Rnd3 is an essential mediator of BDNF-induced synaptogenesis.

<p>DIV6 cultured hippocampal neurons were transfected with m-RFP-βActin ± empty vector (Control and BDNF), ± Rnd3, ± Rnd3+caRhoA, ± si-Rnd3, ± sh-p190GAP, ± sh-p190GAP+Rnd3, ± p190GAP, ± p190GAP+siRnd3 and then treated ±50 ng/mL BDNF on DIV7 until fixed on DIV12. Cultures were then used for electrophysiological recordings, or fixed, mounted, immunostained, and imaged. A) Representative images and quantification of dendritic spine type and filopodia density is shown, with total spine number representing the combination of mushroom and stubby spines (2–3 different dendritic sections (>50 µm) on 24–60 neurons per condition were analyzed in 2 or more experiments). B) Representative traces of mEPSCs recorded from hippocampal neurons. C) Average frequencies of mEPSCs relative to control (20–35 neurons in 2–4 experiments). D) Average spine head width. E) Representative images of neurons immunostained using anti-VGlut1 and anti-Syanapsin1 antibodies. F) Quantification of percent co-localization of presynaptic markers with dendritic spine heads. G) HEK cells transfected with myc-Rnd3± si-Rnd3, and cell lysates analyzed using Western Blot, and stained using anti-Rnd3 and anti-ERK2 antibodies. Representative images of neurons transfected on DIV6 with mRFP βActin ± empty vector, ± myc-Rnd3, or ± myc-Rnd3+si-Rnd3. On DIV12 neurons were fixed and immunostained using anti-myc antibody, and imaged with 60X lens. (± SEM, Statistical analyses utilized ANOVA and Tukey’s post-test, *p<0.001 compared to control, #p<0.001 compared to BDNF, ¥p<0.001 compared to Rnd3).