Extended Data Fig. 7: Pacing was efficient for all mice that were included in analysis of behavioral data.

(a) Volume of opsin expression in MSA (that is, medial septal area and diagonal band of Broca) is plotted against the log of the ratio-based score during 10 Hz, 12 Hz, and 20 Hz stimulation (open circles). Our previously published data (Fig. 1d in ref. ¹⁹) which showed a correlation between expression volume and pacing scores, are plotted for comparison (diamonds), but with expression volume re-quantified to match the methods of the current study. The previously reported correlation between expression volume and pacing efficiency was not observed in the current dataset [ratio-based score: n(10 Hz, 12 Hz, 20 Hz) = 28, 28, 16 mice, rho = -0.016, -0.092, 0.27, P = 0.94, 0.64, 0.32; proportion-based score: n(8 Hz, 10 Hz, 12 Hz, 20 Hz) = 25, 28, 28, 16 mice; rho = 0.075, 0.064, -0.36, -0.29; P = 0.72, 0.75, 0.058, 0.28, Spearman rank correlations). In the current dataset, pacing scores were consistently high, and it is possible that the correlation is not evident without including a sufficient number of cases with low scores. (b) Some mice in (a) showed high pacing efficiency with relatively low expression volumes which may be a consequence of differences in fiber placement in the current study (open circles) compared to the previous study (ref. ¹⁹, diamonds). (c) The previously used ratio-based pacing score (power within 1 Hz of the stimulation frequency divided by power at 7-9 Hz) was not designed to capture pacing efficiency with 8 Hz stimulation. To use a score that can be applied to all frequencies, we therefore considered the observation that efficient pacing results in a narrow frequency distribution around the stimulation frequency. To quantify the frequency distribution, we first measured the predominant LFP oscillation frequency during every 2-s interval and binned these measurements with a 0.2 Hz resolution. The peak proportion (that is, relative frequency) in bins within 1 Hz of the stimulation frequency is taken as the proportion-based pacing efficiency score. Histograms and the corresponding power spectra (inserts) show that pacing was highly efficient for scores >0.2 (examples with scores of 0.45 and 0.22 are shown). (d) Filtered (3-22 Hz, top) and raw traces (bottom) during periods with and without stimulation show efficient pacing of the hippocampal LFP. Traces are from the session for which pacing efficiency is shown in (c). Light pulses are shown as a blue line. (e) For frequencies higher than 8 Hz, where both the ratio-based and the proportion-based scores can be used, the scores are highly correlated [n(10 Hz, 12 Hz, 20 Hz) = 29, 29, 17 mice, rho = 0.90, 0.78, 0.53, P = 4.7 ×10^-7, 2.6 ×10^-6, and 0.029; Spearman rank correlations after confirming with two-sided KS tests that scores were not normally distributed]. (f) Pacing was highly efficient for most animals and stimulation frequencies [n(8 Hz, 10 Hz, 12 Hz, 20 Hz) = 25, 29, 29, 17 ChR2 mice and 10, 10, 10, 6 GFP mice]. Dots are individual data points, open circles and bars are median ± 25th to 75th percentile. When mice with opsin expression did not reach a score of 0.2 (stippled line) at a particular stimulation frequency, the behavioral data at the frequency were excluded from the analysis (n = 1 at 8 Hz, 2 at 12 Hz, and 1 at 20 Hz). A score of 0.2 was not exceeded in any of the GFP controls except for three cases at the 8 Hz frequency. At the 8 Hz frequency, endogenous theta oscillations can result in scores up to 0.3. (g) For 10-trial blocks with the longest sustained stimulation duration (that is, with 10 s delay, average duration for stimulation sessions, 8 Hz: 392.6 s, 10 Hz: 392.5 s; 12 Hz: 410.0 s; 20 Hz: 345.4 s), pacing efficiency did not differ between the first and the second half of the block [n(8 Hz, 10 Hz, 12 Hz, 20 Hz) = 24, 28, 27, 16 mice, dots are individual data points, open circles and bars are median ± 25th to 75th percentile, z = 0.37, -0.30, 0.82, 1.40, P = 0.71, 0.77, 0.41, 0.16, two-sided Wilcoxon signed rank tests].