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Supersensitive Ras activation in dendrites and spines revealed by two-photon fluorescence lifetime imaging

An Erratum to this article was published on 01 March 2006

Abstract

To understand the biochemical signals regulated by neural activity, it is necessary to measure protein-protein interactions and enzymatic activity in neuronal microcompartments such as axons, dendrites and their spines. We combined two-photon excitation laser scanning with fluorescence lifetime imaging to measure fluorescence resonance energy transfer at high resolutions in brain slices. We also developed sensitive fluorescent protein–based sensors for the activation of the small GTPase protein Ras with slow (FRas) and fast (FRas-F) kinetics. Using FRas-F, we found in CA1 hippocampal neurons that trains of back-propagating action potentials rapidly and reversibly activated Ras in dendrites and spines. The relationship between firing rate and Ras activation was highly nonlinear (Hill coefficient 5). This steep dependence was caused by a highly cooperative interaction between calcium ions (Ca2+) and Ras activators. The Ras pathway therefore functions as a supersensitive threshold detector for neural activity and Ca2+ concentration.

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Figure 1: Two-photon fluorescence lifetime imaging microscopy (2pFLIM) system.
Figure 2: FRET sensors for Ras activation (FRas) in HEK293 Cells.
Figure 3: Ras activation in the apical dendrites of CA1 pyramidal neurons in response to depolarization.
Figure 4: Action potential–evoked Ras activation in proximal apical dendrites.
Figure 5: Coupling between Ras activation and [Ca2+].

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Acknowledgements

We thank A. Karpova for mutagenesis of mEGFP; X. Zhang for preparation of cultured slices; R. Tsien (University of California, San Diego), D.W. Piston (Vanderbilt University, Nashville, Tennessee), M. Matsuda (Osaka University, Osaka, Japan), A. Miyawaki (Riken Brain Science Institute, Saitama, Japan) and T. Balla (US National Institutes of Health) for plasmids; and A. Karpova and V. Iyer for critical reading of the manuscript. The research is supported by the Howard Hughes Medical Institute, the US National Institutes of Health, the New York State Office of Science, Technology and Academic Research, the Burroughs Wellcome Fund (R.Y.), the National Alliance for Research on Schizophrenia and Depression (H.Z.) and a David and Fanny Luke Fellowship (C.D.H.).

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Correspondence to Ryohei Yasuda.

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Supplementary information

Supplementary Fig. 1

The number of photons required to detect a change in binding fraction (PAD) from 5% to 15% with signal-to-noise ratio 1 as a function of FRET efficiency (YFRET) (PDF 200 kb)

Supplementary Fig. 2

Fluorescence decay curves. (PDF 419 kb)

Supplementary Note (PDF 41 kb)

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Yasuda, R., Harvey, C., Zhong, H. et al. Supersensitive Ras activation in dendrites and spines revealed by two-photon fluorescence lifetime imaging. Nat Neurosci 9, 283–291 (2006). https://doi.org/10.1038/nn1635

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