RESUMO
The size and structure of the dendritic arbor play important roles in determining how synaptic inputs of neurons are converted to action potential output. The regulatory mechanisms governing the development of dendrites, however, are insufficiently understood. The evolutionary conserved Ste20/Hippo kinase pathway has been proposed to play an important role in regulating the formation and maintenance of dendritic architecture. A key element of this pathway, Ste20-like kinase (SLK), regulates cytoskeletal dynamics in non-neuronal cells and is strongly expressed throughout neuronal development. However, its function in neurons is unknown. We show that, during development of mouse cortical neurons, SLK has a surprisingly specific role for proper elaboration of higher, ≥ third-order dendrites both in male and in female mice. Moreover, we demonstrate that SLK is required to maintain excitation-inhibition balance. Specifically, SLK knockdown caused a selective loss of inhibitory synapses and functional inhibition after postnatal day 15, whereas excitatory neurotransmission was unaffected. Finally, we show that this mechanism may be relevant for human disease, as dysmorphic neurons within human cortical malformations revealed significant loss of SLK expression. Overall, the present data identify SLK as a key regulator of both dendritic complexity during development and inhibitory synapse maintenance.SIGNIFICANCE STATEMENT We show that dysmorphic neurons of human epileptogenic brain lesions have decreased levels of the Ste20-like kinase (SLK). Decreasing SLK expression in mouse neurons revealed that SLK has essential functions in forming the neuronal dendritic tree and in maintaining inhibitory connections with neighboring neurons.
Assuntos
Córtex Cerebral/metabolismo , Dendritos/genética , Inibição Neural/genética , Proteínas Serina-Treonina Quinases/genética , Sinapses/genética , Transmissão Sináptica/fisiologia , Adolescente , Adulto , Idoso , Animais , Córtex Cerebral/patologia , Criança , Pré-Escolar , Dendritos/metabolismo , Dendritos/patologia , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Proteínas Serina-Treonina Quinases/metabolismo , Sinapses/metabolismo , Sinapses/patologia , Adulto JovemRESUMO
Neuregulin 1 (NRG1) and its receptor ERBB4 are schizophrenia (SZ) risk genes that control the development of both excitatory and inhibitory cortical circuits. Most studies focused on the characterization ErbB4 deficient mice. However, ErbB4 deletion concurrently perturbs the signaling of Nrg1 and Neuregulin 3 (Nrg3), another ligand expressed in the cortex. In addition, NRG1 polymorphisms linked to SZ locate mainly in non-coding regions and they may partially reduce Nrg1 expression. Here, to study the relevance of Nrg1 partial loss-of-function in cortical circuits we characterized a recently developed haploinsufficient mouse model of Nrg1 (Nrg1tm1Lex). These mice display SZ-like behavioral deficits. The cellular and molecular underpinnings of the behavioral deficits in Nrg1tm1Lex mice remain to be established. With multiple approaches including Magnetic Resonance Spectroscopy (MRS), electrophysiology, quantitative imaging and molecular analysis we found that Nrg1 haploinsufficiency impairs the inhibitory cortical circuits. We observed changes in the expression of molecules involved in GABAergic neurotransmission, decreased density of Vglut1 excitatory buttons onto Parvalbumin interneurons and decreased frequency of spontaneous inhibitory postsynaptic currents. Moreover, we found a decreased number of Parvalbumin positive interneurons in the cortex and altered expression of Calretinin. Interestingly, we failed to detect other alterations in excitatory neurons that were previously reported in ErbB4 null mice suggesting that the Nrg1 haploinsufficiency does not entirely phenocopies ErbB4 deletions. Altogether, this study suggests that Nrg1 haploinsufficiency primarily affects the cortical inhibitory circuits in the cortex and provides new insights into the structural and molecular synaptic impairment caused by NRG1 hypofunction in a preclinical model of SZ.
