A comparison of viral strategies and model systems to target norepinephrine neurons in the locus coeruleus reveals high variability in transgene expression patterns
Fig 3
Effects of promoter, serotype, and injection volume on viral transduction of LC-NE neurons.
(A) To investigate effects of the promoter on viral transduction, rAAV2/9-hSyn-DIO-eGFP (a construct analog to rAAV2/9-CAG-DIO-eGFP used in Figs 1 and 2) was bilaterally injected into the LC of Dbhcre mice (n = 7 animals). Coronal brain slice image from [37]. (B) Example LC section from an rAAV2/9-hSyn-DIO-eGFP-injected Dbhcre mouse immuno-stained against TH (top left) and GFP (top right), as well as color-coded overlay (bottom left). Cells were identified with CellPose. (C) Efficacy (N(GFP+|TH+)/N(TH+)) and (D) Specificity (N(TH+|GFP+)/N(GFP+)) were comparable upon injection of hSyn-DIO-eGFP (yellow) or CAG-DIO-eGFP (red; re-plotted from Figs 1D and 2) in Dbhcre mice (ns = not significant; two-sample t test, n = 7 each). (E) To investigate effects of the viral serotype on viral transduction, 300 nl of either rAAV2/2-hSyn-eGFP or rAAV2/9-hSyn-eGFP were bilaterally injected into the LC of wild-type mice (n = 6 LC from n = 3 mice each). (F) Example images of hemi-brain slices of an rAAV2/2-injected (orange, left) and an rAAV2/9-injected mouse (blue, center). Images were smoothed and fluorescence was normalized, before thresholding images with an increment of 0.1 and calculating the area above threshold (see example with an increment of 0.2 on the right). (G) Viral spread was quantified at various thresholds, revealing a more restricted spread upon injection of rAAV2/2 as compared to rAAV2/9 (*/**/*** for p < 0.05/0.01/0.001, two-sample t-tests, n = 6 LC per group). (H) To investigate effects of the injection volume on viral transduction, either 300, 100, or 50 nl of rAAV2/9-hSyn-eGFP were bilaterally injected into the LC of wild-type mice (n = 6 LC from n = 3 mice each). (I) Example images of hemi-brain slices of mice injected with 300 nl (dark blue, left), 100 nl (light blue, center) or 50 nl of rAAV2/9-hSyn-eGFP (cyan, right). (J) Viral spread was quantified at various thresholds, revealing a more restricted spread upon injecting 100 and 50 nl of viral suspension as compared to 300 nl of viral suspension (*/**/*** for p < 0.05/0.01/0.001, one-way ANOVA and Tukey’s test, N = 6 LC per group). Asterisks denote significance between the 300 nl group and the 100 nl/50 nl group, respectively, as no significant differences were found between the 100 nl and the 50 nl group. Numerical data underlying panels C, D, G, and J can be found in S1 Data.