Abstract
The structural plasticity of synapses is crucial for regulating brain functions. However, currently available methods for studying synapse organization based on split fluorescent proteins (FPs) have been limited in assessing synaptic dynamics in vivo due to the irreversible binding of split FPs. Here, we develop ‘SynapShot’, a method for visualizing the structural dynamics of intact synapses by combining dimerization-dependent FPs (ddFPs) with engineered synaptic adhesion molecules. SynapShot allows real-time monitoring of reversible and bidirectional changes of synaptic contacts under physiological stimulation. The application of green and red ddFPs in SynapShot enables simultaneous visualization of two distinct populations of synapses. Notably, the red-shifted SynapShot is highly compatible with blue light-based optogenetic techniques, allowing for visualization of synaptic dynamics while precisely controlling specific signaling pathways. Furthermore, we demonstrate that SynapShot enables real-time monitoring of structural changes in synaptic contacts in the mouse brain during both primitive and higher-order behaviors.
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Data availability
The data supporting the findings of this study are available in the paper and its Supplementary Information.
Code availability
The codes for motion correction of imaging data used in this work are available at https://github.com/KanghoonJ/Son_Nagahama_Lee_Jung_NatMethods_2023.
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Acknowledgements
This paper is based on research that has been conducted as part of the KAIST-funded Global Singularity Research Program for 2022 (W.D.H.). This work was also supported by a National Research Foundation of Korea grant funded by the Korean government (MSIT) (no. 2020R1A2C301474213, W.D.H.), National Institute of Health grant DP1MH119428 (H.-B.K.), the Institute for Basic Science, Center for Cognition and Sociality (no. IBS-R001-D2, S.L.) and JSPS Overseas Research Fellowships (60-236, K.N.). Cartoons in Figs. 4a,d and 5a, Extended Data Figs. 8a and 10a and Supplementary Figs. 4 and 5 were created with BioRender.com.
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S.S., K.N., K.J., J.L., J.K., S.L., H.-B.K. and W.D.H. conceived the idea and directed the work. S.S., K.N., J.L., K.J., C.K., J.K., E.K., S.L., H.-B.K. and W.D.H. designed experiments; S.S., K.N., J.L., K.J., C.K. and Y.W.N. performed experiments. S.S., K.N., K.J., J.L., S.L., H.-B.K. and W.D.H. wrote the paper.
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Nature Methods thanks Susana Cohen-Cory and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Rita Strack, in collaboration with the Nature Methods team.
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Extended data
Extended Data Fig. 1 Overall process for the development of SynapShot.
Schematic diagram showing SynapShot constructs and cloning procedures. SP; Signal peptide, Nrx1; Neurexin1, Nlg;Neuroligin. The GFP(1-10) and GFP(11) of split GFP were replaced to copy GA and B3, respectively (#1.1, #2.1). The B3 subunit of ddFP was replaced with the B1 (#1.2, #2.2). In addition, location of GA and B1 was switched (#1.3, #2.3). From the brightest clones (#1.1, #2.3), PDZ domain was deleted of post-constructs protein to produce #1.4, #2.4, which named as SynapShot.
Extended Data Fig. 2 Expression and signals of SynapShot constructs in HEK293T cells.
a,b Confocal images showing expression and signals of each SynapShot construct in HEK293T cells. Copy A (GA) or copy B (B1 or B3) of ddFP was cloned into a SynView (a) or mGRASP (b) backbone vector with different ddFP pairings. Signals of SynapShot in the SynView vector and #2.3 were observed at cell-to-cell contact sites, whereas there were no signals of #2.1 and 2.2. Scale bar, 20 μm. c, Bar graph showing intensities of SynapShot signals at cell-to-cell contact sites. Data represent the mean ± s.e.m. (#1.1, n = 85 puncta; #1.2, n = 148 puncta; #1.3, n = 163 puncta; #2.3, n = 173 puncta; ****p < 0.0001, #1.1 vs. #1.2 to #2.3, one-way ANOVA). d,e, Confocal images showing expression and signals of each SynapShot construct in cultured hippocampal neurons. Copy A (GA) or copy B (B1 or B3) of ddFP was cloned into a SynView (d) or mGRASP (e) backbone vector with different ddFP pairings. Signals of SynapShot in the SynView vector and #2.3 were observed as green puncta, whereas there were no signals of #2.1 and 2.2. Scale bar, 10 μm. f, Bar graph showing intensities of SynapShot signal. Data represent the mean ± s.e.m. (#1.1, n = 99 puncta; #1.2, n = 158 puncta; #1.3, n = 167 puncta; #2.3 n, = 113 puncta).
Extended Data Fig. 3 Confirming alternation of synaptic morphology by deleting the PDZ domain of neuroligin and overexpression of the post-SynapShot or post-SynView component.
a, Confocal images of hippocampal cultured neurons expressing #1.1 SynapShot constructs (left) and #1.4 SynapShot constructs (right). Scale bar, 10 μm. b, Schematic (top) and confocal images (bottom) showing spine morphology, density and size in cultured hippocampal neurons expressing control (mScarlet), SynapShot or SynView construct. Scale bar, 10 μm. c, Bar graph showing the number of spines per 50 μm dendrite length. Data represent the mean ± s.e.m. (Control, n = 8 dendrites; Post-SynapShot, n = 11 dendrites; Post-SynView, n = 11 dendrites; ****p < 0.0001, control vs. post-SynView, Student’s two-tailed t-test). d, Graph showing measured spine size in cultured hippocampal neurons expressing control (mScarlet), SynapShot, or SynView construct. Data represent the mean ± s.e.m. (Control, n = 305 spines; Post-SynapShot, n = 218 spines; Post-SynView, n = 117 spines; ****p < 0.0001, control vs. post-SynView, Student’s two-tailed t-test).
