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. Author manuscript; available in PMC: 2017 Mar 16.
Published in final edited form as: Neuron. 2016 Mar 16;89(6):1317–1330. doi: 10.1016/j.neuron.2016.02.020

Figure 4. Distribution of putative postsynaptic sites is skewed away from output sites.

Figure 4

A) Projection image of the analyzed dendritic processes of a SAC expressing tdTomato (i) and PSD95-YFP (ii). iii is a merged image showing tdTomato in magenta and PSD95-YFP in green; in this image, contrast was adjusted to improve visibility for display. The image was masked to exclude dendritic branches that overlapped with other labeled cells to ensure that only PSD95 puncta from SACs were included in analysis. Magnified regions (dotted and solid boxes) correspond to example proximal and distal regions in C.

B) Skeleton of cell from A with identified PSD95 puncta (colored dots). Colors represent the log ratio of the PSD95 to tdTomato fluorescence intensity within each puncta, which was used for thresholding puncta to include in subsequent analysis (see Methods).

C) Examples of proximal (top) and distal (bottom) regions indicated by the dotted and solid boxes in A. Colors of identified puncta in the 4th column correspond to the heat map in B.

D) Density of PSD95-YFP puncta or putative postsynaptic sites determined by uncaging (see Fig. 3E) as a function of dendritic path length from the soma. For PSD95, the average linear density of PSD95 puncta using a 10 µm sliding window is plotted using a threshold log ratio of 1.0 to select puncta to include (blue line; light blue shading = S.E.M, 8 cells). For uncaging, histogram of the average density of putative postsynaptic sites in 5 µm bins is plotted (orange line; grey bars = S.E.M, 23 dendrites).

E) Histogram of PSD95 puncta from 8 SACs using a threshold log ratio of 1.0 (left axis, blue) as well as the synaptic contacts with DSGCs (outputs, right axis, black) as a function of radial distance from the soma. Orange dotted line: the mean radial distance of the last putative postsynaptic sites detected with uncaging (see Fig. 3E). Outputs were determined from electron microscopy reconstructions of 24 SACs (Briggman et al., 2011) and analyzed as a function of radial distance to compare with PSD95 locations.

See also Figure S4.