ELECTROPHYSIOLOGICAL ALTERATIONS ACCOMPANY NEURON-LIKE PHENOTYPE ACQUISITION IN MAMMALIAN MÜLLER GLIA FOLLOWING REPROGRAMMING

The retina is a neural tissue that functions as a circuit for visual processing and is vulnerable to injury, leading to neuronal damage and vision loss. Müller glia (MG) are the main glial cells of the retina and maintain its structure and homeostasis. In vertebrates such as zebrafish, frog and chicken, MG show regenerative capacity after injury by reprogramming into Müller glia-derived progenitor cells (MGDP) with neurogenic potential. However, in mammals this regenerative potential is strongly limited but present. To further characterize the neurogenic capacity of mammalian cells, we analyzed the morphological and electrophysiological changes in mouse MG after dedifferentiation and differentiation in vitro. Using primary retinal cell cultures from C57BL/6 mice (8˗12 days old), we induced MG reprogramming into MGDP by stimulation with epidermal growth factor (EGF) and fibroblast growth factor (FGF), followed by differentiation induced by gamma-aminobutyric acid (GABA). We characterized cell identity by immunocytochemistry with antibodies against glutamine synthetase, nestin and βIII-tubulin. Moreover, we determined passive membrane properties and currents using whole-cell patch-clamp recordings in current- and voltage-clamp protocols. We found that MGDP expressing nestin showed changes in passive membrane properties, in the magnitude and components of voltage-dependent membrane currents, and the development of spikelet events. Additionally, GABA induced differentiation promoted βIII-tubulin expression and electrophysiological changes that support a functional transition from a glial to a neuron-like excitable phenotype.