(A) Mirror replica image of an AIS double-labeled for Nav1.6 (15 nm gold) and NL-2 (10 nm gold) on the P-face, and the γ2 subunit (10 nm gold) on the complementary E-face of the same fragment of the plasma membrane. (B) High magnification view of the boxed areas shown in (A). (C) Mirror replica images showing a fragment of a somatic plasma membrane containing a GABAergic synapse labeled for NL-2 (10 nm gold) on the P-face and the γ2 subunit on the E-face (10 nm gold). (D) High magnification views of the boxed areas in (C). (E, F) The number of gold particles labeling for NL-2 and the γ2 subunit scales with synaptic area of AIS (E; Spearman correlation, ρ = 0.99, p<0.001 for NL-2 and ρ = 0.77,p=0.072 for γ2, n = 6 synapses, one rat) and somatic (F; ρ = 0.87 for NL-2 and ρ = 0.80 for γ2, p<0.001, n = 74 synapses, one rat) synapses of CA1 PCs. (G) Correlation between gold particle number for NL-2 and the γ2 subunit in AIS and somatic synapses (ρ=0.75, p=0.084 for AIS synapses and ρ = 0.79, p<0.001 for somatic synapses, respectively, one rat). (H, I) Distributions of normalized NL-2 (H; n = 71 AIS and n = 387 somatic synapses, data pooled from five rats) and γ2 subunit densities (I; n = 63 AIS and n = 259 somatic synapses, data pooled from three rats) in AIS (black) and somatic synapses (red). (J) Normalized density of NL-2 was not significantly different in AIS and somatic synapses in the CA3 and CA1 regions (Kruskal-Wallis test, p=0.064; pooled individual synaptic density values from five rats). (K) Normalized density of the γ2 subunit was significantly higher in AIS compared to somatic synapses in both CA3 and CA1 regions (Kruskal-Wallis test, p<0.001; Multiple comparisons of mean ranks, p<0.001 for both CA3 and CA1 regions; pooled individual synaptic density values from three rats). In addition, the γ2 subunit density was significantly higher in CA3 and CA1 AIS synapses compared to CA1 and CA3 somatic synapses (p<0.001 for CA3 AIS vs CA1 soma and p=0.029 for CA1 AIS vs CA3 soma). Plots show individual data (circles), medians (horizontal line) and lower and upper quartiles (Q1, Q3; boxes). (L) The area of AIS synapses was significantly smaller in the CA1 compared to the CA3 region (Kruskal-Wallis test, p<0.001; Multiple comparisons of mean ranks, p=0.005; n = 7 rats), and somatic synapses were significantly larger than AIS synapses in the CA1 region (p= 0.002). Open circles denote median synaptic areas from each rat, and the medians (horizontal line) and lower and upper quartiles (Q1, Q3; boxes) of animals are shown. Scale bars: (A, C) 250 nm; (B, D) 100 nm.