Fig 1: Dysfunctional volume homeostasis in PDGFRa-deficient Müller cells. (A) Scheme of the experimental setup to determine Müller cell volume regulation. Vital retinal slices were stained with Mitotracker Orange and were examined via confocal scanning microscopy while hypotonic solution was washed into the recording chamber. (B) In wildtype Müller cells, PDGF-BB showed a dose-dependent effect, preventing the swelling of Müller cells induced by the co-application of barium ions (1 mM, Ba2+) and exposure to 4 min of hypoosmotic stress (60% osmolarity). (C) AC710, a potent PDGFR family inhibitor (100 µM), abolished the protective action of PDGF-BB (50 ng/mL) in Müller cells from wildtype animals. (D) In the absence of PDGFRa, Müller cells were not able to counterbalance volume changes under hypoosmotic stress as efficiently as their wildtype counterparts. Administration of the PDGFRa ligand PDGF-BB (50 ng/mL) did not prevent cell volume changes in Müller cell-specific PDGFRa KO retinae. In contrast, glutamate (1 mM) efficiently blocked cell swelling of PDGFRa-deficient Müller cells under hypoosmotic stress. Experiments on wildtype littermates were repeated, combining barium ions and PDGF-BB to demonstrate no adverse effect of tamoxifen treatment on key findings observed in (C). (B–D) Bars represent mean ± SEM and data were collected from 15–34 cells isolated from 2–3 animals of each genotype. * p < 0.05, ** p < 0.01; *** p < 0.001. (B) * Changes in soma were tested after 4 min of exposure to hypoosmolar (60% osmolarity) extracellular solution (EC60) with a Mann–Whitney U-test. (E) Representative images of immunofluorescent detection of the water channel aquaporin 4 (AQP4) and the potassium channel Kir4.1 in Müller cell-specific PDGFRa KO retinae and wildtypes. Both proteins were enriched in Müller cell end feet facing the vitreous and blood vessels. There was no discernible phenotype in terms of immunoreactivity or distribution of Kir4.1 and AQP4. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bars, 20 µm.