Ratos Sprague-Dawley , Triptofano Hidroxilase/metabolismoRESUMO
Identifying the factors contributing to the etiology of anxiety and depression is critical for the development of more efficacious therapies. Serotonin (5-HT) is intimately linked to both disorders. The inhibitory serotonin-1A (5-HT(1A)) receptor exists in two separate populations with distinct effects on serotonergic signaling: (1) an autoreceptor that limits 5-HT release throughout the brain and (2) a heteroreceptor that mediates inhibitory responses to released 5-HT. Traditional pharmacologic and transgenic strategies have not addressed the distinct roles of these two receptor populations. Here we use a recently developed genetic mouse system to independently manipulate 5-HT(1A) autoreceptor and heteroreceptor populations. We show that 5-HT(1A) autoreceptors act to affect anxiety-like behavior. In contrast, 5-HT(1A) heteroreceptors affect responses to forced swim stress, without effects on anxiety-like behavior. Together with our previously reported work, these results establish distinct roles for the two receptor populations, providing evidence that signaling through endogenous 5-HT(1A) autoreceptors is necessary and sufficient for the establishment of normal anxiety-like behavior.
Assuntos
Ansiedade/metabolismo , Comportamento Animal/fisiologia , Rede Nervosa/metabolismo , Neurônios/metabolismo , Receptor 5-HT1A de Serotonina/metabolismo , Animais , Ansiedade/genética , Ansiedade/fisiopatologia , Autorradiografia , Catalepsia , Eletrofisiologia , Camundongos , Camundongos Knockout , Microdiálise , Rede Nervosa/fisiopatologia , Receptor 5-HT1A de Serotonina/genéticaAssuntos
Serotonina/fisiologia , Animais , Pesquisa Biomédica/tendências , Humanos , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Receptores de Serotonina/metabolismo , Serotoninérgicos/farmacologia , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Major depression is a debilitating psychiatric disease that may be precipitated by a dysregulation of stress neurocircuitry caused by chronic or severe stress exposure. Moreover, hyperresponsivity to stressors correlates with depressed mood and may contribute to the etiology of major depression. The serotonergic dorsal raphe nucleus (DRN) is an important site in the neurocircuitry underlying behavioral responses to stressors, and is tightly regulated, in part, by a combination of intrinsic cell properties, autoinhibition, and GABAergic synaptic transmission. The stress-related neurotransmitter corticotropin-releasing factor (CRF) modulates DRN neuronal excitability and subsequent 5-HT release in the forebrain. Wistar Kyoto (WKY) rats exhibit exaggerated behavioral responses to stressors, that is, stress hyperresponsivity, and are considered an animal model of depression. To better understand the neurobiological basis of the stress hyperresponsivity, we used a combination of mRNA analysis and whole-cell electrophysiological techniques to measure differences in intrinsic activity and receptor response, in 5-HT- and non-5-HT-containing neurons of the DRN in WKY rats compared with Sprague-Dawley controls. In the WKY rat, there was a decrease in the neuronal excitability of 5-HT neurons coupled with decreased TPH2 production. Additionally, we found that CRF did not increase GABAergic activity in 5-HT neurons as is normally seen in 5-HT neurons of Sprague-Dawley controls. The CRF modulation of 5-HT DRN neurotransmission at the single-cell level is selectively disrupted in the WKY animal model of depression and may be one of the cellular correlates underlying depression.