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.
Astrocytes show large morphological and functional heterogeneity and are involved in many aspects of neural function. Progress in defining astrocyte subpopulations has been hampered by the lack of a suitable antibody for their direct detection and isolation. Here, we describe a new monoclonal antibody, ACSA-1, which was generated by immunization of GLAST1 knockout mice. The antibody specifically detects an extracellular epitope of the astrocyte-specific L-glutamate/L-aspartate transporter GLAST (EAAT1, Slc1a3). As shown by immunohistochemistry, immunocytochemistry, and flow cytometry, ACSA-1 was cross-reactive for mouse, human, and rat. It labeled virtually all astrocytes positive for GFAP, GS, BLBP, RC2, and Nestin, including protoplastic, fibrous, and reactive astrocytes as well as Bergmann glia, Müller glia, and radial glia. Oligodendrocytes, microglia, neurons, and neuronal progenitors were negative for ACSA-1. Using an immunomagnetic approach, we established a method for the isolation of GLAST-positive cells with high purity. Binding of the antibody to GLAST and subsequent sorting of GLAST-positive cells neither interfered with cellular glutamate transport nor compromised astrocyte viability in vitro. The ACSA-1 antibody is not only a valuable tool to identify and track astrocytes by immunostaining, but also provides the possibility of separation and further analysis of pure astrocytes.
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