Mirei Matsumuro, Sotaro Ichinose, Hirohide Iwasaki
Proper coordination of excitatory and inhibitory synaptic inputs is essential for stable neural circuit activity and flexible behavior. Although excitation-inhibition (E/I) imbalance has been implicated in neurodevelopmental disorders, quantitative histological approaches for relating cortical synaptic marker organization to behavioral variation remain limited. Here, using male C57BL/6J mice, we established a standardized depth-aligned histological framework to quantify anterior cingulate cortex (ACC) synaptic marker organization and its relationship to behavioral variation. Using postsynaptic density protein 95 (PSD-95) and gephyrin immunohistochemistry, combined with perspective and logarithmic transformations for anatomical normalization, we compared ACC synaptic marker profiles between control and prenatal valproic acid (VPA)-exposed mice. Prenatal VPA exposure produced a broad reduction in gephyrin-positive inhibitory synaptic markers across the ACC, whereas changes in PSD-95-positive excitatory markers were less pronounced. This inhibitory marker reduction shifted E/I-related synaptic marker indices, primarily reflecting reduced inhibitory synaptic labeling. Behavioral analyses showed altered social interaction and anxiety-related behavior, with modest changes in working memory-related measures. Multivariate analyses further indicated that ACC synaptic marker profiles were associated with behavioral variation. Supplementary intervention analyses suggested that voluntary exercise partially modified selected behavioral measures and was accompanied by modest shifts in ACC synaptic marker profiles (control sedentary, n = 11; control exercise, n = 7; VPA sedentary, n = 11; VPA exercise, n = 7). Together, these findings identify reduced inhibitory synaptic marker organization in the ACC as a structural phenotype associated with prenatal VPA exposure and provide a standardized framework for linking cortical synaptic architecture to behavioral variation.