Brain oscillations have been hypothesized to support cognitive function by coordinating spike timing within and across brain regions, yet it is often not known when timing is either critical for neural computations or an epiphenomenon. The entorhinal cortex and hippocampus are necessary for learning and memory and exhibit prominent theta oscillations (6–9 Hz), which are controlled by pacemaker cells in the medial septal area. Here we show that entorhinal and hippocampal neuronal activity patterns were strongly entrained by rhythmic optical stimulation of parvalbumin-positive medial septal area neurons in mice. Despite strong entrainment, memory impairments in a spatial working memory task were not observed with pacing frequencies at or below the endogenous theta frequency and only emerged at frequencies ≥10 Hz, and specifically when pacing was targeted to maze segments where encoding occurs. Neural computations during the encoding phase were therefore selectively disrupted by perturbations of the timing of neuronal firing patterns.
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We thank M. Robertson, G. De Guia and S. Ahmadi for assistance; B. Lim, V. Lilascharoen and E. Wang for providing viral vectors; and C. Root and K. Miyamoto for assistance with microscopy. This work was supported by NIH grant no. T32 GM007240 to C.R.Q.; a Howard Hughes Medical Institute International Student Research Fellowship to I.Z.; NIH grant no. R01 MH119179 and the Walter F. Heiligenberg Professorship to J.K.L.; and NIH grants no. R01 NS102915, no. R21 MH100354, no. R01 NS084324 and no. R01 NS097772 to S. Leutgeb.
The authors declare no competing interests.
Peer review information Nature Neuroscience thanks James Ainge and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
(a) Pacing efficiency scores for recordings when each mouse explored the open field (top) or rectangular track (bottom). For every 2-s time-window, the peak LFP frequency was calculated for sessions without stimulation (gray bars) and with 12 Hz stimulation (blue bars), and the probability distribution of these peak LFP frequencies was plotted for each animal. Efficient pacing results in a narrow frequency peak at the stimulation frequency, and the pacing efficiency score was therefore defined as the maximum probability (in 0.2 Hz wide bins within 1 Hz of the stimulation frequency) in stimulation sessions. (b) The ratio of the spike waveform peak to trough amplitude (peak-valley ratio) and average firing rate (Hz) for each recorded cell was calculated to determine appropriate cutoff thresholds that separated between putative principal cells (blue circles) and putative interneurons (red circles). For cells recorded from the mEC either on the rectangular track (left) or the open field (middle), we used a rate cutoff of 15 Hz. For cells recorded from the hippocampus (right), we used a combined rate cutoff of 10 Hz and peak-valley cutoff of 1.5. (c) Example of multiple single-unit clusters recorded during the pre-stimulation baseline (baseline 1, left) and during optical stimulation (right). Defined single-unit clusters (red, green and blue) remained stable between sessions without stimulation and with stimulation. (d, top) L-ratio calculated for clusters of single units recorded in the mEC. (d, bottom) Isolation distance values calculated for clusters of single units recorded in the mEC. There was no change in L-ratio [n(B1, 8 Hz, 10 Hz, 12 Hz, B2) = 593, 476, 111, 531, 593 cells from 11 mice, H(4, 2299) = 2.17, P = 0.704, Kruskal-Wallis test] or isolation distance [n(B1, 8 Hz, 10 Hz, 12 Hz, B2) = 580, 462, 109, 518, 579 cells from 11 mice, H(4, 2243) = 7.91, P = 0.095, Kruskal-Wallis test] across stimulation conditions. NS, not significant.
