Fig. 5

Changes in density of excitatory and inhibitory synapses in the CA1 in vivo upon induction of fear memory and functional role of Plexin-B2 thereof. a, b Typical examples (a) and quantitative analysis (b) of the density of excitatory synapses detected as number of anti-PSD-95-immunoreactive puncta of apical dendrites of CA1 pyramidal neurons in CaMK-PB2^−/− mice and PB2^fl/fl littermates in mock-treated group or at 2 days after fear conditioning (corresponding to recent recall). Data are represented as mean of the number of synapses per unit volume, error bars represent SEM. c Scatter-plot representation of the size of PSD-95-immunoreactive puncta on apical dendrites of CA1 pyramidal neurons in CaMK-PB2^−/− mice and PB2^fl/fl littermates in mock-treated group or at 2 days after fear conditioning (red lines represent the mean); examples of automated unbiased size estimation via software are shown above. Scale bar = 1 µm. d, e Typical examples (d) and quantitative analysis (e) of the density of excitatory synapses of apical dendrites of CA1 pyramidal neurons detected via anti-pan-Homer1-immunoreactivity of synaptic puncta on apical dendrites of CA1 pyramidal neurons in the above treatment groups. f, g Typical examples (f) and quantitative analysis (g) of the density of inhibitory synapses of apical dendrites of CA1 pyramidal neurons detected as density of anti-gephyrin-immunoreactive puncta on apical dendrites of CA1 pyramidal neurons in the above treatment groups. a–g n = 3 mice per group, at least 3 different sections per mouse, 3 different ROIs per section were analyzed. Two-way ANOVA was employed in b, e and g, followed by Bonferroni post hoc test. In panel c, Krusker–Wallis test was employed. h Quantitative RT-PCR for Arc mRNA expression in the CA1 area of hippocampus of CaMK-PB2^−/− mice and PB2^fl/fl littermates in mock-treated group or on day 4 after fear conditioning; mice from all groups were subjected to a novel environment one day after recent recall. In panel h, ANOVA was employed followed by Holm–Sidak correction for multiple comparisons. N = 7 mice per group. * represents P < 0.05 when mice with fear conditioning and corresponding mock-treated mice were compared and ^† represents P < 0.05 when mutant and corresponding wild-type littermates were tested. Error bars represent S.E.M. Scale bars represent 10 µm unless otherwise indicated above