DIO-SPOTlight analysis of cellular fluorescence from figure 2-4

Statistics were performed by nesting each individual cell within the mouse from which they were collected. Two-tailed nested t test option in GraphPad Prism 10.3 software (GraphPad Software, Boston, MA) was performed for background subtracted mean RFP, mean GFP, and R:G ratios. Graphical depiction of the data is shown as averages from all cells per mouse in 5-95 percentile box and whisker plots overlayed with circular symbols indicating the mean value from each individual mouse. Graphs depicting Log2 fold changes in red:green (R:G) ratios were performed by normalizing cholinergic neurons regionally within each mouse to the average R:G ratios of regional neighboring neurons across all mice followed by Log2 transformation. Figure 2 data tab: Sub-regional analysis of SPOTlight fluorescence in the striatum: Graphical depiction of RFP and GFP and R:G ratios along with their statistical summary tables is found in “Figure 2 – Supplement 2.” Graphical depiction of Log2 fold change (as described above) can be found in Figure 2D. Figure 3 data tab: Acute tunicamycin exposure modifies SPOTlight readouts: Graphical depiction of RFP and GFP and R:G ratios are found in Figure 3B-D. Statistical summary details are provided in the figure legend. Figure 4 data tab: CNS-wide Atlas of SPOTlight Activity in ChAT-positive neurons: Graphical depiction of R:G ratios and Log2 fold change is are found in Figure 4A-B. Statistical summary tables are provided in Figure 4 – Supplemental 1A. Graphical depiction and statistical summery tables of RFP and GFP fluorescence are located in Figure 4 – Supplemental 1B-C.