RESUMEN
Glial cells release molecules that influence brain development, function, and disease. Calcium-dependent exocytosis has been proposed as potential release mechanism in astroglia, but the physiological relevance of "gliotransmission" in vivo remains controversial. We focused on the impact of glial exocytosis on sensory transduction in the retina. To this end, we generated transgenic mice to block exocytosis by Cre recombinase-dependent expression of the clostridial botulinum neurotoxin serotype B light chain, which cleaves vesicle-associated membrane protein 1-3. Ubiquitous and neuronal toxin expression caused perinatal lethality and a reduction of synaptic transmission thus validating transgene function. Toxin expression in Müller cells inhibited vesicular glutamate release and impaired glial volume regulation but left retinal histology and visual processing unaffected. Our model to study gliotransmission in vivo reveals specific functions of exocytotic glutamate release in retinal glia.
Asunto(s)
Exocitosis/fisiología , Ácido Glutámico/metabolismo , Neuroglía/fisiología , Retina/citología , Animales , Animales Recién Nacidos , Toxinas Botulínicas/genética , Toxinas Botulínicas/metabolismo , Toxinas Botulínicas Tipo A , Carbocianinas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Antagonistas de Estrógenos/farmacología , Exocitosis/efectos de los fármacos , Exocitosis/genética , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Proteínas Fluorescentes Verdes/genética , Integrasas/genética , Integrasas/metabolismo , Luz , Potenciales de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Modelos Biológicos , Neuroglía/ultraestructura , Técnicas de Placa-Clamp , Aglutinina de Mani/metabolismo , Estimulación Luminosa , Tiempo de Reacción/genética , Estadísticas no Paramétricas , Tamoxifeno/farmacología , Tomografía de Coherencia Óptica , Rayos Ultravioleta , Proteína 2 de Membrana Asociada a Vesículas/metabolismo , Proteína 1 de Transporte Vesicular de Glutamato/metabolismoRESUMEN
Astrocytes are thought to exert diverse functions in the brain, but it has been difficult to prove this in vivo because of a scarcity of tools to manipulate these cells. Here, we report the generation of new transgenic mouse lines that allow for conditional gene ablation in astrocytes using the tamoxifen- (TAM-) inducible CreER(T2)/loxP system and bacterial artificial chromosome (BAC)-based transgenesis. In adult transgenic mice, where CreER(T2) expression is driven by the promoter of the sodium-dependent glutamate/aspartate transporter (Glast/Slc1a3) or of connexin 30 (Cx30/Gjb6), intraperitoneal TAM-injection induced Cre-mediated recombination in astroglial cells throughout the brain. Targeting efficacies varied in a region-specific manner from 20 to 90% as indicated by enzyme-based reporter lines and immunohistochemical staining. In addition, the Glast-line allowed to target retinal Müller cells and adult neural stem/progenitor cells in neurogenic regions of the adult brain. Transgenic mice expressing CreER(T2) under the control of the apolipoprotein e (ApoE) or aquaporin 4 (Aqp4) promoter showed inducible recombination in different areas of the central nervous system (CNS) albeit at low levels. Transgenic lines showed TAM-induced recombination in specific peripheral organs. These new mouse lines should help to further explore the relevance of astrocytes for brain function, as well as their contribution to pathological conditions because of aging, disease or injury.
