Figure 3.

Early postnatal retinal cells inhibit maturation of GABA_A responses. (a–c) Representative images of progenitor-derived neuron cultures at 21 DIV (DIC images above) and the regions of interest isolating cells with neuronal morphology (red circles, calcium dye images below), in 3 insert conditions as marked. Scale bar is 50 µm. (d–f) Representative calcium traces as in Fig. 1, showing calcium responses to muscimol under the indicated conditions, with consistently large responses with the P5 insert. (d’–f’) Parallel cultures treated with the KCC2 blocker DIOA restored calcium responses in matured progenitor-derived cultures. (g) P5 and P14 retinal cell survival in inserts was similar at 7 DIV and 21 DIV. P5 and P14 retinas were dissociated, cultured in inserts for 7 DIV and 21 DIV, and then examined and counted as described in Methods. Differences in cell numbers and survival were not significant (n.s). (h) (left panel) mCherry⁺ progenitor-derived neurons and presumptive RGCs; (right panel) fura 2-AM loaded progenitor-derived neurons. Scale bar is 50 µm. (i) Summary statistics demonstrating that by 21 DIV, the majority of progenitor-derived neurons cultured without inserts no longer responded to muscimol application. This maturation was suppressed in the presence of P5 retinal suspension inserts, but not in the presence of P14 retinal suspension inserts. Calcium responses in Thy1-mCherry-expressing neurons are highlighted by blue filled symbols. Red lines are means; error bars are S.D.s. *p < 0.05. (j) In FLIPR voltage-sensitive dye imaging of progenitor-derived cultures treated with muscimol after 14 DIV, approximately 60% of cells exhibited transient decrease in fluorescence, indicative of hyperpolarization (left panel), while the remaining 40% of cells showed elevated fluorescence, indicative of depolarization (right panel). All progenitor-derived cells with neuronal morphology exhibited elevated fluorescence in response to KCl; one example unresponsive cell (as marked) did not have neuronal morphology.