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.统计分析以实验小鼠为嵌套分组单位，将其分离得到的单个细胞纳入对应统计模型。采用GraphPad Prism 10.3软件（GraphPad Software, 波士顿, 马萨诸塞州）中的双侧嵌套t检验模块，对背景校正后的平均RFP（红色荧光蛋白，Red Fluorescent Protein）、平均GFP（绿色荧光蛋白，Green Fluorescent Protein）以及R:G比值进行统计检验。 数据可视化以每只小鼠所有细胞的平均值为基础，采用5%-95%分位数箱线图展示，并叠加圆形标记以代表单只小鼠的平均数值。 针对红/绿（R:G）比值的log₂倍变化量绘图时，首先将每只小鼠内的区域胆碱能神经元标准化为所有小鼠的区域邻近神经元平均R:G比值，随后进行log₂转换。 图2数据集标签页： 纹状体SPOTlight荧光的亚区域分析：RFP、GFP及R:G比值的可视化结果与对应统计汇总表详见「图2补充材料2」。log₂倍变化量的可视化结果详见图2D（如上所述）。 图3数据集标签页： 急性衣霉素暴露可改变SPOTlight检测结果：RFP、GFP及R:G比值的可视化结果详见图3B-D。统计汇总细节详见对应图注。 图4数据集标签页： 胆碱乙酰转移酶（ChAT, Choline Acetyltransferase）阳性神经元的全中枢神经系统SPOTlight活性图谱：R:G比值与log₂倍变化量的可视化结果详见图4A-B。统计汇总表详见「图4补充材料1A」。RFP与GFP荧光的可视化结果及对应统计汇总表详见「图4补充材料1B-C」。