Fig. 3

Nlgn3^−/y neurons display depressed ADBE. A-C) Hippocampal synaptosome lysates from either wild-type (WT) or Nlgn3^−/y rats were probed for the presence of Neuroligin-3 (Nlgn3, A) and total protein (B). (C) Quantification of Nlgn3 levels in both are displayed, normalised to total protein ± SEM, n = 4 independent synaptosome preparations for both WT and Nlgn3^−/y, *** p < 0.0001, unpaired t test. (D-J) Hippocampal neurons derived from either WT or Nlgn3^−/y rat embryos were transfected with synaptophysin-pHluorin (sypHy) after 7 days in vitro (DIV) and imaged at DIV 13–15. (D, H) Mean sypHy fluorescence traces of WT and Nlgn3^−/y hippocampal neurons normalised to either the total SV pool as revealed by NH₄Cl (D) or peak fluorescence during electrical stimulation (H) ± SEM. (E) Mean sypHy surface fraction presented as a percentage of the total SV pool ± SEM. F, G) Mean peak sypHy response in response to either 10 Hz (F) or 40 Hz (G) action potential trains ± SEM. (I, J) Mean sypHy retrieval time constants (τ) in response to either 10 Hz (I) or 40 Hz (J) action potential trains ± SEM. For D-J WT n = 11 coverslips, Nlgn3^−/yn = 14 from 3 independent cultures. (K, L) Primary hippocampal cultures derived either WT or Nlgn3^−/y 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. (K) Representative images of TMR-dextran uptake in WT and Nlgn3^−/y cultures. Scale bar = 50 μm. (L) Mean number of TMR-dextran puncta per field of view normalised to WT ± SEM (WT n = 11 coverslips, Nlgn3^−/yn = 12 from 3 independent cultures). (M) Primary hippocampal cultures derived from either WT or Nlgn3^−/y rat embryos were fixed at DIV13-15 and stained for the presence of SV2A. (N) Mean number of SV2A puncta per field of view normalised to WT ± SEM (WT n = 14 coverslips, Nlgn3^−/yn = 15 coverslips from 3 independent cultures). In all cases an unpaired two-sided students t test was performed, except E, F and J, * p = 0.0112, unpaired t test