FIGURE 4.

Quantitative estimation of the number of gold grains per active zone at mossy fiber synapses in stratum lucidum of CA3 in hippocampal slice cultures subjected to HPF, freeze substitution, embedding in Lowicryl HM20 (no osmication), thin-sectioning, and immunogold labeling for p-cofilin. The number of gold grains at mossy fiber synapses is significantly higher in wild-type control cultures (wt-con) compared to wt cultures treated with TEA (wt-TEA; mean ± SD; **p < 0.01). This difference between control tissue and TEA-treated tissue was not observed in cultures from Munc13-1 mutants (m13-con and m13-TEA, respectively). n.s., not significant. All gold grains within 100 nm of the postsynaptic density were regarded as specific labeling and were counted (60 active zones per experimental group; two-sided Student’s t-test; α = 0.05). See Studer et al. (2014) for details on the method.