Extended Data Figure 5. Controls for Dbh-Cre-based trans-synaptic tracing and TRIO analysis in LC.

a, A Representative coronal section of the LC from a mouse heterozygous for Dbh-Cre and Ai14 Cre reporter transgenes. Sections were labeled with an antibody against tyrosine hydroxylase (TH), an enzyme in the biosynthetic pathway for norepinephrine (green), while cells expressing Cre recombinase are visible by expression of tdTomato (red). b, Quantification of the number of tdTomato+ neurons in the LC that were also labeled by TH antibody (n=3). Every 50-μm section through the LC was collected for quantification. Qualitatively, all TH+ cells expressed tdTomato; however, we cannot determine quantitatively because we could not accurately count TH+ cells due to dense process staining. c, Top, schematic for negative control where AAVs that express Cre-dependent TC and G were injected into the LC of wild-type mice, followed by injection of RVdG. Middle, coronal section of the LC stained with DAPI (blue) shows a small number of GFP+ neurons at the injection site. The dotted rectangle highlights GFP+ neurons magnified in the bottom panel. d, Top, in this negative control, Dbh-Cre mice received Cre-dependent TC (without G) via AAV injection into the LC, followed by RVdG. Middle, a coronal section of the LC stained with DAPI (blue) shows infection of Cre+ LC neurons with TC (red) or TC and RVdG (yellow) at the injection site. The dotted rectangle highlights infected LC neurons magnified in the bottom panel. Most green cells are also red. No GFP+ cells were observed outside the region immediately adjacent to the injection site, indicating that trans-synaptic tracing depends on G. e, Quantification of the number of GFP+ cells (c), or GFP+ cells that did not colocalize with TC (d), that were observed in the experiments described in (c, n=8) and (d, n=6). By comparison, 1381 GFP+ neurons were counted in the same region for an experimental brain that has the median number of starter cells among the 9 brains. For explanation of background labeling, see Extended Data Fig. 1a–c. In either case, no GFP+ neurons were visible > 800 μm away from the injection site. f, Schematic of brain regions quantified for presynaptic GFP+ neurons. Regions approximately 800 μm anterior and posterior to the center of the LC were excluded from analysis due to local background labeling from TC and GFP. Scale, 50 μm (a), 1 mm (c, d, middle panels), 100 μm (c, d, bottom panels). Error bars, s.e.m.