Retinal ganglion cells from NP1/2 KO animals show a delay in the formation of functional synapses in vitro. A, Patch-clamp recording of an RGC cultured with a feeding layer of astrocytes for 2 weeks. Numerous spontaneous mEPSCs are seen (downward deflections). B, Example of recordings of synaptic activity in NP1/2 KO RGC cultures after 2 weeks with astrocyte feeding layers. Fewer synaptic events are seen. Three sets of recordings from KO and WT cultures with between five and six neurons per condition were recorded for a total of 15 neurons per genotype. C, Quantification of the rate of spontaneous synaptic activity seen in RGC cultures from WT or KO mice. In both WT and KO, low rates of activity are seen in purified RGC cultures without astrocyte feeding layers. Astrocyte feeding layers significantly increase the frequency of synaptic activity in WT cultures (WT + Astro), but there is not a corresponding increase in KO cultures (KO + Astro) (WT + Astro, 1105 ± 347 events per minute vs KO + Astro, 293 ± 60 events per minute; p = 0.013; Kruskal–Wallis ANOVA on ranks). D, Frequency histogram of spontaneous synaptic events recorded in WT and KO RGC cultures after 2 weeks. The KO events (open bars) are much smaller amplitude compared with WT events (black bars; KO + Astro, 2 weeks in culture, 26 ± 0.2 pA vs WT + Astro, 2 weeks in culture, 44 ± 0.8 pA; Mann–Whitney rank sum test). The largest events likely represent multiquantal release (Paulsen and Heggelund, 1996). E, Example of synaptic activity measured in WT cultures with feeding layers of astrocytes after 4 weeks. High rates of spontaneous synaptic activity are still seen. F, Example of spontaneous synaptic activity seen in KO RGCs with feeding layers of astrocytes at 4 weeks in culture. High-frequency events are now seen in contrast to the cultures after 2 weeks. G, Quantification of the rate of synaptic activity seen in WT and KO RGC cultures with astrocyte feeding layers. No statistically significant differences in the rates of activity are seen between WT and KO cultures (WT + Astro, 1135 ± 570 events per minute vs KO + Astro, 756 ± 98 events per minute; p > 0.5; Kruskal–Wallis ANOVA on ranks). H, Frequency histograms of spontaneous synaptic activity measured in WT or KO cultures after 4 weeks. Astro, Astrocyte.