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. 2022 Oct 26;11:e81248. doi: 10.7554/eLife.81248

Figure 4. Quantifying connectivity using ExLLSM.

Figure 4.

(A) Unexpanded female brain with SAG (and off-target) neurons (magenta), pC1 (green) neurons, and BRP presynaptic sites (nc82, gray) labeled. (B–F) Representative examples of quantifying connectivity via pC1 postsynaptic site contact with SAG presynaptic sites. (B) Segmented SAG mask and the automatically detected pre- (cyan) and postsynaptic sites (green) of SAG to pC1 connections. (C) Max intensity projection of 200 z-slices from region approximately in the rectangle in (B). (D–F) Single z-slice of region in the rectangle from (C). (G) Quantification of SAG to pC1 connections. SAG to pC1 connections as quantified by the number of pC1 postsynaptic sites that contact SAG presynaptic sites in 11 ExLLSM samples (post ExLLSM) and the number of pC1 postsynaptic sites that connect to SAG presynaptic sites in one electron microscopy (EM) sample (connections, post EM). SAG presynaptic sites that make connections to pC1 as quantified by the number of SAG presynaptic sites that contact pC1 postsynaptic sites in 11 ExLLSM samples (pre recep. ExLLSM), number of SAG presynaptic sites that contact the pC1 membrane in six ExLLSM samples (pre mem. ExLLSM), and number of SAG presynaptic sites that make connections to pC1 postsynaptic sites in one EM sample (pre EM). (H) Quantification of SAG presynaptic sites and SAG to pC1 connections in virgin and mated female ExLLSM samples. Number of SAG presynaptic sites in five virgin samples (SAG pre virgin) and six mated samples (SAG pre mated). SAG to pC1 connections as quantified by the number of pC1 postsynaptic sites that contact SAG presynaptic sites in five virgin samples (SAG to pC1 post virgin) and in six mated samples (SAG to pC1 post mated). SAG presynaptic sites that make connections to pC1 as quantified by the number of SAG presynaptic sites that contact pC1 postsynaptic sites in five virgin samples (SAG to pC1 pre virgin) and six mated samples (SAG to pC1 pre mated). Virgin pre- and postsynaptic ExLLSM counts are significantly different from mated pre- and postsynaptic ExLLSM counts (t-tests). Individuals and mean ± SD plotted in (G, H). (I–O) Quantifying connectivity via pC1 membrane contact with SAG presynaptic sites. (I) Segmented masks of SAG and pC1 and SAG to pC1 connections approximately in rectangle in (A). (J–L) 100 z-slices of region approximately in the rectangle in (I). (M–O) Single z-slice of data from (J–L). Mask outlines shown.