(a) Running speed in either the open field (left) or on the rectangular track (right) was not altered when oscillations were paced by MSA stimulation [open field: grey and colored dots are individual data points, horizontal bars are medians, top and bottom of boxes are 25th and 75th percentile, n = 11 mice, H(4,487) = 7.2, P = 0.13, Kruskal Wallis test; linear track, grey and colored dots are individual data points, baseline vs stimulation, 8 Hz: n = 55 sessions in 6 mice, mean (±SEM), 12.08 (±0.44) and 11.51 (±0.40), t(54) = 2.31, P = 0.025 (0.074, Holm-Bonferroni corrected); 10 Hz: n = 9 sessions in 3 mice, median (25th, 75th percentile), 9.31 (8.77, 9.56) and 9.40 (9.33, 12.44), W(10) = 37, P = 0.065; 12 Hz: n = 54 sessions in 6 mice, mean (±SEM), 11.87 (±0.41) and 11.36 (±0.43), t(53) = 1.67, P = 0.10, two-sided paired t-test for normally distributed data, otherwise two-sided Wilcoxon signed rank test]. n.s., not significant. (b) There were no consistent effects on running speed in any of the maze zones during light stimulation in the spatial alternation task. During no delay trials, there was a minor increase in the delay zone with 8 Hz stimulation (note that the mice are running through the delay zone in the no delay trials) and in the reward zone with 10 Hz stimulation [delay, 8 Hz: n = 22 mice, z = -3.13, P = 0.0017 (0.027, Holm-Bonferroni corrected); reward, 10 Hz: n = 25 mice, z = -3.11, P = 0.0019 (0.028, Holm-Bonferroni corrected), two-sided Wilcoxon signed rank tests]. In 2 s delay trials, there was a minor decrease in running speed with 10 Hz stimulation on the stem [n = 25 mice, z = 3.92, P = 9.0 ×10-5 (0.0014, Holm-Bonferroni corrected), two-sided Wilcoxon signed rank test]. For all other comparisons, we do not find any differences in running speed on any of the maze segments.
Extended Data Fig. 3 The hippocampal depth profile during paced oscillations corresponded to the depth profile during endogenous theta oscillations.
(a) Sixteen-site linear silicon probes were chronically implanted in the hippocampus, and LFP recordings were performed in freely moving mice (n = 3 with one mouse excluded from analysis because of inadequate pacing; average pacing efficiency scores: 0.37 for mouse 89, 0.16 for mouse 95, and 0.04 for the excluded mouse 97). Schematic and brightfield histology images of the dorsal hippocampus for mouse 89 and 95. (b) Example raw LFP traces from all 16 recording sites during movement and rest. Ripple amplitude is known to be maximal in the pyramidal cell layer (encircled with red line), and alignment of the probe with the pyramidal layer was performed by visual inspection of sharp wave ripple amplitude and matched with the position of the probe in the histological reconstruction in (a). Scale bar is 250 ms. (c) Average current source density across hippocampal layers from recordings without stimulation (no stimulation) and with rhythmic stimulation at 8, 10, and 12 Hz. Periods from -125 to +125 ms are shown to include approximately two theta cycles (mouse 89, first and second column; mouse 95, third and fourth column). Note that there is a shortening of the theta cycle with higher stimulation frequencies, but during periods with stimulation the most pronounced current source-sink pair remained in slm, which is the termination zone of direct projections from entorhinal cortex. The distribution of less pronounced sink-source pairs across other hippocampal layers is also consistent between stimulation and no stimulation sessions. so, stratum oriens; sp, stratum pyramidale; sr, stratum radiatum; slm, stratum lacunosum moleculare.
(a) Example spike time autocorrelations for mEC interneurons during periods without stimulation (gray, left) and with stimulation (blue, middle) at 8 and 12 Hz. Right, FFT analysis of spike trains shows accelerated frequencies during stimulation compared to no stimulation sessions. Red vertical lines denote the peak oscillation frequency without and with light stimulation. (b) Same as in (a) but for hippocampal interneurons in the CA1 area. (c) Cumulative distribution function of the peak power (from the FFT analysis of spike trains) for mEC interneurons (left) and hippocampal interneurons (right). Interneurons generally showed high amplitude theta modulation at baseline, and a further increase compared to baseline was only observed at a subset of stimulation frequencies (mEC, 8, 10 and 12 Hz: K = 0.50, 0.30, 0.16; P = 1.7 ×10-5, 0.20, 0.44; hippocampus, 8, 10, and 12 Hz: K = 0.50, 0.80, 0.56, P = 0.014, 0.036, 0.078, two-sided KS tests). (d) Cumulative distribution function of the peak oscillation frequency of mEC interneurons (left) and hippocampal interneurons (right). Interneurons in mEC and hippocampus were similarly entrained [8, 10, and 12 Hz stimulation, mEC: n = 43, 23, 57; difference between stimulation frequency and cells’ oscillation frequency, median (±iqr) = 0.04 (±0.08), 0.10 (±0.23), 0.09 (±0.16); hippocampus: n = 18, 5, 9; median (±iqr): 0.19 (±2.64), 0.25 (±0.29), 0.11 (±1.21); mEC vs hippocampus: z = -0.14, -2.04, -0.36, P = 0.89, 0.04, 0.72, two-sided Wilcoxon rank sum tests].
