Figure 14.

L-NAME treatment does not affect TrkA levels in aged BFCNs. Aged rat BFCNs (DIV21) were treated with 1 mM L-NAME for 24 h prior to analysis of TrkA levels using immunocytochemistry. TrkA is shown in green and DAPI is shown in blue. (A,E) DIV8 BFCNs treated with vehicle; (B,F) DIV21 BFCNs treated with vehicle; (C,G) DIV21 BFCNs treated with L-NAME; (D) Quantification of the images shown in A–C. TrkA levels at the cell bodies were decreased in aged BFCNs relative to young BFCNs regardless of treatment condition. No differences in TrkA levels were observed between aged BFCNs treated with L-NAME and aged vehicle-treated control BFCNs. For the DIV8 vehicle, n = 35 images, DIV21 vehicle n = 30 images, DIV21 L-NAME n = 29 images. One-way ANOVA and post hoc Tukey test, **p < 0.01, ***p < 0.001, (H) Quantification of the images shown in E–G. No differences in TrkA levels were observed between any of the groups at the axon terminals. For the DIV8 vehicle-treated group, n = 35 images, for both aged groups, n = 30 images. All images were obtained from three chambers in three independent experiments. One-way ANOVA, p > 0.05. The highest point in each group was detected as an outlier using ROUT (Q = 1%). Removal of these outliers did not affect the significance of the comparison (p = 0.182). Error bars: SEM. DIV, days in vitro. (I,J) Immunocytochemistry results were confirmed using western blotting for TrkA in DIV21 BFCNs treated with either vehicle or 1 mM L-NAME. (I) Representative western blot comparing relative intensity of TrkA expression normalized to β-actin expression. 16.8 μg of total protein was loaded in each BFCN sample well; 5.88 μg protein for PC12nnr5 and 1.68 μg protein for PC12nnr5B5 were loaded as negative and positive controls, respectively. (J) There were no differences in TrkA expression between BFCNs treated with L-NAME (n = 4) and vehicle treatment (n = 5; p = 0.85). Error bars: SEM. DIV, days in vitro. Scale bars: 10 μm.