In adult zebrafish, Müller glia are the source of new retinal neurons (57). While adult mammals also possess Müller glial cells, they do not typically possess the same regenerative abilities found in zebrafish. However, recently, this lab has provided evidence that Müller glia induce Müller glia-derived progenitor cells (MDPC) in adult rodents. Specifically, eye drop treatment with the selective alpha7 nicotinic acetylcholine receptor (nAChR) agonist, PNU-282987, has been shown to cause dedifferentiation of Müller glia and neurogenesis in the adult rodent retina (15-19). This current R15 grant proposal is designed to test the hypothesis that PNU-282987- induced neurogenesis of retinal neurons in adult mice occurs through indirect activation of Müller glia. Two specific aims have been designed to address this hypothesis. The first specific aim is designed to quantify the specific retinal cell populations that undergo mitosis and proliferation after application of PNU-282987 in the adult mammalian retina using flow cytometry. Previous results from this lab demonstrated that eye drop application of PNU-282987 induced neurogenesis of photoreceptors and RGCs that originated from Müller glia (19). Specific aim #1 is designed to determine and quantify what specific retinal cell populations are affected by eye drop application of PNU-282987 using flow cytometry. Does PNU-282987 induce neurogenesis in all retinal cells? The Fortessa flow cytometer will be used to detect and count markers associated with various retinal cells, cell components associated with progenitor cells, Müller glia and mitotically active markers before and after PNU- 282987 treatment. The second specific aim is designed to examine the transcriptome gene changes that occur in primary cultures of Muller glia that lead to formation of progenitor cells after treatment with an alpha7 nAChR agonist. In this proposal, primary cell cultures of RPE and Müller glia will be obtained from adult mice to determine which Müller glia genes are affected by eye drop application of PNU-282987. Primary Muller glia cells will be cultured in conditioned media obtained from primary cultures of RPE cells obtained from adult mice that are treated with PNU-282987. It is hypothesized that PNU-282987 binds to alpha7 nAChRs on retinal pigment epithelium to release signaling molecules onto Muller glia that induce dedifferentiation. After mRNAseq analysis, up- or down-regulated genes will be compared with published literature of Müller glia genes involved in development and dedifferentiation. Identified genes will be quantified and verified with RT-PCR, RNAscope technology (ACDBio) and immunocytochemistry. Once specific genes are identified and potential pathways are proposed, gene knockout (KO) studies and gene overexpression studies will be performed in primary Muller glia using CRISPR technology to demonstrate the necessity of these genes in Muller glia-derived neurogenesis induced by PNU-282987 in adult mice. ...