FIGURE 1.

Action potential blockade and up-regulation produce opposite effects on proteasome activity in hippocampal neurons. A, schematic of the pa UPS reporter Sindbis constructs. The CL1 degradation signal (degron) is fused to the carboxyl terminus of paGFP (non CL1 containing control reporter), converting it into an ubiquitin-dependent proteasomal degradation reporter (paGFPu). mCherry is co-expressed using an IRES signal. B, representative image of a hippocampal neuron infected with paGFPu before (PRE) and after (POST) photoactivation. mCherry is expressed to locate infected cells before photoactivation. C, CL1 degron promotes degradation of paGFPu. Straightened dendrites from representative time-lapse experiments of cultured hippocampal neurons expressing paGFPu alone, paGFPu plus MG132 (25 μm), or paGFP. paGFPu fluorescence decay is observed in dendrites and is blocked by the addition of the proteasome inhibitor MG132. No significant fluorescence loss is observed in paGFP (no degron control)-expressing dendrites. D, representative straightened dendrites from time-lapse experiments in which cultured hippocampal neurons expressing paGFPu were treated with TTX (2 μm), BIC (40 μm), or BIC plus MG132 (25 μm), and imaged every 2 min for 20 min. Time of treatment is indicated by black arrows. E, grouped analysis (plotted as line graphs, mean ± S.E.) of dendritic fluorescent intensity normalized to time 0 for control (paGFPu alone), MG132, TTX, BIC, BIC plus MG132-treated neurons, or paGFP (no degron). F, bar graph depicting the mean degradation rate ± S.E. (AU) per treated group normalized to the control (paGFPu) rate of fluorescence decay. BIC dramatically increased paGFPu rate of degradation, most significantly in the first 10 min, whereas TTX blocks paGFPu degradation (*, p < 0.05; **, p < 0.001, relative to paGFPu (control)). See Table 1 for rates of degradation and p and n values. The scale bar for all images is 10 μm.