RESUMO
We report that the apical dendrites of CA3 hippocampal pyramidal neurons are increased during labor and birth in the valproate model of autism but not in control animals. Using the iDISCO clearing method, we show that hippocampal, especially CA3 region, and neocortical volumes are increased and that the cerebral volume distribution shifts from normal to lognormal in valproate-treated animals. Maternal administration during labor and birth of the NKCC1 chloride transporter antagonist bumetanide, which reduces [Cl-]i levels and attenuates the severity of autism, abolished the neocortical and hippocampal volume changes and reduced the whole-brain volume in valproate-treated animals. These results suggest that the abolition of the oxytocin-mediated excitatory-to-inhibitory shift of GABA actions during labor and birth contributes to the pathogenesis of autism spectrum disorders by stimulating growth during a vulnerable period.
Assuntos
Transtorno do Espectro Autista/tratamento farmacológico , Transtorno do Espectro Autista/fisiopatologia , Bumetanida/uso terapêutico , Hipocampo/metabolismo , Parto/metabolismo , Células Piramidais/metabolismo , Inibidores de Simportadores de Cloreto de Sódio e Potássio/uso terapêutico , Animais , Animais Recém-Nascidos , Transtorno do Espectro Autista/induzido quimicamente , Dendritos/efeitos dos fármacos , Dendritos/metabolismo , Modelos Animais de Doenças , Feminino , GABAérgicos/farmacologia , Gravidez , Células Piramidais/efeitos dos fármacos , Ratos , Ratos Wistar , Ácido Valproico/farmacologiaRESUMO
We report that half striatal cholinergic interneurons are dual transmitter cholinergic and GABAergic interneurons (CGINs) expressing ChAT, GAD65, Lhx7, and Lhx6 mRNAs, labeled with GAD and VGAT, generating monosynaptic dual cholinergic/GABAergic currents and an inhibitory pause response. Dopamine deprivation increases CGINs ongoing activity and abolishes GABAergic inhibition including the cortico-striatal pause because of high [Cl-]i levels. Dopamine deprivation also dramatically increases CGINs dendritic arbors and monosynaptic interconnections probability, suggesting the formation of a dense CGINs network. The NKCC1 chloride importer antagonist bumetanide, which reduces [Cl-]i levels, restores GABAergic inhibition, the cortico-striatal pause-rebound response, and attenuates motor effects of dopamine deprivation. Therefore, most of the striatal cholinergic excitatory drive is balanced by a concomitant powerful GABAergic inhibition that is impaired by dopamine deprivation. The attenuation by bumetanide of cardinal features of Parkinson's disease paves the way to a novel therapeutic strategy based on a restoration of low [Cl-]i levels and GABAergic inhibition.
Assuntos
Neurônios Colinérgicos/metabolismo , Corpo Estriado/metabolismo , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Interneurônios/metabolismo , Doença de Parkinson Secundária/metabolismo , Ácido gama-Aminobutírico/metabolismo , Acetilcolina/metabolismo , Acetilcolina/farmacologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Bumetanida/farmacologia , Cloretos/metabolismo , Colinérgicos/metabolismo , Colinérgicos/farmacologia , Neurônios Colinérgicos/efeitos dos fármacos , Neurônios Colinérgicos/patologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/patologia , Dopamina/deficiência , Regulação da Expressão Gênica , Glutamato Descarboxilase/genética , Glutamato Descarboxilase/metabolismo , Humanos , Interneurônios/efeitos dos fármacos , Interneurônios/patologia , Transporte de Íons , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Plasticidade Neuronal/efeitos dos fármacos , Doença de Parkinson Secundária/genética , Doença de Parkinson Secundária/patologia , Técnicas de Patch-Clamp , Inibidores de Simportadores de Cloreto de Sódio e Potássio/farmacologia , Membro 2 da Família 12 de Carreador de Soluto/genética , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/genética , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismo , Ácido gama-Aminobutírico/farmacologiaRESUMO
Progress in elucidating the molecular and cellular pathophysiology of neuropsychiatric disorders has been hindered by the limited availability of living human brain tissue. The emergence of induced pluripotent stem cells (iPSCs) has offered a unique alternative strategy using patient-derived functional neuronal networks. However, methods for reliably generating iPSC-derived neurons with mature electrophysiological characteristics have been difficult to develop. Here, we report a simplified differentiation protocol that yields electrophysiologically mature iPSC-derived cortical lineage neuronal networks without the need for astrocyte co-culture or specialized media. This protocol generates a consistent 60:40 ratio of neurons and astrocytes that arise from a common forebrain neural progenitor. Whole-cell patch-clamp recordings of 114 neurons derived from three independent iPSC lines confirmed their electrophysiological maturity, including resting membrane potential (-58.2±1.0 mV), capacitance (49.1±2.9 pF), action potential (AP) threshold (-50.9±0.5 mV) and AP amplitude (66.5±1.3 mV). Nearly 100% of neurons were capable of firing APs, of which 79% had sustained trains of mature APs with minimal accommodation (peak AP frequency: 11.9±0.5 Hz) and 74% exhibited spontaneous synaptic activity (amplitude, 16.03±0.82 pA; frequency, 1.09±0.17 Hz). We expect this protocol to be of broad applicability for implementing iPSC-based neuronal network models of neuropsychiatric disorders.
Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Neurogênese/fisiologia , Potenciais de Ação/fisiologia , Astrócitos/fisiologia , Células Cultivadas , Técnicas de Cocultura , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Redes Neurais de Computação , Células-Tronco Neurais/fisiologia , Neurônios/fisiologia , Técnicas de Patch-Clamp/métodosRESUMO
Memories are encoded within sparsely distributed neuronal ensembles. However, the defining cellular properties of neurons within a memory trace remain incompletely understood. Using a fluorescence-based Arc reporter, we were able to visually identify the distinct subset of lateral amygdala (LA) neurons activated during auditory fear conditioning. We found that Arc-expressing neurons have enhanced intrinsic excitability and are preferentially recruited into newly encoded memory traces. Furthermore, synaptic potentiation of thalamic inputs to the LA during fear conditioning is learning-specific, postsynaptically mediated and highly localized to Arc-expressing neurons. Taken together, our findings validate the immediate-early gene Arc as a molecular marker for the LA neuronal ensemble recruited during fear learning. Moreover, these results establish a model of fear memory formation in which intrinsic excitability determines neuronal selection, whereas learning-related encoding is governed by synaptic plasticity.