Fig. 3.

Synapse specificity of SA-Ch expression at potentiated synapses. SA-Ch is specifically expressed at potentiated synapses. DIV 8-10 neurons were focally stimulated by uncaging glutamate in close proximity to selected spines. a Neurons were maintained in standard Mg²⁺-free ACSF in the presence of forskolin and TTX for 20 min before uncaging with or without MNI-caged glutamate. Following two-photon uncaging, medium was changed to 1 mM Mg²⁺ ACSF supplemented with B27. b Local release of glutamate stimulates SA-Ch translation at stimulated (s), but not nearby (n) spines. This effect was specific to glutamate release, as it was absent when MNI-glutamate was not added to the medium. Following stimulation, S-Ch change was much lower and is an effect of spine enlargement. ***P < 0.001, one-way ANOVA, Bonferroni comparison of means. ### P < 0.001 unpaired samples Student’s t-test, two-tailed. Bars are mean ± SEM. c Translation inhibition with anysomicin blocked SA-Ch accumulation at stimulated synapses. Representative images of stimulated dendrites in neurons transfected with SA-Ch. Red dots in the EGFP channel indicate the location of two-photon uncaging. Experimental conditions are indicated on top of images. Scale bar, 2 μm. d Time course of relative changes in volume (ΔV/V, top graphs, measured by the EGFP intensity) and SA-Ch intensity (ΔCh/Ch, bottom graphs) following uncaging of stimulated (red) and near spines (blue). Stimulation induced a long-lasting volume change, paralleled by a slowly rising accumulation of SA-Ch; in the presence of anysomicin, volume change was transient and no accumulation of SA-Ch was evident. Bold lines represent mean ± EM, whereas narrow lines are single traces for depicted data for stimulated (light red) and nonstimulated (light blue) spines. d Corresponding conditions in c above: from left to right, samples with MNI-Glu/forskolin, MNI-Glu/forskolin/anysomicin, forskolin only/no MNI-Glu. Open circles are corresponding ΔV/V values at 60 min for SA-Ch spines in b