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Local, persistent activation of Rho GTPases during plasticity of single dendritic spines.

Murakoshi H, Wang H, Yasuda R - Nature (2011)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, Duke University Medical Center, Durham, NC 27710, USA.

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Spatiotemporal dynamics of RhoA activation during long-term structural plasticity induced in single spinesa, Fluorescence lifetime images of RhoA activation during spine structural plasticity induced by 2-photon glutamate uncaging. Arrow heads indicate the stimulated spine. Warmer colours indicate shorter lifetimes and higher RhoA activity. Scale bar, 5 μm.b, Time course of RhoA activation measured as a change in the fraction of mEGFP-RhoA bound to mCherry-RBD-mCherry in stimulated spines (stim), the dendritic shaft beside the stimulated spines (dend; within 1 μm), and adjacent spines (adj; between 3–5 μm of the stimulated spines). Data using pharmacological inhibitors (Ctrl, control condition; KN62, CaMKII inhibitor; AP5, NMDA receptor inhibitor) are also shown. Inset: closer view. The numbers of samples (spines/neurons) are 35/29 for stimulated spines and dendrites, 29/26 for adjacent spines, 16/10 for KN62 and 8/5 AP5. Error bars are s.e.m.c, Transient (averaged over 16–64 s) and sustained (averaged over 20–38 min) RhoA activation. Stars denote statistically significant difference (< 0.05) from the value in the stimulated spines under control condition. Wilcoxon signed-rank test was used for dendrites and adjacent spines, and ANOVA followed by post-hoc tests using the least significant difference was used for experiments with pharmacological inhibitors.d, Averaged time course of spine volume change in the same experiments in b.e, Transient (volume change averaged over 1.5–2 min subtracted by that over 20–38 min) and sustained volume change (volume change averaged over 20–38 min).
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Figure 1: Spatiotemporal dynamics of RhoA activation during long-term structural plasticity induced in single spinesa, Fluorescence lifetime images of RhoA activation during spine structural plasticity induced by 2-photon glutamate uncaging. Arrow heads indicate the stimulated spine. Warmer colours indicate shorter lifetimes and higher RhoA activity. Scale bar, 5 μm.b, Time course of RhoA activation measured as a change in the fraction of mEGFP-RhoA bound to mCherry-RBD-mCherry in stimulated spines (stim), the dendritic shaft beside the stimulated spines (dend; within 1 μm), and adjacent spines (adj; between 3–5 μm of the stimulated spines). Data using pharmacological inhibitors (Ctrl, control condition; KN62, CaMKII inhibitor; AP5, NMDA receptor inhibitor) are also shown. Inset: closer view. The numbers of samples (spines/neurons) are 35/29 for stimulated spines and dendrites, 29/26 for adjacent spines, 16/10 for KN62 and 8/5 AP5. Error bars are s.e.m.c, Transient (averaged over 16–64 s) and sustained (averaged over 20–38 min) RhoA activation. Stars denote statistically significant difference (< 0.05) from the value in the stimulated spines under control condition. Wilcoxon signed-rank test was used for dendrites and adjacent spines, and ANOVA followed by post-hoc tests using the least significant difference was used for experiments with pharmacological inhibitors.d, Averaged time course of spine volume change in the same experiments in b.e, Transient (volume change averaged over 1.5–2 min subtracted by that over 20–38 min) and sustained volume change (volume change averaged over 20–38 min).

Mentions: 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).


Local, persistent activation of Rho GTPases during plasticity of single dendritic spines.

Murakoshi H, Wang H, Yasuda R - Nature (2011)

Spatiotemporal dynamics of RhoA activation during long-term structural plasticity induced in single spinesa, Fluorescence lifetime images of RhoA activation during spine structural plasticity induced by 2-photon glutamate uncaging. Arrow heads indicate the stimulated spine. Warmer colours indicate shorter lifetimes and higher RhoA activity. Scale bar, 5 μm.b, Time course of RhoA activation measured as a change in the fraction of mEGFP-RhoA bound to mCherry-RBD-mCherry in stimulated spines (stim), the dendritic shaft beside the stimulated spines (dend; within 1 μm), and adjacent spines (adj; between 3–5 μm of the stimulated spines). Data using pharmacological inhibitors (Ctrl, control condition; KN62, CaMKII inhibitor; AP5, NMDA receptor inhibitor) are also shown. Inset: closer view. The numbers of samples (spines/neurons) are 35/29 for stimulated spines and dendrites, 29/26 for adjacent spines, 16/10 for KN62 and 8/5 AP5. Error bars are s.e.m.c, Transient (averaged over 16–64 s) and sustained (averaged over 20–38 min) RhoA activation. Stars denote statistically significant difference (< 0.05) from the value in the stimulated spines under control condition. Wilcoxon signed-rank test was used for dendrites and adjacent spines, and ANOVA followed by post-hoc tests using the least significant difference was used for experiments with pharmacological inhibitors.d, Averaged time course of spine volume change in the same experiments in b.e, Transient (volume change averaged over 1.5–2 min subtracted by that over 20–38 min) and sustained volume change (volume change averaged over 20–38 min).
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Related In: Results  -  Collection

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Figure 1: Spatiotemporal dynamics of RhoA activation during long-term structural plasticity induced in single spinesa, Fluorescence lifetime images of RhoA activation during spine structural plasticity induced by 2-photon glutamate uncaging. Arrow heads indicate the stimulated spine. Warmer colours indicate shorter lifetimes and higher RhoA activity. Scale bar, 5 μm.b, Time course of RhoA activation measured as a change in the fraction of mEGFP-RhoA bound to mCherry-RBD-mCherry in stimulated spines (stim), the dendritic shaft beside the stimulated spines (dend; within 1 μm), and adjacent spines (adj; between 3–5 μm of the stimulated spines). Data using pharmacological inhibitors (Ctrl, control condition; KN62, CaMKII inhibitor; AP5, NMDA receptor inhibitor) are also shown. Inset: closer view. The numbers of samples (spines/neurons) are 35/29 for stimulated spines and dendrites, 29/26 for adjacent spines, 16/10 for KN62 and 8/5 AP5. Error bars are s.e.m.c, Transient (averaged over 16–64 s) and sustained (averaged over 20–38 min) RhoA activation. Stars denote statistically significant difference (< 0.05) from the value in the stimulated spines under control condition. Wilcoxon signed-rank test was used for dendrites and adjacent spines, and ANOVA followed by post-hoc tests using the least significant difference was used for experiments with pharmacological inhibitors.d, Averaged time course of spine volume change in the same experiments in b.e, Transient (volume change averaged over 1.5–2 min subtracted by that over 20–38 min) and sustained volume change (volume change averaged over 20–38 min).
Mentions: 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).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, Duke University Medical Center, Durham, NC 27710, USA.

Show MeSH
Related in: MedlinePlus