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. 2022 Feb 8;38(6):110347. doi: 10.1016/j.celrep.2022.110347

Figure 6.

Figure 6

Gephyrin dynamics after single-spine LTP protocol

(A) Representative timelapse of pseudocolored FingR-gephyrin-GFP clusters in a dendritic portion of a Homer1c-DsRed+ cultured neuron (white outline). Yellow arrowhead: stimulated spine (LFS + uncaging). White arrowhead: a potentiated cluster at d > 3 μm from the stimulated spine. White arrow: depressed cluster at d < 3 μm. Scale bar, 1 μm.

(B) Gephyrin fluorescence intensity is depressed in clusters at d < 3μm from the stimulated spine (n = 13, F4,48 = 4.8, p < 0.01, RM one-way ANOVA followed by Tukey's post-test) (left panel) and potentiated in clusters at d > 3μm (n = 13; F4,48 = 2.7, p < 0.05, RM one-way ANOVA followed by Tukey's post-test) (right panel). Data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01.

(C) Left: Representative confocal image (z stack projection) of an acute slice of a patched Teal-gephyrin+ (green) neuron filled with Alexa Fluor 647 (A647, red). Scale bar, 20μm. Right: magnification of the two dendritic portions framed on the left, showing gephyrin clusters, spines (arrowhead), and the merge of the two channels. Scale bar, 1μm.

(D) Top. Schematics of the single-spine LTP protocol in acute slices. Bottom. Multiple representative averaged traces of uEPSCs recorded from stimulated spines before and 30 min after the protocol.

(E) Differential changes in the fluorescence intensity of gephyrin clusters, depending on the distance from the stimulated spine. Each data point represents at least three synapses (max 5) from six neurons in six slices, F5,17 = 6.7, p < 0.01, one-way ANOVA followed by Tukey's post-test).

(F) Representative confocal image of a dendritic portion of a Teal-gephyrin+ neuron (white outline) in acute slice, showing pseudocolored Teal-gephyrin clusters at different time points before (baseline) and after the delivery of the single-spine protocol (yellow arrowhead). White arrowhead: potentiated cluster at d > 4 μm from the stimulated spine. White arrow: depressed clusters at d < 4 μm. Scale bar, 1 μm.

(G) Gephyrin fluorescence intensity is depressed in clusters at d < 4 μm from the stimulated spine (n = 6, F3,15 = 5.5, p < 0.01, RM one-way ANOVA followed by Tukey's post-test) and potentiated at d > 4 μm (n = 6; F3,15 = 6.5, p < 0.01, RM one-way ANOVA followed by Tukey's post-test). Data are presented as mean ± SEM. ∗∗p < 0.01.