Extended Data Fig. 5 Light-induced remapping was observed in the mEC of control mice on the rectangular track, but not in the open field.
(a) Rectangular track, Firing statistics of mEC cells in GFP control mice (n = 6) expressing mEmerald in the medial septal area. As described for Fig. 5, the mice ran on a rectangular track with alternating light-off and light-on laps. During light-on laps, stimulation frequency was either 8 Hz or 12 Hz. The mean firing rate and peak firing rate of principal cells did not differ between no stimulation and stimulation laps for either stimulation frequency [Average firing rate, 8 and 12 Hz: n = 153 and 150 cells, two-way ANOVA, stim frequency*light-off vs on, interaction, F(1,602) = 0.037, P = 0.85, stim frequency, F(1,602) = 0.10, P = 0.76, light-off vs on, F(1,602) = 2.83, P = 0.093. Peak firing rate, 8 Hz and 12 Hz: n = 153 and 150 cells, two-way ANOVA, stim frequency*light-off vs on, interaction, F(1,602) = 0.15, P = 0.70, stim frequency, F(1,602) = 0.79, P = 0.37, light-off vs on, F(1,602) = 0.03, P = 0.86]. (b) A decrease in the number of place fields per cell as a result of light-induced remapping was observed across both stimulation frequencies in control mice [8 Hz and 12 Hz: n = 153 and 150 cells, two-way ANOVA, stim frequency*light-off vs on, interaction, F(1,602) = 0.28, P = 0.60, stim frequency, F(1,602) = 1.54, P = 0.21, light-off vs on, F(1,602) = 10.6, P = 0.0012]. Letters above columns (a, b) indicate significant differences between light-on and light-off conditions, two-sided Tukey-Kramer posthoc tests. (c) Light delivery in GFP control mice resulted in a significant decrease in place field stability within light-on trials and resulted in remapping, measured as the correlation between light-on and light-off trials. Both measures remained higher than a shuffled distribution suggesting partial light-induced remapping. There were no differences between stimulation frequencies for rate map stability condition [within light-off, within light-on, between light-on and light-off, shuffled; 8 and 12 Hz: n = 153 and 150 cells, two-way ANOVA, stim frequency*condition, interaction, F(3,1201) = 0.29, p = 0.83, stim frequency, F(1,1201) = 0.35, p = 0.56, condition, F(3,1201) = 328.2, p < 0.001]. Different letters (a-d) above columns indicate significant differences (P < 0.001) between conditions, two-sided Tukey-Kramer posthoc tests. (d) There was no change in spatial information of mEC fields on the linear track despite significant remapping [8 Hz and 12 Hz: n = 153 and 150 cells, two-way ANOVA, stim frequency*light-off vs on, interaction, F(1,602) = 0.26, P = 0.61, stim frequency, F(1,602) = 0.04, P = 0.84, light-off vs on, F(1,602) = 3.18, P = 0.075]. (e) Positions of the spatial fields of mEC principal cells on the rectangular track are linearized and ordered left to right for each stimulation frequency according to the position during light-off sessions. The reorganization across light-off and light-on trials arises as a result of blue light delivery in control mice without pacing. (f) Open field, MEC cells were recorded from GFP control mice (n = 6) in a random foraging task, as described in Fig. 6. Light delivery (at either 8 Hz, 10 Hz, or 12 Hz) was performed during 1-2 sessions in-between two baseline sessions without light delivery. There was no change in the mean firing rate of principal cells and interneurons during stimulation sessions. (g) Light stimulation did not have consistent effects on irregular spatial cells as measured by the spatial information, spatial field stability (within-session correlation), and the extent of remapping (rate map correlation across sessions) [n(B1, 8 Hz, 10 Hz, 12 Hz, B2) = 39, 27, 29, 21, 39 cells from 6 mice]. In (a-d), (f), and (g), grey and colored dots are individual data points, horizontal bars are medians, top and bottom of boxes are 25th and 75th percentile. See Supplementary Table 2 for detailed statistics.