Assuntos
Córtex Cerebral/metabolismo , Neurônios GABAérgicos/metabolismo , Hipocampo/metabolismo , Potenciais Pós-Sinápticos Inibidores/genética , Interneurônios/metabolismo , Inibição Neural/genética , Neuregulina-1/genética , Proteína Vesicular 1 de Transporte de Glutamato/metabolismo , Animais , Calbindina 2/metabolismo , Córtex Cerebral/diagnóstico por imagem , Córtex Cerebral/patologia , Córtex Cerebral/fisiopatologia , Neurônios GABAérgicos/patologia , Expressão Gênica , Haploinsuficiência , Hipocampo/diagnóstico por imagem , Hipocampo/patologia , Hipocampo/fisiopatologia , Interneurônios/patologia , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Camundongos , Parvalbuminas/metabolismo , RNA Mensageiro/metabolismo , Receptor ErbB-4/genética , Ácido gama-Aminobutírico/metabolismoRESUMO
BACKGROUND: In an early stage of Alzheimer's disease (AD), before the formation of amyloid plaques, neuronal network hyperactivity has been reported in both patients and animal models. This suggests an underlying disturbance of the balance between excitation and inhibition. Several studies have highlighted the role of somatic inhibition in early AD, while less is known about dendritic inhibition. OBJECTIVE: In this study we investigated how inhibitory synaptic currents are affected by elevated Aß levels. METHODS: We performed whole-cell patch clamp recordings of CA1 pyramidal neurons in organotypic hippocampal slice cultures after treatment with Aß-oligomers and in hippocampal brain slices from AppNL-F-G mice (APP-KI). RESULTS: We found a reduction of spontaneous inhibitory postsynaptic currents (sIPSCs) in CA1 pyramidal neurons in organotypic slices after 24âh Aß treatment. sIPSCs with slow rise times were reduced, suggesting a specific loss of dendritic inhibitory inputs. As miniature IPSCs and synaptic density were unaffected, these results suggest a decrease in activity-dependent transmission after Aß treatment. We observed a similar, although weaker, reduction in sIPSCs in CA1 pyramidal neurons from APP-KI mice compared to control. When separated by sex, the strongest reduction in sIPSC frequency was found in slices from male APP-KI mice. Consistent with hyperexcitability in pyramidal cells, dendritically targeting interneurons received slightly more excitatory input. GABAergic action potentials had faster kinetics in APP-KI slices. CONCLUSION: Our results show that Aß affects dendritic inhibition via impaired action potential driven release, possibly due to altered kinetics of GABAergic action potentials. Reduced dendritic inhibition may contribute to neuronal hyperactivity in early AD.
Assuntos
Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Dendritos/metabolismo , Potenciais Pós-Sinápticos Inibidores/genética , Fragmentos de Peptídeos/metabolismo , Células Piramidais/metabolismo , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/farmacologia , Amiloidose , Animais , Região CA1 Hipocampal/citologia , Dendritos/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Imuno-Histoquímica , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Inibição Neural/efeitos dos fármacos , Inibição Neural/genética , Técnicas de Patch-Clamp , Células Piramidais/efeitos dos fármacosRESUMO
Microglia, the brain's resident macrophages, help to regulate brain function by removing dying neurons, pruning non-functional synapses, and producing ligands that support neuronal survival1. Here we show that microglia are also critical modulators of neuronal activity and associated behavioural responses in mice. Microglia respond to neuronal activation by suppressing neuronal activity, and ablation of microglia amplifies and synchronizes the activity of neurons, leading to seizures. Suppression of neuronal activation by microglia occurs in a highly region-specific fashion and depends on the ability of microglia to sense and catabolize extracellular ATP, which is released upon neuronal activation by neurons and astrocytes. ATP triggers the recruitment of microglial protrusions and is converted by the microglial ATP/ADP hydrolysing ectoenzyme CD39 into AMP; AMP is then converted into adenosine by CD73, which is expressed on microglia as well as other brain cells. Microglial sensing of ATP, the ensuing microglia-dependent production of adenosine, and the adenosine-mediated suppression of neuronal responses via the adenosine receptor A1R are essential for the regulation of neuronal activity and animal behaviour. Our findings suggest that this microglia-driven negative feedback mechanism operates similarly to inhibitory neurons and is essential for protecting the brain from excessive activation in health and disease.
Assuntos
Retroalimentação Fisiológica , Microglia/fisiologia , Inibição Neural , Neurônios/fisiologia , 5'-Nucleotidase/metabolismo , Potenciais de Ação , Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Antígenos CD/metabolismo , Apirase/metabolismo , Cálcio/metabolismo , Corpo Estriado/citologia , Corpo Estriado/fisiologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/citologia , Inibição Neural/genética , Receptor A1 de Adenosina/metabolismo , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/metabolismo , Fatores de TempoRESUMO
The balance of excitation and inhibition is essential for cortical information processing, relying on the tight orchestration of the underlying subcellular processes. Dynamic transcriptional control by DNA methylation, catalyzed by DNA methyltransferases (DNMTs), and DNA demethylation, achieved by ten-eleven translocation (TET)-dependent mechanisms, is proposed to regulate synaptic function in the adult brain with implications for learning and memory. However, focus so far is laid on excitatory neurons. Given the crucial role of inhibitory cortical interneurons in cortical information processing and in disease, deciphering the cellular and molecular mechanisms of GABAergic transmission is fundamental. The emerging relevance of DNMT and TET-mediated functions for synaptic regulation irrevocably raises the question for the targeted subcellular processes and mechanisms. In this study, we analyzed the role dynamic DNA methylation has in regulating cortical interneuron function. We found that DNMT1 and TET1/TET3 contrarily modulate clathrin-mediated endocytosis. Moreover, we provide evidence that DNMT1 influences synaptic vesicle replenishment and GABAergic transmission, presumably through the DNA methylation-dependent transcriptional control over endocytosis-related genes. The relevance of our findings is supported by human brain sample analysis, pointing to a potential implication of DNA methylation-dependent endocytosis regulation in the pathophysiology of temporal lobe epilepsy, a disease characterized by disturbed synaptic transmission.
