RESUMEN
Corticotropin-releasing hormone (CRH), which is synthesized in the paraventricular nucleus (PVN) of the hypothalamus, plays an important role in the endocrine stress response. The excitability of CRH neurons is regulated by γ-aminobutyric acid (GABA)-containing neurons projecting to the PVN. We investigated the role of GABA in the regulation of CRH release. The release of CRH was impaired, accumulating in the cell bodies of CRH neurons in heterozygous GAD67-GFP (green fluorescent protein) knock-in mice (GAD67(+/GFP)), which exhibited decreased GABA content. The GABAA receptor (GABAAR) and the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1), but not the K(+)-Cl(-) cotransporter (KCC2), were expressed in the terminals of the CRH neurons at the median eminence (ME). In contrast, CRH neuronal somata were enriched with KCC2 but not with NKCC1. Thus, intracellular Cl(-) concentrations ([Cl(-)]i) may be increased at the terminals of CRH neurons compared with concentrations in the cell body. Moreover, GABAergic terminals projecting from the arcuate nucleus were present in close proximity to CRH-positive nerve terminals. Furthermore, a GABAAR agonist increased the intracellular calcium (Ca(2+)) levels in the CRH neuron terminals but decreased the Ca(2+) levels in their somata. In addition, the increases in Ca(2+) concentrations were prevented by an NKCC1 inhibitor. We propose a novel mechanism by which the excitatory action of GABA maintains a steady-state CRH release from axon terminals in the ME.
Asunto(s)
Hormona Liberadora de Corticotropina/metabolismo , Receptores de GABA-A/metabolismo , Miembro 2 de la Familia de Transportadores de Soluto 12/metabolismo , Simportadores/genética , Ácido gamma-Aminobutírico/metabolismo , Animales , Axones/metabolismo , Señalización del Calcio , Neuronas GABAérgicas/metabolismo , Regulación de la Expresión Génica , Técnicas de Sustitución del Gen , Glutamato Descarboxilasa/genética , Proteínas Fluorescentes Verdes/genética , Hipotálamo/metabolismo , Eminencia Media/metabolismo , Ratones , Núcleo Hipotalámico Paraventricular/metabolismo , Receptores de GABA-A/genética , Miembro 2 de la Familia de Transportadores de Soluto 12/genética , Simportadores/metabolismo , Cotransportadores de K ClRESUMEN
Fluorescent membrane voltage indicators that enable optical imaging of neuronal circuit operations in the living mammalian brain are powerful tools for biology and particularly neuroscience. Classical voltage-sensitive dyes, typically low molecular-weight organic compounds, have been in widespread use for decades but are limited by issues related to optical noise, the lack of generally applicable procedures that enable staining of specific cell populations, and difficulties in performing imaging experiments over days and weeks. Genetically encoded voltage indicators (GEVIs) represent a newer alternative that overcomes several of the limitations inherent to classical voltage-sensitive dyes. We critically review the fundamental concepts of this approach, the variety of available probes and their state of development.