Figure 10.
Regulation of F-actin dynamics by Vav-Rac1 signaling is required for activity-dependent synaptic plasticity and RP mobilization. (A–F) Acute blockade of Vav-Rac1 signaling phenocopies the defects of Vav or Rac1 loss-of-function mutants in tetanus-induced synaptic plasticity and RP mobilization. (A–C) Effects of acute Vav-Rac1 blockade on synaptic augmentation and PTP. WT larval preparations were preincubated for 30 min in 0.3 mM Ca2+ saline containing an inhibitor of Vav-Rac1 signaling (20 μM EHop-016 or 50 μM EHT 1864 in 0.1% DMSO), and PTP was assayed using the 10 Hz, 1 min PTP induction protocol. (A) Plot of mean EJP amplitudes normalized to mean initial amplitude. Each point in the ordinate represents the mean normalized amplitude for every 10 s. (B and C) Bar graphs of mean normalized EJP amplitudes at the end of (B) and at 60 s after (C) tetanic stimulation. n = 12 larvae. (D) Effect of EHop-016 on synaptic depression. Plot of mean EJP amplitudes normalized to mean initial amplitude is shown for 0.1% DMSO- or EHop-016–treated WT larvae. Synaptic depression was induced using a 10 Hz, 10 min train in 10 mM Ca2+. Each point in the ordinate represents the mean normalized amplitude for every 30 s. n = 12 larvae. (E and F) Effect of EHop-016 on RP mobilization. (E) WT boutons loaded with FM1-43 using the ECP-RP loading protocol as described in Fig. 5 C were quickly washed with Ca2+-free saline and imaged (E1). RP-loaded boutons were subsequently stimulated at 30 Hz for 5 min and reimaged (E2). (F) FM1-43 fluorescence intensity in boutons before (height of whole columns) and after (height of black columns) 30 Hz stimulation. n = 12 boutons from six different larvae. (G–L) Application of jasplakinolide rescues the defects of Vav mutants in tetanus-induced synaptic plasticity and RP mobilization. Experimental paradigms and data presentation are the same as in A–F except that WT and Vav2/Y larval preparations were preincubated with 10 μM jasplakinolide in 0.1% DMSO. (G–I) Rescue of reduced PTP in Vav2/Y mutants by jasplakinolide. n = 15 larvae. (J) Rescue of enhanced synaptic depression in Vav2/Y mutants by jasplakinolide. n = 12 larvae. (K and L) Rescue of reduced RP mobilization in Vav2/Y mutants by jasplakinolide. n = 12 boutons from six different larvae. Data represent mean ± SEM. Comparisons are with WT (*, P < 0.05; **, P < 0.01, ***, P < 0.001). Dashed lines represent mean pre- or post-tetanic WT + DMSO values. Scale bars: 5 μm.

Regulation of F-actin dynamics by Vav-Rac1 signaling is required for activity-dependent synaptic plasticity and RP mobilization. (A–F) Acute blockade of Vav-Rac1 signaling phenocopies the defects of Vav or Rac1 loss-of-function mutants in tetanus-induced synaptic plasticity and RP mobilization. (A–C) Effects of acute Vav-Rac1 blockade on synaptic augmentation and PTP. WT larval preparations were preincubated for 30 min in 0.3 mM Ca2+ saline containing an inhibitor of Vav-Rac1 signaling (20 μM EHop-016 or 50 μM EHT 1864 in 0.1% DMSO), and PTP was assayed using the 10 Hz, 1 min PTP induction protocol. (A) Plot of mean EJP amplitudes normalized to mean initial amplitude. Each point in the ordinate represents the mean normalized amplitude for every 10 s. (B and C) Bar graphs of mean normalized EJP amplitudes at the end of (B) and at 60 s after (C) tetanic stimulation. n = 12 larvae. (D) Effect of EHop-016 on synaptic depression. Plot of mean EJP amplitudes normalized to mean initial amplitude is shown for 0.1% DMSO- or EHop-016–treated WT larvae. Synaptic depression was induced using a 10 Hz, 10 min train in 10 mM Ca2+. Each point in the ordinate represents the mean normalized amplitude for every 30 s. n = 12 larvae. (E and F) Effect of EHop-016 on RP mobilization. (E) WT boutons loaded with FM1-43 using the ECP-RP loading protocol as described in Fig. 5 C were quickly washed with Ca2+-free saline and imaged (E1). RP-loaded boutons were subsequently stimulated at 30 Hz for 5 min and reimaged (E2). (F) FM1-43 fluorescence intensity in boutons before (height of whole columns) and after (height of black columns) 30 Hz stimulation. n = 12 boutons from six different larvae. (G–L) Application of jasplakinolide rescues the defects of Vav mutants in tetanus-induced synaptic plasticity and RP mobilization. Experimental paradigms and data presentation are the same as in A–F except that WT and Vav2/Y larval preparations were preincubated with 10 μM jasplakinolide in 0.1% DMSO. (G–I) Rescue of reduced PTP in Vav2/Y mutants by jasplakinolide. n = 15 larvae. (J) Rescue of enhanced synaptic depression in Vav2/Y mutants by jasplakinolide. n = 12 larvae. (K and L) Rescue of reduced RP mobilization in Vav2/Y mutants by jasplakinolide. n = 12 boutons from six different larvae. Data represent mean ± SEM. Comparisons are with WT (*, P < 0.05; **, P < 0.01, ***, P < 0.001). Dashed lines represent mean pre- or post-tetanic WT + DMSO values. Scale bars: 5 μm.

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