Assuntos
Metilação de DNA/genética , Endocitose/genética , Neurônios GABAérgicos/metabolismo , Interneurônios/metabolismo , Inibição Neural/genética , Sinapses/metabolismo , Animais , Clatrina , Proteínas do Citoesqueleto/genética , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dioxigenases/genética , Dioxigenases/metabolismo , Epigenoma , Epilepsia do Lobo Temporal/genética , Humanos , Potenciais Pós-Sinápticos Inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Técnicas de Patch-Clamp , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vesículas Sinápticas/metabolismo , TranscriptomaRESUMO
Neurofibromatosis type 1 (NF1) is an autosomal dominant disease caused by loss-of-function mutations in NF1 gene, which encodes a GTPase activating protein for RAS. NF1 affects multiple systems including brain and is highly associated with cognitive deficits such as learning difficulties and attention deficits. Previous studies have suggested that GABAergic inhibitory neuron is the cell type primarily responsible for the learning deficits in mouse models of NF1. However, it is not clear how NF1 mutations selectively affect inhibitory neurons in the central nervous system. In this study, we show that the expression level of Nf1 is significantly higher in inhibitory neurons than in excitatory neurons in mouse hippocampus and cortex by using in situ hybridization. Furthermore, we also found that NF1 is enriched in inhibitory neurons in the human cortex, confirming that the differential expressions of NF1 between two cell types are evolutionarily conserved. Our results suggest that the enriched expression of NF1 in inhibitory neurons may underlie inhibitory neuron-specific deficits in NF1.
Assuntos
Inibição Neural , Neurofibromina 1/genética , Neurônios/metabolismo , Animais , Encéfalo/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Inibição Neural/genética , Neurofibromina 1/metabolismo , Transdução de SinaisRESUMO
Inhibitory neurons are crucial for shaping and regulating the dynamics of the entire network, and disturbances in these neurons contribute to brain disorders. Despite the recent progress in genetic labeling techniques, the heterogeneity of inhibitory neurons requires the development of highly characterized tools that allow accurate, convenient, and versatile visualization of inhibitory neurons in the mouse brain. Here, we report a novel genetic technique to visualize the vast majority and/or sparse subsets of inhibitory neurons in the mouse brain without using techniques that require advanced skills. We developed several lines of Cre-dependent tdTomato reporter mice based on the vesicular GABA transporter (VGAT)-BAC, named VGAT-stop-tdTomato mice. The most useful line (line #54) was selected for further analysis based on two characteristics: the inhibitory neuron-specificity of tdTomato expression and the transgene integration site, which confers efficient breeding and fewer adverse effects resulting from transgene integration-related genomic disruption. Robust and inhibitory neuron-specific expression of tdTomato was observed in a wide range of developmental and cellular contexts. By breeding the VGAT-stop-tdTomato mouse (line #54) with a novel Cre driver mouse line, Galntl4-CreER, sparse labeling of inhibitory neurons was achieved following tamoxifen administration. Furthermore, another interesting line (line #58) was generated through the unexpected integration of the transgene into the X-chromosome and will be used to map X-chromosome inactivation of inhibitory neurons. Taken together, our studies provide new, well-characterized tools with which multiple aspects of inhibitory neurons can be studied in the mouse.
Assuntos
Integrases/metabolismo , Proteínas Luminescentes/metabolismo , Inibição Neural/genética , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismo , Animais , Encéfalo/citologia , Antagonistas de Estrogênios/farmacologia , Regulação da Expressão Gênica/genética , Glutamato Descarboxilase/genética , Glutamato Descarboxilase/metabolismo , Integrases/genética , Antígeno Ki-67/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/genética , N-Acetilgalactosaminiltransferases/genética , N-Acetilgalactosaminiltransferases/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fator de Transcrição PAX2/metabolismo , Proteínas RGS/genética , Proteínas RGS/metabolismo , Somatostatina/metabolismo , Tamoxifeno/farmacologia , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/genética , Polipeptídeo N-AcetilgalactosaminiltransferaseRESUMO
Schizophrenia is a neurodevelopmental disorder characterized by abnormal processing of information and attentional deficits. Schizophrenia has a high genetic component but is precipitated by environmental factors, as proposed by the 'two-hit' theory of schizophrenia. Here we compared latent inhibition as a measure of learning and attention, in CHL1-deficient mice, an animal model of schizophrenia, and their wild-type littermates, under no-stress and chronic mild stress conditions. All unstressed mice as well as the stressed wild-type mice showed latent inhibition. In contrast, CHL1-deficient mice did not show latent inhibition after exposure to chronic stress. Differences in neuronal activation (c-Fos-positive cell counts) were noted in brain regions associated with latent inhibition: Neuronal activation in the prelimbic/infralimbic cortices and the nucleus accumbens shell was affected solely by stress. Neuronal activation in basolateral amygdala and ventral hippocampus was affected independently by stress and genotype. Most importantly, neural activation in nucleus accumbens core was affected by the interaction between stress and genotype. These results provide strong support for a 'two-hit' (genes x environment) effect on latent inhibition in CHL1-deficient mice, and identify CHL1-deficient mice as a model of schizophrenia-like learning and attention impairments.
