Figure 1: CaMKv protein synthesis at synapses is regulated by neuronal activity.

(a) Western blot analysis of CaMKv expression in the mouse forebrain at the indicated stages. (b) Co-localization between CaMKv (red) and PSD-95 (green) puncta (arrowheads) in the dendrites of cultured hippocampal neurons (23 days in vitro (DIV)). Scale bars, 20 μm (upper); 10 μm (lower panels). (c) CaMKv expression in the mouse visual cortex before (postnatal day (P) 10) and after eye-opening (P13). Arc protein increase upon eye-opening served as a positive control. (d) Quantification of CaMKv protein normalized to that of actin (n=3 mice per condition, *P<0.05, Student’s t-test). (e) CaMKv expression in the mouse visual cortex was reduced after dark rearing (DR). (f) CaMKv protein was quantified after normalization to that of GAPDH (n=11 mice per condition, *P<0.05, Student’s t-test). (g,h) Cortical neurons (14–16 DIV) were treated with Bic (40 μM) or TTX (2 μM) for the indicated durations. (i) CaMKv expression (normalized to actin) was increased by Bic and decreased by TTX (n=3 independent experiments). (j,k) Bic-induced CaMKv expression required protein synthesis. Cortical neurons (14 DIV) were pretreated with anisomycin (40 μM) for 1 h and incubated with Bic (40 μM) or vehicle (Veh) for 1 h (*P<0.05, one-way analysis of variance, Bonferroni’s multiple comparison test; n=3 independent experiments). (l) camkv mRNA was localized at the distal dendrites (>100 μm from cell bodies) of dissociated hippocampal neurons (23 DIV) as indicated by fluorescence in situ hybridization. Arrowheads indicate camkv mRNA (green) along MAP2-positive dendrites (red). Scale bars, 20 μm (upper); 10 μm (lower). (m) The postsynaptic marker PSD-95 was highly enriched in the synaptoneurosome fraction (SNS) but absent in the cytosolic fraction (Cyto). (n) CaMKv was locally translated in the synaptoneurosome as revealed by metabolic labelling; its synthesis was abolished by cycloheximide (CHX, 10 ng μl⁻¹).