Figure 2. Characterization of AHA-tagged proteins in processes of NAc MSNs.

Co-cultured NAc and PFC neurons were incubated with methionine (Met, 1 mM), the non-canonical amino acid azidohomoalanine (AHA, 1 mM), or 1 mM AHA plus the protein synthesis inhibitor anisomycin (40 μM) (AHA + Aniso) for 2 hours and then tagged using click chemistry with 20 nM DBCO-Cy5 for visualization of AHA. A, Top panels show representative images of NAc MSN processes under phase contrast and lower panels show Cy5 signal detected with fluorescence microscopy. Co-incubation of AHA with anisomycin reduced Cy5 fluorescence in NAc MSN processes nearly to the level detected in the Met control group, confirming that Cy5 fluorescence reflects AHA incorporation into newly translated proteins. We note that Cy5 signal was detected in the soma of all groups (not shown). Scale bar = 50 μm. B, Quantification of Cy5 fluorescence in processes of NAc MSNs for the experimental groups shown in panel A (Met, AHA, and AHA + Aniso). In addition, data are shown for a negative control group treated with AHA but no DBCO-Cy5 (No Click). The Cy5 signal was markedly higher in the AHA group relative to all other groups (**** p<0.0001). Data are presented as arbitrary fluorescent units (au). C, Blocking microtubule-dependent transport of newly synthesized proteins from the soma, using the microtubule polymerization inhibitor colchicine (Colc), did not impact basal protein translation in NAc MSN dendritic segments measured using AHA incorporation (left bars). Colchicine also failed to affect bicuculline (Bicuc)-stimulated protein translation in processes of NAc MSNs (right bars). D, A trend towards a decrease in the AHA signal in NAc MSN dendritic segments was observed with increasing distance from the soma (one-way ANOVA, p=0.12). Numbers correspond to measures made starting 15, 30 or 45 μm from the soma. The number of processes analyzed is indicated within each bar. *p<0.05 indicates main effect of bicuculline on the AHA signal.