Assuntos
Encéfalo/patologia , Moléculas de Adesão Celular/deficiência , Inibição Psicológica , Neurônios/metabolismo , Esquizofrenia/patologia , Estresse Psicológico/patologia , Animais , Moléculas de Adesão Celular/genética , Condicionamento Psicológico/fisiologia , Modelos Animais de Doenças , Congelamento , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Inibição Neural/genética , Proteínas Proto-Oncogênicas c-fos/metabolismoRESUMO
Rapid eye movement (REM) sleep behavior disorder in humans is often accompanied by a reduced ability to smell and detect odors, and olfactory bulbectomized rats exhibit increased REM sleep, suggesting that the olfactory bulb (OB) is involved in REM-sleep regulation. However, the molecular mechanism of REM-sleep regulation by the OB is unknown. Adenosine promotes sleep and its A2A receptors (A2AR) are expressed in the OB. We hypothesized that A2AR in the OB regulate REM sleep. Bilateral microinjections of the A2AR antagonist SCH58261 into the rat OB increased REM sleep, whereas microinjections of the A2AR agonist CGS21680 decreased REM sleep. Similar to the A2AR antagonist, selective A2AR knockdown by adeno-associated virus carrying short-hairpin RNA for A2AR in the rat OB increased REM sleep. Using chemogenetics on the basis of designer receptors exclusively activated by designer drugs, we demonstrated that the inhibition of A2AR neurons increased REM sleep, whereas the activation of these neurons decreased REM sleep. Moreover, using a conditional anterograde axonal tract-tracing approach, we found that OB A2AR neurons innervate the piriform cortex and olfactory tubercle. These novel findings indicate that adenosine suppresses REM sleep via A2AR in the OB of rodents.
Assuntos
Bulbo Olfatório/fisiologia , Receptor A2A de Adenosina/metabolismo , Sono REM/fisiologia , Adenosina/análogos & derivados , Adenosina/farmacologia , Agonistas do Receptor A2 de Adenosina/farmacologia , Antagonistas do Receptor A2 de Adenosina/farmacologia , Análise de Variância , Animais , Dependovirus/genética , Relação Dose-Resposta a Droga , Eletroencefalografia , Eletromiografia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Inibição Neural/efeitos dos fármacos , Inibição Neural/genética , Bulbo Olfatório/efeitos dos fármacos , Técnicas de Patch-Clamp , Fenetilaminas/farmacologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Pirimidinas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor A2A de Adenosina/genética , Sono REM/genética , Transdução Genética , Triazóis/farmacologiaRESUMO
GABAergic interneurons are positioned to powerfully influence the dynamics of neural activity, yet the interneuron-mediated circuit mechanisms that control spontaneous and evoked neocortical activity remains elusive. Vasoactive intestinal peptide (VIP+) interneurons are a specialized cell class which synapse specifically on other interneurons, potentially serving to facilitate increases in cortical activity. In this study, using in vivo Ca(2+) imaging, we describe the interaction between local network activity and VIP+ cells and determine their role in modulating neocortical activity in mouse visual cortex. VIP+ cells were active across brain states including locomotion, nonlocomotion, visual stimulation, and under anesthesia. VIP+ activity correlated most clearly with the mean level of population activity of nearby excitatory neurons during all brain states, suggesting VIP+ cells enable high-excitability states in the cortex. The pharmacogenetic blockade of VIP+ cell output reduced network activity during locomotion, nonlocomotion, anesthesia, and visual stimulation, suggesting VIP+ cells exert a state-independent facilitation of neural activity in the cortex. Collectively, our findings demonstrate that VIP+ neurons have a causal role in the generation of high-activity regimes during spontaneous and stimulus evoked neocortical activity.
