(A and B) Hevin induces presynaptically active synapses between cultured RGCs.

(A) Representative images of RGC dendrites from cells treated with growth media (GM) only or hevin (80nM) or ACM, live stained with luminal synaptotagmin antibody (magenta) followed by synaptic staining with anti-bassoon (red) and anti-homer-1 (green) (Scale bar = 10 μm). Presynaptically active synapses can be visualized by the triple colocalization (white arrows) of synaptotagmin, bassoon, and homer-1. Only a small population of bassoon and homer-1 positive synapses lacked synaptotagmin (yellow arrows) and these “ghost” synapses were present in all conditions.

(B) Quantification of triple co-localized synaptic puncta reveals that hevin induces synapses that recycle presynaptic vesicles. Data is presented as fold increase from that of RGCs grown in GM. n > 20 cell/condition. ∗p < 0.05, one way ANOVA followed by Fisher’s LSD Post hoc test, error bars ± SEM.

(C–K) Hevin induces a strong increase in NMDA currents in autaptic RGC cultures.

(C) Representative image of an autaptic RGC making synapses between its own axon and dendritic arbors (bassoon in red and homer-1 in green) (Scale bar = 20 μm).

(D) Representative NMDA (100 μM)-induced currents at −70mV in GM- (growth media), hevin- and ACM-treated RGCs.

(E) Membrane capacitance of RGCs was not significantly different in GM-, hevin- and ACM-treated cultures.

(F and G) The NMDA-induced whole-cell currents (F) and the current densities (G) were significantly larger in hevin- and ACM-treated RGCs compared to GM-treated RGCs (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; one-way ANOVA with Bonferroni’s Multiple Comparison Test).

(H) Representative NMDA (100 μM)-induced currents before (black traces) and after treatment (red traces) with the GluN2B-specific antagonist ifenprodil (5 μM) in GM-, hevin- and ACM-treated RGCs.

(I) Repeated NMDA application did not alter the currents amplitude. Inhibition of NMDA-induced currents by Ifenprodil (5 μM) was significantly increased in hevin- and ACM-treated RGCs compared to GM-treated RGCs.

(J and K) NR2B component and NR2B current density were significantly larger in hevin- and ACM-treated RGCs compared to GM-treated RGCs. ∗p < 0.05, ∗∗∗p < 0.001; one-way ANOVA with Bonferroni’s Multiple Comparison Test, error bars represent ± SEM.