Fig. 7

Rasal1 promotes synapse development and NMDA-mediated synaptic transmission. A Representative ratiometric measurements of Fura-2 fluorescence, indicative of intracellular Ca²⁺ concentrations, in cultured EGFP-control, Rasal1-OE, and Rasal1-shRNA transduced hippocampal neurons following perfusion with depolarizing buffer. B Quantification of Fura-2 ratios in EGFP-control (n-41), Rasal1-OE (n = 47), and Rasal1-shRNA (n = 40) transduced hippocampal neurons following perfusion with depolarizing buffer (40 mM KCl). Mean ± SEM plotted. ****p < 0.0001. C Fura-2 color map of loaded neuron. (D1) Punctate expression of endogenous Rasal1 and perinuclear expression in a mature Div16 neuron. (D2-4) Confocal images of synapse morphology stained for FM43 (red) and Rasal1 (cyan) in depolarized EGFP-control, Rasal1-OE, and Rasal1-shRNA transduced hippocampal neurons at DIV 16 day. Scale bar: 10 µM. E Quantification of fluorescent FM43 puncta as a measure of functional synapses per µM of dendrite in EGFP-control (n = 45), Rasal1-OE (n = 50), and Rasal1-shRNA (n = 50) transduced hippocampal neurons perfused in depolarizing medium (60 mM KCL). Quantified using SynD software. Data presented as Mean ± SEM. *p < 0.05. F The frequency plot (left) and Gaussian distribution (right) of miniature EPSCs (mEPSCs) recorded from EGFP-control, Rasal1-OE, and Rasal1-shRNA transduced hippocampal neurons. G Representative traces of isolated NMDA-mediated mEPSCs in EGFP-control, Rasal1-OE, and Rasal1-shRNA transduced hippocampal neurons. H NMDA-mediated mEPSC amplitude (pA) of EGFP-control (n = 28), Rasal1-OE (n = 29), and Rasal1-shRNA (n = 19) transduced hippocampal neurons. Mean ± SEM. *p < 0.05. (I) NMDA-mediated mEPSC frequency (Hz) of EGFP-control (n = 28), Rasal1-OE (n = 29), and Rasal1-shRNA (n = 19) transduced hippocampal neurons. Mean ± SEM. *p < 0.05, ***p < 0.001. Data information: In (B, E, G, H) data was analyzed using two-tailed unpaired student t-test