Assuntos
Interneurônios/fisiologia , Neocórtex/fisiologia , Inibição Neural/fisiologia , Peptídeo Intestinal Vasoativo/metabolismo , Córtex Visual/citologia , Animais , Cálcio/metabolismo , Clozapina/análogos & derivados , Clozapina/farmacologia , Feminino , Interneurônios/efeitos dos fármacos , Locomoção/fisiologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Rede Nervosa/fisiologia , Inibição Neural/efeitos dos fármacos , Inibição Neural/genética , Estimulação Luminosa , Receptor Muscarínico M4/genética , Receptor Muscarínico M4/metabolismo , Somatostatina/genética , Somatostatina/metabolismo , Transdução Genética , Peptídeo Intestinal Vasoativo/genética , Ácido gama-AminobutíricoRESUMO
It is well known that the neuronal effects of vascular endothelial growth factor (VEGF) include modulating learning and memory, plasticity of mature neurons, and synaptic transmission in addition to neurogenesis. However, there is conflicting evidence particularly of its role in the regulation of excitatory synaptic activity. In this study, application of the patch-clamp technique revealed that lower doses (10 and 50 ng/mL) of VEGF enhanced excitatory neurotransmission in hippocampal slices of mice through both presynaptic and postsynaptic mechanisms. However, the effects were reversed by higher doses of VEGF (>100 ng/mL), which inhibited excitatory neurotransmission via a presynaptic mechanism. These competing, concentration-dependent effects of VEGF suggested that different pathways were involved. The involvement of the Notch1 receptor was tested in the modulation of VEGF on synaptic activity by using heterozygous Notch1(+/-) mice. Notch1 knockdown did not influence the inhibitory effect of high VEGF doses (200 ng/mL) but reduced the enhancement effects of low concentration of VEGF (50 ng/mL) at the postsynaptic level, which might be due to the decreased level of VEGF receptor. The results indicate that the Notch1 receptor plays a role in VEGF-induced modulation of synaptic activity, which provides new insights into a complex VEGF/Notch signaling cross-talk. These findings set the groundwork for understanding new mechanisms of Notch signaling and the neurotrophic effects of VEGF, which is beneficial to develop new therapeutic targets to the VEGF/Notch axis and improve current treatments for neural diseases.
Assuntos
Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/genética , Hipocampo/citologia , Neurônios/efeitos dos fármacos , Receptor Notch1/deficiência , Fator A de Crescimento do Endotélio Vascular/farmacologia , Animais , Animais Recém-Nascidos , Fenômenos Biofísicos/efeitos dos fármacos , Fenômenos Biofísicos/genética , Relação Dose-Resposta a Droga , Estimulação Elétrica , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Inibição Neural/efeitos dos fármacos , Inibição Neural/genética , Neurônios/fisiologia , Receptor Notch1/genética , Estatísticas não Paramétricas , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Interference between similar or overlapping memories formed at different times poses an important challenge on the hippocampal declarative memory system. Difficulties in managing interference are at the core of disabling cognitive deficits in neuropsychiatric disorders. Computational models have suggested that, in the normal brain, the sparse activation of the dentate gyrus granule cells maintained by tonic inhibitory control enables pattern separation, an orthogonalization process that allows distinct representations of memories despite interference. To test this mechanistic hypothesis, we generated mice with significantly reduced expression of the α5-containing GABAA (α5-GABAARs) receptors selectively in the granule cells of the dentate gyrus (α5DGKO mice). α5DGKO mice had reduced tonic inhibition of the granule cells without any change in fast phasic inhibition and showed increased activation in the dentate gyrus when presented with novel stimuli. α5DGKO mice showed impairments in cognitive tasks characterized by high interference, without any deficiencies in low-interference tasks, suggesting specific impairment of pattern separation. Reduction of fast phasic inhibition in the dentate gyrus through granule cell-selective knock-out of α2-GABAARs or the knock-out of the α5-GABAARs in the downstream CA3 area did not detract from pattern separation abilities, which confirms the anatomical and molecular specificity of the findings. In addition to lending empirical support to computational hypotheses, our findings have implications for the treatment of interference-related cognitive symptoms in neuropsychiatric disorders, particularly considering the availability of pharmacological agents selectively targeting α5-GABAARs. SIGNIFICANCE STATEMENT: Interference between similar memories poses a significant limitation on the hippocampal declarative memory system, and impaired interference management is a cognitive symptom in many disorders. Thus, understanding mechanisms of successful interference management or processes that can lead to interference-related memory problems has high theoretical and translational importance. This study provides empirical evidence that tonic inhibition in the dentate gyrus (DG), which maintains sparseness of neuronal activation in the DG, is essential for management of interference. The specificity of findings to tonic, but not faster, more transient types of neuronal inhibition and to the DG, but not the neighboring brain areas, is presented through control experiments. Thus, the findings link interference management to a specific mechanism, proposed previously by computational models.
