RESUMEN
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.
Asunto(s)
Trastorno del Espectro Autista/tratamiento farmacológico , Trastorno del Espectro Autista/fisiopatología , Bumetanida/uso terapéutico , Hipocampo/metabolismo , Parto/metabolismo , Células Piramidales/metabolismo , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico/uso terapéutico , Animales , Animales Recién Nacidos , Trastorno del Espectro Autista/inducido químicamente , Dendritas/efectos de los fármacos , Dendritas/metabolismo , Modelos Animales de Enfermedad , Femenino , GABAérgicos/farmacología , Embarazo , Células Piramidales/efectos de los fármacos , Ratas , Ratas Wistar , Ácido Valproico/farmacologíaRESUMEN
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.
Asunto(s)
Neuronas Colinérgicas/metabolismo , Cuerpo Estriado/metabolismo , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Interneuronas/metabolismo , Enfermedad de Parkinson Secundaria/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Acetilcolina/metabolismo , Acetilcolina/farmacología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Bumetanida/farmacología , Cloruros/metabolismo , Colinérgicos/metabolismo , Colinérgicos/farmacología , Neuronas Colinérgicas/efectos de los fármacos , Neuronas Colinérgicas/patología , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/patología , Dopamina/deficiencia , Regulación de la Expresión Génica , Glutamato Descarboxilasa/genética , Glutamato Descarboxilasa/metabolismo , Humanos , Interneuronas/efectos de los fármacos , Interneuronas/patología , Transporte Iónico , Proteínas con Homeodominio LIM/genética , Proteínas con Homeodominio LIM/metabolismo , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Plasticidad Neuronal/efectos de los fármacos , Enfermedad de Parkinson Secundaria/genética , Enfermedad de Parkinson Secundaria/patología , Técnicas de Placa-Clamp , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico/farmacología , Miembro 2 de la Familia de Transportadores de Soluto 12/genética , Miembro 2 de la Familia de Transportadores de Soluto 12/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas del Transporte Vesicular de Aminoácidos Inhibidores/genética , Proteínas del Transporte Vesicular de Aminoácidos Inhibidores/metabolismo , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
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.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/fisiología , Neurogénesis/fisiología , Potenciales de Acción/fisiología , Astrocitos/fisiología , Células Cultivadas , Técnicas de Cocultivo , Humanos , Células Madre Pluripotentes Inducidas/fisiología , Redes Neurales de la Computación , Células-Madre Neurales/fisiología , Neuronas/fisiología , Técnicas de Placa-Clamp/métodosRESUMEN
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.