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. Author manuscript; available in PMC: 2020 Jan 16.
Published in final edited form as: Neuron. 2018 Dec 10;101(2):260–273.e6. doi: 10.1016/j.neuron.2018.11.028

Figure 2. CaV2.1 α1 OE Leads to Increase in CaV2.1 Channels and Clusters at Putative AZ at P7.

Figure 2.

(A) Representative SDS-digested freeze-fracture immunogold-labeled replicas of calyx P-faces (pseudocolored blue) of control and CaV2.1 α1 OE at P7. (Top) CaV2.1 distribution. Large gold particles (12 nm) label CaV2.1, small gold particles (6 nm) label mEGFP (CaV2.1 α1 OE only). (Bottom) High-magnification images of the boxed areas showing clustering of gold particles. A 30 nm circle indicates spatial uncertainty of CaV2.1 (light gray). Note that 12 nm gold particles have been enhanced for better visibility.

(B) Cumulative frequency distribution of gold particles per cluster; (insets) closeup of 0–10 particle clusters and corresponding bar graph (p < 0.0001, Mann-Whitney U test, n = 428 for control and n = 957 for CaV2.1 α1 OE).

(C) Cumulative frequency distribution of cluster area; (inset) closeup of 0–0.01 μm2 and corresponding bar graph (p = 0.0006, Mann-Whitney U test, n = 428 for control and n = 957 for CaV2.1 α1 OE).

(D) Cumulative frequency distribution of gold particle density per μm2 cluster area; (inset) bar graph (p < 0.0001, Mann-Whitney U test, n = 428 for control and n = 957 for CaV2.1 α1 OE).

(E) Relative frequency of single channels to channel clusters at P7 (p < 0.0001, Fisher’s exact test, n = 2,290 particles in 8 replicas for control and n = 7,314 particles in 7 replicas for CaV2.1 α1 OE).

(F) Representative SDS-digested freeze-fracture immunogold-labeled replicas of calyx P-faces (blue) of control and CaV2.1 α1 OE. Light gray circles indicate 30 nm radius, and dark gray circles indicate 100 nm radius around gold particles.

(G) (Top) Scheme of putative AZ categories. Cluster defined as overlap of at least two 30 nm circles and putative AZ as overlap of all 100 nm circles of clusters. (Bottom) Cumulative frequency distribution of channel cluster assembly within a putative AZ. (Inset) Number of clusters per putative AZ (p < 0.0001, Mann-Whitney U test, n = 271 for control and n = 390 for CaV2.1 α1 OE). All data are shown as mean ± SEM. EM images are montage of multiple images assembled from the calyx P-face area containing CaV2.1. See also Table S2.