Assuntos
Giro Denteado/citologia , Memória/fisiologia , Inibição Neural/genética , Neurônios/fisiologia , Receptores de GABA-A/metabolismo , Animais , Discriminação Psicológica/fisiologia , Comportamento Exploratório/fisiologia , Agonistas GABAérgicos/farmacocinética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Imidazóis/farmacocinética , Técnicas In Vitro , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Potenciais Pós-Sinápticos Inibidores/genética , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Receptores de GABA-A/genética , Reconhecimento Psicológico/fisiologia , Natação/psicologiaRESUMO
The amygdala is a key region for the processing of information underlying fear, anxiety, and fear extinction. Within the local neuronal networks of the amygdala, a population of inhibitory, intercalated neurons (ITCs) modulates the flow of information among various nuclei of amygdala, including the basal nucleus (BA) and the centromedial nucleus (CeM) of the amygdala. These ITCs have been shown to be important during fear extinction and are target of a variety of neurotransmitters and neuropeptides. Here we provide evidence that the activation of µ-opioid receptors (MORs) by the specific agonist DAMGO ([D-Ala2,N-Me-Phe4,Gly5-ol]-Enkephalin) hyperpolarizes medially located ITCs (mITCs) in acute brain slices of mice. Moreover, we use whole-cell patch-clamp recordings in combination with local electrical stimulation or glutamate uncaging to analyze the effect of MOR activation on local microcircuits. We show that the GABAergic transmission between mITCs and CeM neurons is attenuated by DAMGO, whereas the glutamatergic transmission on CeM neurons and mITCs is unaffected. Furthermore, MOR activation induced by theta burst stimulation in BA suppresses plastic changes of feedforward inhibitory transmission onto CeM neurons as revealed by the MOR antagonist CTAP d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH2. In summary, the mITCs constitute a target for the opioid system, and therefore, the activation of MOR in ITCs might play a central role in the modulation of the information processing between the basolateral complex of the amygdala and central nuclei of the amygdala.
Assuntos
Núcleo Central da Amígdala/citologia , Inibição Neural/fisiologia , Neurônios/fisiologia , Receptores Opioides mu/fisiologia , Transmissão Sináptica/fisiologia , Analgésicos Opioides/farmacologia , Animais , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Glutamato Descarboxilase/genética , Glutamato Descarboxilase/metabolismo , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Antagonistas de Entorpecentes/farmacologia , Inibição Neural/efeitos dos fármacos , Inibição Neural/genética , Neurônios/efeitos dos fármacos , Peptídeos/farmacologia , Quinoxalinas/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/genética , Tetrodotoxina/farmacologia , Valina/análogos & derivados , Valina/farmacologiaRESUMO
Cognitive impairments are a major clinical feature of the common neurogenetic disease neurofibromatosis type 1 (NF1). Previous studies have demonstrated that increased neuronal inhibition underlies the learning deficits in NF1, however, the molecular mechanism underlying this cell-type specificity has remained unknown. Here, we identify an interneuron-specific attenuation of hyperpolarization-activated cyclic nucleotide-gated (HCN) current as the cause for increased inhibition in Nf1 mutants. Mechanistically, we demonstrate that HCN1 is a novel NF1-interacting protein for which loss of NF1 results in a concomitant increase of interneuron excitability. Furthermore, the HCN channel agonist lamotrigine rescued the electrophysiological and cognitive deficits in two independent Nf1 mouse models, thereby establishing the importance of HCN channel dysfunction in NF1. Together, our results provide detailed mechanistic insights into the pathophysiology of NF1-associated cognitive defects, and identify a novel target for clinical drug development.
Assuntos
Transtornos Cognitivos/tratamento farmacológico , Transtornos Cognitivos/metabolismo , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Neurofibromatose 1/complicações , Canais de Potássio/metabolismo , Animais , Transtornos Cognitivos/etiologia , Transtornos Cognitivos/genética , Modelos Animais de Doenças , Antagonistas de Aminoácidos Excitatórios/uso terapêutico , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/genética , Hipocampo/citologia , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Lamotrigina , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Atividade Motora/efeitos dos fármacos , Atividade Motora/genética , Mutação/genética , Inibição Neural/efeitos dos fármacos , Inibição Neural/genética , Neurofibromatose 1/genética , Neurofibromina 1/genética , Neurofibromina 1/metabolismo , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/genética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Canais de Potássio/genética , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Triazinas/uso terapêuticoRESUMO
In cortical networks, different types of inhibitory interneurons control the activity of glutamatergic principal cells and GABAergic interneurons. Principal neurons represent the major postsynaptic target of most interneurons; however, a population of interneurons that is dedicated to the selective innervation of GABAergic cells exists in the CA1 area of the hippocampus. The physiological properties of these cells and their functional relevance for network computations remain unknown. Here, we used a combination of dual simultaneous patch-clamp recordings and targeted optogenetic stimulation in acute mouse hippocampal slices to examine how one class of interneuron-specific (IS) cells controls the activity of its GABAergic targets. We found that type 3 IS (IS3) cells that coexpress the vasoactive intestinal polypeptide (VIP) and calretinin contact several distinct types of interneurons within the hippocampal CA1 stratum oriens/alveus (O/A), with preferential innervation of oriens-lacunosum moleculare cells (OLMs) through dendritic synapses. In contrast, VIP-positive basket cells provided perisomatic inhibition to CA1 pyramidal neurons with the asynchronous GABA release and were not connected with O/A interneurons. Furthermore, unitary IPSCs recorded at IS3-OLM synapses had a small amplitude and low release probability but summated efficiently during high-frequency firing of IS3 interneurons. Moreover, the synchronous generation of a single spike in several IS cells that converged onto a single OLM controlled the firing rate and timing of OLM interneurons. Therefore, dendritic inhibition originating from IS cells is needed for the flexible activity-dependent recruitment of OLM interneurons for feedback inhibition.
