Figure 7.

Anterograde viral labeling strategies for the projections of NI/Rln3 neurons. A, B, Injection of AAV:FLEX-GFP into Rln3^Cre mice bearing the Gt(ROSA)26-targeted tdTomato reporter allele Ai14. A1,B1, tdTomato signal; A2,B2, GFP signal; A3,B3, merged image. This genetic labeling of Rln3^tdT neurons allows assessment of the efficiency of viral labeling. The injected AAV efficiently labels the entire rostrocaudal extent of the NI, with labeling predominantly on the left. Targeted coordinates: AP: −5.40, ML: 0.10, DV: 3.95. Anterograde tracing data appear in Figures 8, 9. C–E, Co-injection of AAV:FLEX-sypGFP and AAV:FLEX -tdTomato into Rln3^Cre mice. Expression is largely restricted to the right side. C1, tdTomato signal; C2, GFP signal; C3, merged image. In D, E, individual labeled fibers can be seen in red, with presumptive synaptic areas labeled in green. Targeted coordinates: AP: −5.40, ML: 0.10, DV: 4.15. Anterograde tracing data appear in Figures 10, 11. F, Injection of AAV:FLEX-GFP and AAV:FAS-tdTomato into Rln3^Cre mice. F1, tdTomato signal; F2, GFP signal; F3, merged image. In this “Cre-on, Cre-off” strategy, the expression cassette in the FAS-tdTomato virus is inactivated rather than activated by Cre recombinase. This allows the specific labeling of the Rln3^Cre-negative neurons in the injected area, and allows the efferents of the Cre-expressing and Cre-non-expressing NI neurons to be distinguished. Labeling of Rln3^Cre neurons is bilateral, but labeling of the surrounding area with FAS-tdT is more extensive on the left. In some Cre-expressing neurons, the inactivation of FAS-tdT was incomplete, and these neurons appear yellow in the merged version. Targeted coordinates: AP: −5.40, ML: 0.10, DV: 4.15. Anterograde tracing data appear in Figure 12. DTg, dorsal tegmental nucleus; LDTg, laterodorsal tegmental nucleus; NI, nucleus incertus. Scale bar: 200 μm.