Extended Data Fig. 5: In vivo two-photon imaging of OPC-synapse interactions.
From: Oligodendrocyte precursor cells engulf synapses during circuit remodeling in mice
(a) Schematic of the in vivo two-photon imaging paradigm using AAV-hSYN-eGFP to label thalamocortical (TC) inputs in the NG2-CreERT2tdTomato reporter line. (b) In vivo two-photon image of a tdTomato+ OPC (green) and eGFP+ TC inputs (magenta). Scale bar, 10 μm. (c) Reconstruction of OPC shown in (B) (white) with inputs color coded based upon their distance to the OPC surface (engulfed in red, contacting in cyan, and non-contacting in magenta). Scale bar, 10 μm. (d) Inset of reconstruction shown in (C). Yellow arrow, engulfed input. Scale bar, 3 μm. (e,f) The number of TC inputs contacting individual OPCs normalized to OPC volume and the number of TC inputs contacting individual OPCs (13 OPCs from 5 mice). (g) In vivo two-photon image of a tdTomato+ OPC and eGFP+ TC inputs including XZ and YZ orthogonal projections. Scale bar, 5 μm. (h) Reconstruction of OPC shown in (g). OPC in green and inputs color coded based upon overlap between OPC and eGFP input signals as demonstrated by the volume overlap scale on the right. Scale bar, 3 μm. (i) In vivo two-photon image of a tdTomato+ OPC and eGFP+ TC inputs including XZ and YZ projections. Scale bar, 10 μm. (j) Cumulative frequency distribution of average input size of non-contacting, contacting, or engulfed inputs. Two-tailed Kruskal-Wallis test with Dunn’s post-hoc comparisons (engulfed vs. contacting P = 0.0001, engulfed vs. non-contacting P = 0.0354, contacting vs. non-contacting P > 0.05). (k) Quantification of the average volumes of synaptic inputs depending upon whether they contact OPCs, are engulfed by OPCs, or do not interact with OPCs. Two-tailed One-way ANOVA, P <0.0001  with Tukey post-hoc test (engulfed vs. contacting P = 0.0038, engulfed vs. non-contacting P = 0.5258, contacting vs. non-contacting P = 0.0412). n = 10, 12, and 12 OPCs from 5 mice for engulfed, contacting and non-contacting volumes. Data represented in histograms as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001.
