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. Author manuscript; available in PMC: 2009 Oct 31.
Published in final edited form as: Cell. 2008 Oct 31;135(3):535–548. doi: 10.1016/j.cell.2008.09.057

Figure 6.

Figure 6

MyoVb is Required for LTP-Induced Exocytosis, AMPA Receptor Insertion, and Spine Growth

(A) Hippocampal neurons expressing MyoVb shRNA were transfected with TfR-SEP either alone (MyoVb RNAi) or together with RNAi-resistant MyoVb-WT (Rescue). Exocytosis from REs was monitored by TfR-SEP fluorescence before and 15 min after glycine. Fluorescence intensity is indicated as a pseudocolor scale. White and red arrows indicated spines with increased and unchanged/decreased TfR-SEP signal after glycine stimulation, respectively. Scale bars, 2 μm and 1 μm. See Movies S9-S11.

(B) Quantitative analysis of surface appearing TfR-SEP in spines. Data represent means ± SEM of the fractional increase in fluorescence intensity (ΔF/F0). The black bar indicates the period of glycine application. Vec, vector control; Scram, scrambled shRNA.

(C) Data presented as in (B) for neurons expressing MyoVb shRNA along with RNAi-resistant versions of MyoVb-WT, MyoVb-CCtr, or MyoVb-ΔRBD. Note the ordinate scale difference between the graphs in (B) and (C).

(D) Hippocampal neurons expressing MyoVb shRNA were transfected with SEP-GluR1 either alone (MyoVb RNAi) or together with RNAi-resistant MyoVb-WT (Rescue). Stimulus-induced AMPA receptor insertion was monitored by increased SEP-GluR1 fluorescence before and 15 min after glycine stimulation. Yellow and red arrows indicate spines with increased and decreased SEP-GluR1 after glycine respectively. Yellow boxes indicated corresponding magnified regions. Scale bars, 2 μm.

(E) Quantitative analysis of SEP-GluR1 in spines after glycine stimulation. Data represent means ± SEM of the normalized SEP-GluR1 fluorescence intensity in spines (F/F0). SEP-GluR1 F/F0 in spines: Vec, 1.87 ± 0.10; Scram, 1.93 ± 0.13; RNAi, 1.07 ± 0.03; WT, 1.97 ± 0.22; CCtr, 1.99 ± 0.14; ΔRBD, 1.22 ± 0.05; Vec + APV (APV), 0.85 ± 0.02. n = 703, 900, 337, 661, 945, and 497 spines from 14, 14, 12, 16, 21, 14 and 10 cells for Vec, RNAi, Scram, WT, CCtr, ΔRBD and APV, respectively; **p<0.001 relative to Vec or Scram; t-test.

(F) Depleting endogenous MyoVb inhibits stimulus-induced spine growth. Yellow and red arrows indicate dendritic protrusions that appeared and disappear after glycine respectively. Scale bars, 2 μm.

(G) Quantitative analysis of spine formation following LTP stimulation. New protrusions per 100 μm of dendrite: Vec, 10.9 ± 2.4; Scram, 10.8 ± 2.1; RNAi, -4.0 ± 2.6; Rescue, 8.9 ± 2.2; Vec + APV, 2.2 ± 1.9. n = 12, 15, 27, 11 and 8 cells for Vec, Scram, RNAi, Rescue, and Vec + APV, respectively. **p <0.001, *p<0.05 relative to Vec or Scram; t-test.

(H) Quantitative analysis of spine growth following LTP stimulation. Normalized spine size S/S0: Vec, 1.52 ± 0.07; Scram, 1.81 ± 0.15; RNAi, 0.88 ± 0.04; Rescue, 1.91 ± 0.17; Vec + APV, 0.84 ± 0.05. n = 104, 74, 241, 132, and 105 spines from 6, 9, 14, 8 and 5 cells for Vec, Scram, RNAi, Rescue and Vec + APV, respectively. **p <0.001 relative to Vec or Scram; t-test.