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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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Spatial profile of RhoA and Cdc42 activitiesa, Fluorescence lifetime images of RhoA activity before and after glutamate uncaging. Arrow heads indicate stimulated spine.b, Averaged spatial profile of RhoA activation. Red circles indicate the activity in the stimulated spine, and black circles indicate the activity in dendrite, plotted as a function of the distance along the dendrite from the simulated spine. The number of samples (dendrites/neurons) is 20/18.c, Fluorescence lifetime images of Cdc42 activity.d, Averaged spatial profile of Cdc42 activation. The number of samples(dendrites/neurons) is 30/26.e, The fluorescence images of paGFP-RhoA (left) and paGFP-Cdc42 (right) after spine-head photoactivation (green, paGFP-Rho GTPases; red, tandem mCherry).f, Averaged timecourse of fluorescence decay in spines after photoactivation of paGFP tagged proteins in the spines. The fluorescence intensity was normalized to the peak. The numbers of samples (spines/neurons) are 63/6 for paGFP, 38/4 for paGFP-HRas (G12V), 41/5 for paGFP-Cdc42 (WT), 83/9 for paGFP-Cdc42 (Q61L), 40/4 for paGFP- RhoA (WT) and 79/10 for paGFP-RhoA (Q63L). HRas (G12V), RhoA (Q63L) and Cdc42 (Q61L) are constitutively active mutants.g,h, Decay time constants (g) and the fraction remaining at 20 s (h) of paGFP fluorescence in the photoactivated spines. Horizontal bars, mean.
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Figure 3: Spatial profile of RhoA and Cdc42 activitiesa, Fluorescence lifetime images of RhoA activity before and after glutamate uncaging. Arrow heads indicate stimulated spine.b, Averaged spatial profile of RhoA activation. Red circles indicate the activity in the stimulated spine, and black circles indicate the activity in dendrite, plotted as a function of the distance along the dendrite from the simulated spine. The number of samples (dendrites/neurons) is 20/18.c, Fluorescence lifetime images of Cdc42 activity.d, Averaged spatial profile of Cdc42 activation. The number of samples(dendrites/neurons) is 30/26.e, The fluorescence images of paGFP-RhoA (left) and paGFP-Cdc42 (right) after spine-head photoactivation (green, paGFP-Rho GTPases; red, tandem mCherry).f, Averaged timecourse of fluorescence decay in spines after photoactivation of paGFP tagged proteins in the spines. The fluorescence intensity was normalized to the peak. The numbers of samples (spines/neurons) are 63/6 for paGFP, 38/4 for paGFP-HRas (G12V), 41/5 for paGFP-Cdc42 (WT), 83/9 for paGFP-Cdc42 (Q61L), 40/4 for paGFP- RhoA (WT) and 79/10 for paGFP-RhoA (Q63L). HRas (G12V), RhoA (Q63L) and Cdc42 (Q61L) are constitutively active mutants.g,h, Decay time constants (g) and the fraction remaining at 20 s (h) of paGFP fluorescence in the photoactivated spines. Horizontal bars, mean.

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)

Spatial profile of RhoA and Cdc42 activitiesa, Fluorescence lifetime images of RhoA activity before and after glutamate uncaging. Arrow heads indicate stimulated spine.b, Averaged spatial profile of RhoA activation. Red circles indicate the activity in the stimulated spine, and black circles indicate the activity in dendrite, plotted as a function of the distance along the dendrite from the simulated spine. The number of samples (dendrites/neurons) is 20/18.c, Fluorescence lifetime images of Cdc42 activity.d, Averaged spatial profile of Cdc42 activation. The number of samples(dendrites/neurons) is 30/26.e, The fluorescence images of paGFP-RhoA (left) and paGFP-Cdc42 (right) after spine-head photoactivation (green, paGFP-Rho GTPases; red, tandem mCherry).f, Averaged timecourse of fluorescence decay in spines after photoactivation of paGFP tagged proteins in the spines. The fluorescence intensity was normalized to the peak. The numbers of samples (spines/neurons) are 63/6 for paGFP, 38/4 for paGFP-HRas (G12V), 41/5 for paGFP-Cdc42 (WT), 83/9 for paGFP-Cdc42 (Q61L), 40/4 for paGFP- RhoA (WT) and 79/10 for paGFP-RhoA (Q63L). HRas (G12V), RhoA (Q63L) and Cdc42 (Q61L) are constitutively active mutants.g,h, Decay time constants (g) and the fraction remaining at 20 s (h) of paGFP fluorescence in the photoactivated spines. Horizontal bars, mean.
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Figure 3: Spatial profile of RhoA and Cdc42 activitiesa, Fluorescence lifetime images of RhoA activity before and after glutamate uncaging. Arrow heads indicate stimulated spine.b, Averaged spatial profile of RhoA activation. Red circles indicate the activity in the stimulated spine, and black circles indicate the activity in dendrite, plotted as a function of the distance along the dendrite from the simulated spine. The number of samples (dendrites/neurons) is 20/18.c, Fluorescence lifetime images of Cdc42 activity.d, Averaged spatial profile of Cdc42 activation. The number of samples(dendrites/neurons) is 30/26.e, The fluorescence images of paGFP-RhoA (left) and paGFP-Cdc42 (right) after spine-head photoactivation (green, paGFP-Rho GTPases; red, tandem mCherry).f, Averaged timecourse of fluorescence decay in spines after photoactivation of paGFP tagged proteins in the spines. The fluorescence intensity was normalized to the peak. The numbers of samples (spines/neurons) are 63/6 for paGFP, 38/4 for paGFP-HRas (G12V), 41/5 for paGFP-Cdc42 (WT), 83/9 for paGFP-Cdc42 (Q61L), 40/4 for paGFP- RhoA (WT) and 79/10 for paGFP-RhoA (Q63L). HRas (G12V), RhoA (Q63L) and Cdc42 (Q61L) are constitutively active mutants.g,h, Decay time constants (g) and the fraction remaining at 20 s (h) of paGFP fluorescence in the photoactivated spines. Horizontal bars, mean.
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