Figure 8. Bidirectional control of presynaptic Rac1 signaling modulates short-term synaptic depression.

(A) Schematic of constructs created to control the firing of presynaptic neurons with reduced or enhanced Rac1 signaling. ChrimsonR-tdTomato was expressed alone as a control (WT), or co-expressed with HA-tagged photoactivatable Rac1 (PA-Rac1) with dominant negative (DN) or constitutively active (CA) mutations. Insets are representative images of WT, DN, and CA cultures fixed on DIV14 and stained for tdTomato (magenta) and HA (blue). Scale bars, 50 μm. (B) Schematic of experimental design. Whole-cell patch clamp recordings were conducted on non-fluorescent neurons with light delivered through the objective by an LED. The ‘Before’ 20 Hz train was evoked by 525–660 nm light. After waiting 1 min for recovery, PA-Rac1 was brought into the open configuration by 460 nm light to modulate presynaptic Rac1 signaling. Then, the ‘After’ 20 Hz train was evoked by 525–660 nm light. (C–E) Representative traces and quantification of before and after EPSC trains in (C) WT cultures (black, gray, n=21 neurons/3 cultures), (D) DN cultures (blue, cyan, n=15/3), and (E) CA cultures (green, lime, n=16/3). (F–H) Estimates from cumulative EPSCs in all cultures of: (F) Release probability; WT (U=217, p=0.9355), DN (t₂₈=0.1803, p=0.8582), CA (U=108, p=0.4677). (G) RRP size; WT (U=217, p=0.9355), DN (t₂₈=0.1081, p=0.9147), and CA (U=124, p=0.8965). (H) Replenishment rate; WT (U=182, p=0.3394), DN (t₂₈=2.800, p=0.0092), CA (U=48, p=0.0019). All data are mean ± SEM. **p<0.01, n.s. not significant. t values are t-tests, and U values are Mann-Whitney U-tests.