Extended Data Fig. 4 Effects of the expression level of proteins on the SynapShot signals in cultured hippocampal neurons.
a, Confocal images showing expression of Synapshot constructs and ddGFP signals carried by CAG promoter(left) or CamKIIa(right) in cultured hippocampal neurons. Scale bar, 5 μm. b-c, Bar graph showing size (b) and mean intensity (c) of the SynapShot signal. Data represent the mean ± s.e.m. (CAG, n = 32 puncta; CamKIIa, n = 70 puncta; n.s., not significant; **** p < 0.0001, Student’s two-tailed t-test).
Extended Data Fig. 5 Colocalization analysis of SynapShot signals with endogenous synaptic markers.
a, Representative confocal images and pie chart showing co-localization of SynapShot and Bassoon as a synaptic marker detected by immunofluorescence. Scale bar, 5 μm. Percentage of co-localization of Bassoon with SynapShot signals (right; Bassoon total n = 443). b, Confocal images and pei charts showing co-localization of SynapShot with vGLUT or Gephyrin in cultured hippocampal neurons. Scale bar, 5 μm. Percentage of co-localization of vGLUT or Gephyrin with SynapShot signals (right; vGLUT1 total n = 440, Gephyrin total n = 623). b, Representative confocal images showing mGRASP-based SynapShot signals with immunostained synaptic markers and pie charts summarizing proportion of punctum clusters of SynapShot with synaptic markers. (Total number for Homer1 n = 1624, Synapsin1 n = 1767, Bassoon n = 1637, Gephyrin n = 781.) Scale bar, 5 μm.
Extended Data Fig. 6 Monitoring signals of mGRASP-based SynapShot upon the inhibition of NMDA receptors.
Representative time-lapse images and graphs showing mGRASP-based SynaspShot signal dynamics in the presence of APV (50 μM) in cultured hippocampal neurons. Scale bar, 5 μm. A normalized reduction in the size and intensity of the mGRASP-based spines was detected. Data represent the mean ± s.e.m. (n = 15 puncta; ****p < 0.0001, Welch’s t-test).
Extended Data Fig. 7 Measurement of the exchange of reporter molecules in ddFP or split-FP signals in a single synapse.
a, Time-lapse images showing the ddFP (top) or split-FP (bottom) signal changes after photobleaching in cultured hippocampal neurons. Scale bar, 5 μm. b, Graph showing recovery curves after photobleaching of each green signal in synapses (ddGFP, n = 16 puncta; split-GFP, n = 18 puncta).
Extended Data Fig. 8 SynapShot labeling in the CA3-CA1 circuit.
a, Scheme showing acute slice imaging of a brain with SynapShot virus expressed in the CA3-CA1 circuit of the hippocampus. b, Representative acute slice images of a brain with SynapShot virus expressed in the contralateral CA1 (top), ipsilateral CA1 (middle), and post-SynapShot virus expressed in CA1 only (bottom).
Extended Data Fig. 9 Visualizing synaptic signals of mGRASP in the somatosensory-motor circuit of behaving mice.
a, Time-lapse imaging of GFP fluorescent from mGRASP on a dendrite of layer 2/3 M1 neurons. An example of a GFP punctum showing gradually-decrease of the signal, indicated by the white arrow. Scale bar, 5 µm. b, Graph summarizing the normalized ratio of signal intensity from GFP (green; G) over dTomato (red; R) (n = 111 puncta from 3 mice). c, Pie chart showing the proportion of puncta with increased (increase; > 130%), stable (stable; ≤ 130%, ≥ 80%), and decreased (decrease; < 80%) GFP signals.
Extended Data Fig. 10 Electrophysiological recordings of M1 neurons expressing SynapShot component.
a, Schematics showing SynapShot virus injection for mEPSC recording and image of the recording site. b, Graphs showing the mEPSC frequency and mEPSC amplitude in the Synapshot SynapShot-expressing (n = 12 cells) and non-expressing neurons (n = 11). Data represent the mean ± s.e.m. (n.s., not significant, Student’s two-tailed t-test).
Supplementary information
Supplementary Information
Supplementary Figs. 1–5.
Supplementary Video 1
Reversibility of SynapShot in HEK293T cells. Reversibility of the ddFP-based sensor in HEK2939T cells in the presence of EGTA (8 mM), related to Fig. 1b. Time is given in min:s.
Supplementary Video 2
Spontaneous synapse enlargement in a hippocampal cultured neuron. Spontaneous synapse formation accompanied by enlargement of a dendritic spine, related to Fig. 3e. Time is given in h:min.
Supplementary Video 3
Spontaneous synapse shrinkage in a hippocampal cultured neuron. Spontaneous synapse elimination accompanied by shrinkage of a dendritic spine, related to Fig. 3f. Time is given in h:min.
Supplementary Video 4
Change of dendritic spines and SynapShot signals after activation of OptoTrkB. The dendritic spine of a postsynaptic neuron (purple) and R-SynapShot signals (red) 12 h after activation of OptoTrkB, related to Fig. 3k. Time is given in h:min.
Supplementary Data
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Son, S., Nagahama, K., Lee, J. et al. Real-time visualization of structural dynamics of synapses in live cells in vivo. Nat Methods 21, 353–360 (2024). https://doi.org/10.1038/s41592-023-02122-4
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DOI: https://doi.org/10.1038/s41592-023-02122-4
