We next confirmed the effectiveness of the BAP removal algorithm. First, BAP removed spine signals showed sharp orientation tuning (OSI = 0.84 ± 0.14, mean ± s.d., n=190 spines) comparable to V1 excitatory neurons (Supplementary Fig. 9, p>>0.05, Wilcoxon rank sum). This holds even for spines preferring the orthogonal orientation compared to the soma (OSI=0.82 ± 0.16, n=24 spines), indicating that the BAP contamination was cleanly removed. Second, the majority (79.7%) of visually responsive spines showed little trial-to-trial correlation with the dendritic shaft signal after BAP subtraction. 20.3% spines still displayed significant correlation with shaft responses compared to trial shuffled controls (p<0.01). This could reflect synchronously active pre-synaptic cells, or imperfect BAP action potential signal subtraction. Because we were unable to distinguish between these possibilities, these spines were excluded from further analysis. In two cells (cell 4 and cell 5; Fig. 5), we collected larger numbers of trials (15 trials per orientation), which made it possible to further exclude trials with detectable dendritic responses (ΔF/F0 > 6%). The result (i.e., the preferred orientation of the summed spine responses) was identical as with using the subtraction procedure alone.
