Local, persistent activation of Rho GTPases during plasticity of single dendritic spines.
Inhibition of the Rho-Rock pathway preferentially inhibited the initial spine growth, whereas the inhibition of the Cdc42-Pak pathway blocked the maintenance of sustained structural plasticity.RhoA and Cdc42 activation depended on Ca(2+)/calmodulin-dependent kinase (CaMKII).Thus, RhoA and Cdc42 relay transient CaMKII activation to synapse-specific, long-term signalling required for spine structural plasticity.
Affiliation: Department of Neurobiology, Duke University Medical Center, Durham, NC 27710, USA.
- Dendritic Spines/enzymology*/physiology*
- Neuronal Plasticity/physiology*
- rho GTP-Binding Proteins/antagonists & inhibitors/metabolism*
- Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism
- Enzyme Activation
- GTPase-Activating Proteins/antagonists & inhibitors/metabolism
- Long-Term Potentiation/physiology
- Microscopy, Fluorescence
- Phosphoproteins/antagonists & inhibitors/metabolism
- Pyramidal Cells/physiology
- Signal Transduction
- Time Factors
- rhoA GTP-Binding Protein/antagonists & inhibitors/metabolism
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Figure 4: The effect of Rho GTPase inhibition for structural plasticity of spine head enlargementa-h, Averaged time course of spine volume change in stimulated spines in neurons under manipulations of Rho GTPase signalling. Neurons were transfected with shRNAs against RhoA and RhoB (sh-RhoA/B) and mEGFP (a), sh-RhoA/B, mEGFP-shRNA resistant RhoA (mEGFP-RhoA res) and tandem mCherry (b), mCherry-C3 (C3) and mEGFP (c), mEGFP (d, h), shRNA against Cdc42 (sh-Cdc42) and mEGFP (e), sh-Cdc42, mEGFP-shRNA resistant Cdc42 (mEGFP-Cdc42 res) and tandem mCherry (f) or mCherry-Wasp(210–321) (Wasp) and mEGFP (g) (red). Paired control experiments (black) were performed in the same batch of slices using a scrambled shRNA instead of targeted shRNAs (a, e), mEGFP alone (b, f) or mCherry instead of C3 and Wasp (c, g). Pharmacology experiments (d, h) were performed before (paired control, black) and 30–40 min (red) after applying drugs to the bath. Fluorescence intensity of mEGFP (a, c, d, e, g, h) or tandem mCherry (b, f) was used to measure the spine volume change. The numbers of samples (spines/neurons) are indicated in the figures (same numbers for control and experiment groups).i, Transient volume change (volume change averaged over 1.5–2 min subtracted by that averaged over 20–36 min). Stars denote statistical significance (p < 0.05, paired t-test).j, Sustained volume change (volume change averaged over 20–36 min).k, A model of Cdc42 and RhoA activation.l, Superimposed time courses of spine volume change and activation of RhoA (Fig. 1b), Cdc42 (Fig. 2b) and CaMKII13 in spines undergoing structural plasticity. The time courses were normalized to the peak. Right, closer view.
Using these sensors, we measured the activity of RhoA and Cdc42 during spine structural plasticity associated with LTP (Figs. 1, 2 and 3). Pyramidal neurons in the CA1 region of cultured hippocampal slices were ballistically18 transfected with the RhoA or Cdc42 sensor, and the FRET signal was imaged under 2pFLIM. The spine volume was monitored using the red fluorescence of mCherry-RBD-mCherry (Supplementary Fig. 3)12. To induce structural plasticity in a single dendritic spine, we applied a low frequency train of two-photon glutamate uncaging pulses (30 pulses at 0.5 Hz) to the spine in zero extracellular Mg2+ (Ref 13,14,19). The spine volume increased rapidly by ~300% following glutamate uncaging (transient phase) and relaxed to an elevated level of 70–80% for more than 30 min (sustained phase) (Figs. 1d, 2d)12–14. The time course of spine enlargement in neurons expressing the FRET sensor was similar to that in neurons expressing only EGFP (Fig. 4)14, suggesting that the overexpression of FRET sensors causes almost no effects on spine structural plasticity (Supplementary note).