Assuntos
Potenciais de Ação/fisiologia , Dendritos/fisiologia , Hipocampo/citologia , Interneurônios/fisiologia , Rede Nervosa/fisiologia , Inibição Neural/fisiologia , Potenciais de Ação/genética , Animais , Animais Recém-Nascidos , Dendritos/efeitos dos fármacos , Feminino , Antagonistas GABAérgicos/farmacologia , Proteínas de Fluorescência Verde/genética , Técnicas In Vitro , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Potenciais Pós-Sinápticos Inibidores/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Rede Nervosa/efeitos dos fármacos , Inibição Neural/efeitos dos fármacos , Inibição Neural/genética , Piridazinas/farmacologia , Fatores de Tempo , Peptídeo Intestinal Vasoativo/genéticaRESUMO
OBJECTIVE: To develop a constitutively active K(+) leak channel using TREK-1 (TWIK-related potassium channel 1; TREK-M) that is resistant to compensatory down-regulation by second messenger cascades, and to validate the ability of TREK-M to silence hyperactive neurons using cultured hippocampal neurons. To test if adenoassociated viral (AAV) delivery of TREK-M could reduce the duration of status epilepticus and reduce neuronal death induced by lithium-pilocarpine administration. METHODS: Molecular cloning techniques were used to engineer novel vectors to deliver TREK-M via plasmids, lentivirus, and AAV using a cytomegalovirus (CMV)-enhanced GABRA4 promoter. Electrophysiology was used to characterize the activity and regulation of TREK-M in human embryonic kidney (HEK-293) cells, and the ability to reduce spontaneous activity in cultured hippocampal neurons. Adult male rats were injected bilaterally with self-complementary AAV particles composed of serotype 5 capsid into the hippocampus and entorhinal cortex. Lithium-pilocarpine was used to induce status epilepticus. Seizures were monitored using continuous video-electroencephalography (EEG) monitoring. Neuronal death was measured using Fluoro-Jade C staining of paraformaldehyde-fixed brain slices. RESULTS: TREK-M inhibited neuronal firing by hyperpolarizing the resting membrane potential and decreasing input resistance. AAV delivery of TREK-M decreased the duration of status epilepticus by 50%. Concomitantly it reduced neuronal death in areas targeted by the AAV injection. SIGNIFICANCE: These findings demonstrate that TREK-M can silence hyperexcitable neurons in the brain of epileptic rats and treat acute seizures. This study paves the way for an alternative gene therapy treatment of status epilepticus, and provides the rationale for studies of AAV-TREK-M's effect on spontaneous seizures in chronic models of temporal lobe epilepsy.
Assuntos
Técnicas de Transferência de Genes , Neurônios/patologia , Canais de Potássio de Domínios Poros em Tandem/genética , Estado Epiléptico/genética , Estado Epiléptico/prevenção & controle , Animais , Morte Celular/genética , Polaridade Celular/genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Células HEK293 , Humanos , Masculino , Inibição Neural/genética , Neurônios/fisiologia , Canais de Potássio de Domínios Poros em Tandem/administração & dosagem , Ratos , Ratos Sprague-Dawley , Estado Epiléptico/patologiaRESUMO
The influence of motor activity on sensory processing is crucial for perception and motor execution. However, the underlying circuits are not known. To unravel the circuit by which activity in the primary vibrissal motor cortex (vM1) modulates sensory processing in the primary somatosensory barrel cortex (S1), we used optogenetics to examine the long-range inputs from vM1 to the various neuronal elements in S1. We found that S1-projecting vM1 pyramidal neurons strongly recruited vasointestinal peptide (VIP)-expressing GABAergic interneurons, a subset of serotonin receptor-expressing interneurons. These VIP interneurons preferentially inhibited somatostatin-expressing interneurons, neurons that target the distal dendrites of pyramidal cells. Consistent with this vM1-mediated disinhibitory circuit, the activity of VIP interneurons in vivo increased and that of somatostatin interneurons decreased during whisking. These changes in firing rates during whisking depended on vM1 activity. Our results suggest previously unknown circuitry by which inputs from motor cortex influence sensory processing in sensory cortex.