(a) There was a minor decrease in the peak firing rate of principal cells during MSA stimulation compared to baseline (grey and colored dots are individual data points, horizontal bars are medians, top and bottom of boxes are 25th and 75th percentile). Peak rate with 8 and 12 Hz stimulation was decreased compared to both the pre- and post-stimulation baseline. Peak rate at 10 Hz was decreased only compared to the pre-stimulation baseline [n(B1, 8 Hz, 10 Hz, 12 Hz, B2) = 593, 477, 170, 589, 592 cells from 11 mice, H(4,2416) = 32.09, P = 1.8 ×10-6, Kruskal Wallis test]. (b) There was a significant decrease in the peak firing rate of interneurons during 10 Hz and 12 Hz stimulation sessions compared to the pre-stimulation baseline (grey and colored dots are individual data points, horizontal bars are medians, top and bottom of boxes are 25th and 75th percentile). The rates with 12 Hz stimulation also differed from the post-stimulation baseline [n(B1, 8 Hz, 10 Hz, 12 Hz, B2) = 58, 42, 23, 55, 58 cells from 11 mice, H(4,231) = 12.04, P = 0.017, Kruskal Wallis test]. *** P < 0.001, * P < 0.05. See Supplementary Table 2 for detailed statistics.
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].
Extended Data Fig. 8 Pacing of oscillations at frequencies below 8 Hz did not result in memory impairment.
(a) Pacing was efficient with 4 Hz and 6 Hz stimulation in the majority of mice but harmonics were frequently observed. Distribution of the predominant frequency (left), power spectrum (insert), and example raw and filtered (3-22 Hz) waveforms (right) without stimulation and with 4 Hz stimulation in one mouse with weak harmonics (top) and one mouse with strong harmonics (bottom). (b) Proportion-based pacing scores at stimulation frequencies <8 Hz, and for comparison, at stimulation frequencies of 8 Hz and 12 Hz [n(4 Hz, 6 Hz, 8 Hz, 12 Hz) = 12, 12, 25, 29 mice]. Dots are individual data points, open circles and bars are median ± 25th to 75th percentile. When harmonics at 8 and 12 Hz were more pronounced the pacing efficiency score at 4 Hz was reduced [for example, 0.16 in the bottom example in (a) is below the threshold of 0.2], such that these sessions were excluded from analysis of behavioral data (n = 3 at 4 Hz, 3 at 6 Hz). (c) Similar to the effect with 8 Hz stimulation, stimulation at 4 and 6 Hz did not result in impaired memory performance in the delayed spatial alternation task (No delay: n = 9 and 8, W = 14.0 and 6.5, P = 0.53 and 0.94; 2 s delay: W = 12.5 and 18.5, P = 0.48 and 0.59; 10 s delay: W = 25.0 and 20.5, P = 0.094 and 0.34, two-sided Wilcoxon signed rank tests). Dots are individual data points, open circles and bars are median ± 25th to 75th percentile. Data with 8 Hz and 12 Hz pacing are shown for comparison and correspond to the data shown in Fig. 8. * P < 0.05, ** P < 0.01. See Supplementary Table 3 for detailed statistics.
Extended Data Fig. 9 There was no relation between pacing efficiency and the decreased behavioral performance in the 10 s delay condition.
