Fig. 5
SyngapΔ − GAP/Δ−GAP neurons display depressed ADBE. Hippocampal neurons derived from either wild-type (WT), Syngap+/Δ−GAP or SyngapΔ − GAP/Δ−GAP rat embryos were transfected with synaptophysin-pHluorin (sypHy) after 7 days in vitro (DIV) and imaged at DIV 13–15. A, E) Mean sypHy fluorescence traces of WT, Syngap+/Δ−GAP or SyngapΔ − GAP/Δ−GAP hippocampal neurons normalised to either the total SV pool as revealed by NH4Cl (A) or peak fluorescence during electrical stimulation (E) ± SEM. B) Mean sypHy surface fraction presented as a percentage of the total SV pool ± SEM. C, D) Mean peak sypHy response in response to either 10 Hz (C) or 40 Hz (D) action potential trains ± SEM. F, G) Mean sypHy retrieval time constants (τ) in response to either 10 Hz (F) or 40 Hz (G) action potential trains ± SEM. For A-G, WT n = 14 coverslips, Syngap+/Δ−GAPn = 12 and SyngapΔ − GAP/Δ−GAPn = 10 from 3 independent cultures. H-I) Primary hippocampal cultures derived either wild-type (WT), Syngap+/Δ−GAP or SyngapΔ − GAP/Δ−GAP rat embryos were challenged with a train of action potentials (40 Hz, 10 s) in the presence of tetramethylrhodamine (TMR)-dextran (50 µM). TMR-dextran was immediately washed away and the number of TMR-dextran puncta were counted. H) Representative images of TMR-dextran uptake in WT, Syngap+/Δ−GAP or SyngapΔ − GAP/Δ−GAP cultures. Scale bar = 50 μm. I) Mean number of TMR-dextran puncta per field of view normalised to WT ± SEM (WT n = 8 coverslips, Syngap+/Δ−GAP and SyngapΔ − GAP/Δ−GAPn = 9 from 3 independent cultures). J) Primary hippocampal cultures derived from either WT, Syngap+/Δ−GAP or SyngapΔ − GAP/Δ−GAP rat embryos were fixed at DIV13-15 and stained for the presence of SV2A. K) Mean number of SV2A puncta per field of view normalised to WT ± SEM (WT n = 10 coverslips, Syngap+/Δ−GAPn = 11 and SyngapΔ − GAP/Δ−GAPn = 13 from 3 independent cultures). In all cases a one-way ANOVA was performed, * p = 0.017