title: Supersensitive Ras activation in dendrites and spines revealed by two-photon fluorescence lifetime imaging.
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 approximately 5). This steep dependence was caused by a highly cooperative interaction between calcium ions (Ca(2+)) and Ras activators. The Ras pathway therefore functions as a supersensitive threshold detector for neural activity and Ca(2+) concentration.
authors.affiliation: Howard Hughes Medical Institute. yasuda@neuro.duke.edu
authors.forename: Ryohei
authors.initials: R
authors.lastname: Yasuda
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Harvey
authors.forename: Haining
authors.initials: H
authors.lastname: Zhong
authors.forename: Aleksander
authors.initials: A
authors.lastname: Sobczyk
authors.forename: Linda
authors.initials: L
authors.lastname: van Aelst
authors.forename: Karel
authors.initials: K
authors.lastname: Svoboda
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D049229:Dendritic Spines / Q000502:physiology*; D003952:Diagnostic Imaging / Q000295:instrumentation / Q000379:methods; D004789:Enzyme Activation; D006624:Hippocampus / Q000502:physiology*; D006801:Humans; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D009924:Organ Culture Techniques; D018408:Patch-Clamp Techniques; D017785:Photons; D051381:Rats; D018631:ras Proteins / Q000378:metabolism*
chemical_list: D018631:ras Proteins; D002118:Calcium