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. 2019 Jan 14;10:211. doi: 10.1038/s41467-018-08217-3

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — Spatiotemporal visualization of small GTPase activation in dendrites and single spines upon optoTrkB activation and LTP induction. a Schematic of Ras activation by blue light-mediated OptoTrkB activation. b Cultured hippocampal neuron (DIV-9) showing fluorescence change of R-HRas upon blue light-induced whole cell activation of OptoTrkB. Whole-cell activation; light was globally illuminated in the whole field of view. c Time-lapse measurement showing fluorescence change of R-HRas upon light stimulation. n = 4 (red), 6 (blue). Scale bar, 20 μm. d (left) images showing local fluorescence increase of R-HRas intensity upon OptoTrkB activation. Local stimulation was applied to a small region of dendrite (indicated by red circle) of hippocampal neuron (DIV-12) at t = 1 min. (right) Enlarged images of the indicated regions (white boxes) showing fluorescence change of R-HRas at the region of illumination and a distal dendrite. Scale bar, 20 μm. e Time-lapse graph showing fluorescence changes of R-HRas within regions indicated by white circles in d (magenta; nearby stimulation, green; distal from stimulation). f Time-lapse images of dendrites from layer 2/3 pyramidal neurons co-expressing tdTomato (red) and small GTPases sensors, G-Cdc42 (left) and G-HRas (right). Target spines (arrowheads) were exposed to HFU (blue crosses). g Time-lapse measurement showing fluorescence changes of G-Cdc42 and G-HRas in HFU-stimulated spines (top) and nearby dendrites (bottom, indicated by white circles in f, < 2.5 μm from target spines). Filled circles, G-Cdc42: **P < 0.01 at all post-HFU time points; n = 26 spines, 22 cells; G-HRas: **P < 0.01 at all post-HFU time points; n = 22 spines, 19 cells. Open circles, G-Cdc42: n = 10 spines, 10 cells; G-HRas: n = 11 spines, 11 cells. Open squares, G-Cdc42: n = 23 spines, six cells; G-HRas: n = 23 spines, six cells. Triangles (G-Cdc42), n = 26 spines, 22 cells. Triangles (G-HRas), **P < 0.01 up to 6 min; n = 22 spines, 19 cells. h Graph showing relative changes of fluorescence of G-Cdc42 and G-HRas in dendrites in different time periods (transient and sustained) following HFU. **P < 0.01. Statistical analysis were performed by Student’s two-tailed t test; n.s., not significant. *HFU: high-frequency uncaging; RFU: relative fluorescence unit; AU: arbitrary unit; Error bars: s.e.m. Images or quantified data are representatives of multiple experiments (N > 3)