Figure 4.
Reduced axonal transport of TH mRNA decreases axonal catecholamine levels in SCG axons. A, Intra-axonal DA and NE levels were measured using immunocytochemistry in axons and soma of SCG neurons infected with with lentiCRISPR (control) or sgRNA 1 and sgRNA 2 containing lentiCRSPR (CRISPR). Arrows denote axonal DA and NE. B–H, Fluorescence intensity as a measure of DA (B, D) and NE (C, E) levels was quantified in the soma and distal axons of SCG neurons, respectively, using ImageJ, and fluorescence levels are indicated as relative fluorescence intensity. Reduced DA and NE levels are detected in distal axons of CRISPR-treated neurons, whereas the parental soma DA and NE levels remained at control levels. Data are the mean ± SEM from the measurement of neurons cultured in six Campenot chambers from three independent experiments. F, Distal axons located in the lateral compartment of Campenot chambers were treated for 10 min with 100 mM KCl. NE release into culture media was subsequently measured using an ELISA immunoassay, and NE concentration in the culture media was calculated using linear regression analysis. Reduced NE levels are detected in distal axons of CRISPR neurons, as compared with NE levels measured in the axons of control neurons. Values are mean ± SEM of four different experiments. Student’s t test, **p ≤ 0.003.