Assuntos
Vias Aferentes/fisiologia , Interneurônios/fisiologia , Rede Nervosa/fisiologia , Inibição Neural/fisiologia , Córtex Somatossensorial/citologia , Vias Aferentes/efeitos dos fármacos , Anestésicos Locais/farmacologia , Animais , Animais Recém-Nascidos , Channelrhodopsins , Estimulação Elétrica , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/genética , Proteínas de Homeodomínio/genética , Técnicas In Vitro , Interneurônios/classificação , Camundongos , Camundongos Transgênicos , Inibição Neural/genética , Parvalbuminas/genética , Técnicas de Patch-Clamp , Estimulação Física , Receptores 5-HT3 de Serotonina/genética , Somatostatina/genética , Tetrodotoxina/farmacologia , Fatores de Transcrição/genética , Transdução Genética , Peptídeo Intestinal Vasoativo/genética , Vibrissas/inervaçãoRESUMO
Sensory experience alters cortical circuitry by parallel processes of axon outgrowth and pruning, but the mechanisms that control these rearrangements are poorly understood. Using in vivo 2-photon longitudinal imaging, we found a marked reduction in axonal pruning in somatosensory cortex of mice with a knock-out of the DR6 gene, which codes for Death Receptor 6. This effect was seen for both long-range horizontal excitatory connections and for the axons of inhibitory neurons. These results identify a new pathway governing axonal plasticity associated with experience-dependent changes in cortical maps.
Assuntos
Plasticidade Neuronal/fisiologia , Neurônios/fisiologia , Receptores do Fator de Necrose Tumoral/metabolismo , Córtex Somatossensorial , Vibrissas/inervação , Animais , Axônios/fisiologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Dependovirus , Glutamato Descarboxilase/genética , Glutamato Descarboxilase/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Knockout , Inibição Neural/genética , Plasticidade Neuronal/genética , Estimulação Física , Terminações Pré-Sinápticas/fisiologia , Receptores do Fator de Necrose Tumoral/deficiência , Córtex Somatossensorial/citologia , Córtex Somatossensorial/metabolismo , Córtex Somatossensorial/fisiologia , Transdução GenéticaRESUMO
Systems of coupled oscillators abound in nature. How they establish stable phase relationships under diverse conditions is fundamentally important. The mammalian suprachiasmatic nucleus (SCN) is a self-sustained, synchronized network of circadian oscillators that coordinates daily rhythms in physiology and behavior. To elucidate the underlying topology and signaling mechanisms that modulate circadian synchrony, we discriminated the firing of hundreds of SCN neurons continuously over days. Using an analysis method to identify functional interactions between neurons based on changes in their firing, we characterized a GABAergic network comprised of fast, excitatory, and inhibitory connections that is both stable over days and changes in strength with time of day. By monitoring PERIOD2 protein expression, we provide the first evidence that these millisecond-level interactions actively oppose circadian synchrony and inject jitter into daily rhythms. These results provide a mechanism by which circadian oscillators can tune their phase relationships under different environmental conditions.
Assuntos
Ritmo Circadiano/fisiologia , Rede Nervosa/fisiologia , Neurônios/fisiologia , Periodicidade , Ácido gama-Aminobutírico/metabolismo , Potenciais de Ação/genética , Animais , Mapeamento Encefálico , Ritmo Circadiano/genética , Colchicina/farmacologia , Antagonistas GABAérgicos/farmacologia , Luciferases/genética , Medições Luminescentes , Camundongos , Camundongos Transgênicos , Rede Nervosa/efeitos dos fármacos , Inibição Neural/efeitos dos fármacos , Inibição Neural/genética , Técnicas de Cultura de Órgãos , Técnicas de Patch-Clamp , Proteínas Circadianas Period/genética , Piridazinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Núcleo Supraquiasmático/citologia , Fatores de Tempo , Moduladores de Tubulina/farmacologia , Peptídeo Intestinal Vasoativo/deficiênciaRESUMO
Tonic inhibitory GABA(A) receptor-mediated currents are observed in numerous cell types in the CNS, including thalamocortical neurons of the ventrobasal thalamus, dentate gyrus granule cells, and cerebellar granule cells. Here we show that in rat brain slices, activation of postsynaptic GABA(B) receptors enhances the magnitude of the tonic GABA(A) current recorded in these cell types via a pathway involving G G proteins, adenylate cyclase, and cAMP-dependent protein kinase. Using a combination of pharmacology and knockout mice, we show that this pathway is independent of potassium channels or GABA transporters. Furthermore, the enhancement in tonic current is sufficient to significantly alter the excitability of thalamocortical neurons. These results demonstrate for the first time a postsynaptic crosstalk between GABA(B) and GABA(A) receptors.