(a) Comparisons of the decrease in behavioral performance in the 10 s delay condition with either the pacing efficiency (left, proportion-based score; middle, ratio-based score) or the volume of viral vector expression in MSA (right) did not reveal any correlations at any stimulation frequency [ratio-based score, n(10, 12, 20 Hz) = 25, 24, 14; rho = 0.19, -0.086, -0.55; P = 0.35, 0.68, 0.033 (0.10, Holm-Bonferroni corrected); proportion-based score: n(8, 10, 12, 20 Hz) = 20, 25, 24, 14; rho = -0.12, 0.22, -0.01, -0.06; P = 0.60, 0.30, 0.96, 0.83; EYFP volume, n(8, 10, 12, 20 Hz) = 22, 26, 24, 14; rho = -0.16, 0.0096, -0.34, -0.59; P = 0.48, 0.96, 0.10, 0.027 (0.11, Holm-Bonferroni corrected), Spearman rank correlations]. (b) Behavioral data for no stimulation and stimulation sessions at each frequency. Dots are individual data points, open circles and bars are median ± 25th to 75th percentile. The experimental design included, for each of the three delay conditions on each day, blocks of 10 trials with stimulation and 10 trials without stimulation, and the difference in performance between stimulation and no-stimulation blocks was calculated to control for day-to-day variations in behavioral performance (as shown in Fig. 8c and e). Statistical analysis was thus performed on the difference in percent correct trials between blocks with and without stimulation [ChR2/oChIEF: n(8 Hz, 10 Hz, 12 Hz, 20 Hz) = 22, 27, 25, 15 mice, 2 s delay, P = 0.61, 0.021, 0.037, 0.037; 10 s delay, P = 0.86, 0.026, 0.0012, 0.0043, Holm-Bonferroni corrected, two-sided Wilcoxon signed rank tests; GFP/ChR2 green light: n = 10, 14, 14, 10 mice, all P values, n.s., two-sided Wilcoxon signed rank tests). See main text and Supplementary Table 3 for detailed statistics. (c) Behavioral data for sessions with stimulation that were targeted to maze zones [n(Reward, Delay + Stem, Delay + Stem + Reward, Return) = 12, 12, 10, 10 mice; 10 s delay/Return: P = 0.0078, Holm-Bonferroni corrected, two-sided Wilcoxon signed rank test; all other comparisons, n.s.]. Dots are individual data points, open circles and bars are median ± 25th to 75th percentile. Occupancy times in the stimulated zones were higher in the delay + stem + reward condition than in the return condition (Extended Data Fig. 10) such that time of stimulation cannot explain the selective effect of return-arm stimulation on behavioral performance. n.s., not significant, * P < 0.05, ** P < 0.01, two-sided Wilcoxon signed rank tests, see main text and Supplementary Table 3 for detailed statistics.
Extended Data Fig. 10 Length of light stimulation and magnitude of LFP power across maze segments did not explain the selective effect of return-arm stimulation on behavioral performance during 10 s delay trials.
(a) Time in each of the maze zones did not differ between no stimulation and stimulation trials [n(no stimulation, stimulation) = [36,39], [36,39], [36,39], and [36,39] sessions for Delay, Stem, Reward, and Return, z = 0.93, 1.12, 1.12, 0.53, P = 0.35, 0.26, 0.26, 0.59, two-sided Wilcoxon rank sum test]. Data are presented as boxplots (horizontal line in box: median, bottom and top of box: 25th and 75th percentiles, whiskers: most extreme values that are not outliers). Importantly, the median time in the return arms (7.52 s) was substantially lower than in the condition when stimulation was on during the delay + stem + reward (17.68 s), which indicates that the selective effect of return-arm stimulation on memory performance (Fig. 8e and Extended Data Fig. 9c) cannot be attributed to longer stimulation durations. (b) Baseline theta power differed between maze segments [H(3) = 22.76, P = 4.5 ×10-5, Kruskal-Wallis test; delay (n = 24 sessions) vs reward (n = 22 sessions): P = 3.0 ×10-4 (0.0015, Holm-Bonferroni corrected), stem (n = 24 sessions) vs reward (n = 24 sessions): P = 3.4 ×10-5 (0.00020, Holm-Bonferroni corrected), stem (n = 24 sessions) vs return (n = 33 sessions): P = 0.0053 (0.02, Holm-Bonferroni corrected); all other comparisons, n.s., two-sided Wilcoxon rank sum tests], but oscillation power corresponded across maze segments during 12 Hz pacing [n(Delay, Return, Reward, Stem) = 20, 11, 22, 20 sessions, H(3) = 0.77, P = 0.86, Kruskal-Wallis test]. Moreover, oscillation frequency was reliably shifted to 12 Hz in each maze segment. Differences in pacing efficiency across maze segments therefore do not appear to contribute to the selective effect of return-arm stimulation on memory performance (Fig. 8e and Extended Data Fig. 9c). Dots are individual data points, open circles and bars are mean ± SEM.
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Quirk, C.R., Zutshi, I., Srikanth, S. et al. Precisely timed theta oscillations are selectively required during the encoding phase of memory. Nat Neurosci (2021). https://doi.org/10.1038/s41593-021-00919-0