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Unique contributions of parvalbumin and cholinergic interneurons in organizing striatal networks during movement

Abstract

Striatal parvalbumin (PV) and cholinergic interneurons (CHIs) are poised to play major roles in behavior by coordinating the networks of medium spiny cells that relay motor output. However, the small numbers and scattered distribution of these cells have hindered direct assessment of their contribution to activity in networks of medium spiny neurons (MSNs) during behavior. Here, we build on recent improvements in single-cell calcium imaging combined with optogenetics to test the capacity of PVs and CHIs to affect MSN activity and behavior in mice engaged in voluntary locomotion. We find that PVs and CHIs have unique effects on MSN activity and dissociable roles in supporting movement. PV cells facilitate movement by refining the activation of MSN networks responsible for movement execution. CHIs, in contrast, synchronize activity within MSN networks to signal the end of a movement bout. These results provide new insights into the striatal network activity that supports movement.

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Fig. 1: Experimental framework and imaging protocol.
Fig. 2: Striatal population activity correlates with discrete aspects of movement.
Fig. 3: Optogenetic stimulation of interneuron populations modulates movement.
Fig. 4: Anatomical clustering and coordinated activity within each cell population.
Fig. 5: Coordinated activity between interneurons and MSNs by anatomical distance.
Fig. 6: PVs, but not CHIs, are strong predictors of speed and MSN population activity.
Fig. 7: Interneurons regulate MSN activity and movement state.

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Data availability

The raw data that support the findings of this study are available from the corresponding author upon request.

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Acknowledgements

We thank members of the Han Lab for suggestions on the manuscript. We would also like to thank J. Li and E. Kolaczyk for their useful insights on data analysis and statistical analysis. We would also like to acknowledge C. Harvey and D. Dombeck for their help on the construction of the 3D spherical tracking system. This work was supported by the NIH Director’s Office (No. 1DP2NS082126 to X.H.), NINDS (Nos. 1R01NS081716 and 1R01NS087950 to X.H.), the Grainger Foundation (to X.H.) and the Pew Foundation (to X.H.).

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Authors and Affiliations

Authors

Contributions

W.M.H. and H.J.G. performed all experiments. M.R. and D.Z. analyzed the data. M.B. contributed software for video processing and data analysis. X.H. supervised the study. W.M.H, H.J.G, M.R., A.G.D and X.H. wrote the manuscript and contributed to the interpretation of the results. A.G.D. and M.K. provided consultation on both statistical analysis and permutation tests. V.S. provided consultation on both calcium imaging data analysis and generalized linear models.

Corresponding author

Correspondence to Xue Han.

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The authors declare no competing interests.

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Journal peer review informationNature Neuroscience thanks Tianyi Mao and other anonymous reviewer(s) for their contribution to the peer review of this work.

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Integrated supplementary information

Supplementary Figure 1 Experimental timeline and post-experiment histology.

(a) Experimental timeline. (b) Post-study representative confocal photomicrograph examples from two individual PV-Cre mice used in this study. Each example includes merged images along with separate channel images for GCaMP6f (green), tdTomato (red), and anti-PV immunofluorescence (blue). White arrows indicate cells with co-localized expression of tdTomato and anti-PV. Red arrows highlight cells expressing anti-PV that were not tdTomato labeled. (c) Box plots showing quantification of viral specificity from a subset of PV-Cre mice recorded from cohort 1 and cohort 2 of this study (n = 7; see methods). 74.0 ± 0.8% (mean ± s.e.m.) of PV immunoreactive cells co-expressed tdTomato and 96.1 ± 0.2% (mean ± s.e.m.) of tdTomato expressing cells were co-immunoreactive for PV. (d) Same as (b), but for two representative Chat-Cre mice, with anti-Chat immunofluorescence in blue. (e) Box plots showing quantification of viral specificity from a subset of Chat-Cre mice recorded from cohort 1 and cohort 2 of this study (n = 7; see methods). 78.8 ± 0.4% (mean ± s.e.m.) of Chat immunoreactive cells co-expressed tdTomato and 97.2 ± 0.2% (mean ± s.e.m.) of tdTomato expressing cells were immunoreactive for Chat. In a separate analysis, comparison of tdTomato images taken during all imaging sessions (n = 28 sessions in 6 PV-cre mice, and 6 Chat-cre mice), revealed that 63.2 ± 5.1% (mean ± s.e.m.) of all tdTomato positive cells, also co-expressed GCaMP6f. For all box plot figures, middle lines indicate the median, lower and upper edges of the box indicate quartiles below and above the median, and upper and lower whiskers indicate maximum or minimum values respectively.

Supplementary Figure 2 Changes in calcium event amplitude, rise time, and width during movement; and correlations with movement acceleration, velocity, and rotation.

(a) The peak amplitude of calcium events in MSNs, PVs, and CHIs, during periods of high or low speed movement. MSN events were characterized by the largest amplitude. Further, the amplitude of events in all cell types tended to increase from periods of low movement to high, however only events in MSNs exhibited a statistically significant change (Kruskal-Wallis, main effect of cell type, X 2(2)=38.4, P = 4.49e-09, n MSN=7727 neurons, n PV=78 neurons, n CHI=50; mean ranks: MSN: 3.95e+03, PV: 2.95e+03, CHI: 2.38e+03; Tukey’s HSD post hoc, MSN vs PV: P = 3.16e-04; MSN vs CHI: P = 3.30e-06; PV vs CHI: P = 0.35; high speed versus low speed; two-sided sign-test, MSN: sign=1122, n = 1813, P = 5.59e-24; CHI: sign=6.0, n = 11, P = 1.0; PV: sign=6, n = 13, P = 1.0). (b) The rise time of calcium events in MSNs, PVs and CHIs, detected during periods of high or low speed movement. Calcium events in PVs exhibited the slowest rise time when compared to those in CHIs and MSNs, which were similar to one another (Kruskal-Wallis, main effect of cell type, X 2(2)=60.8, P = 6.26e-14, n MSN=7727 neurons, n PV=78 neurons, n CHI=50 neurons; mean ranks: MSN: 3.9e+03, PV: 5.9e+03, CHI: 4.3e+03; Tukey’s HSD post hoc, MSN vs PV: P = 9.56e-10; MSN vs CHI: P = 0.38; PV vs CHI: P = 4.46e-04; high speed versus low speed; two-sided sign-test, MSN: sign=1139, n = 1778, ties=35 P = 2.60e-32; CHI: sign=8, n = 11, P = 0.23; PV: sign=9, n = 13, P = 0.27). (c) The width of calcium events detected during periods of high or low speed movement. Like rise times, calcium events in PVs are longest when compared to those in CHIs and MSNs, which were similar (Kruskal-Wallis, main effect of cell type, X 2(2)=53.2, P = 2.75e-12, n MSN=7727 neurons, n PV=78 neurons, n CHI=50 neurons; mean ranks: MSN: 3.91e+03, PV: 5.78e+03, CHI: 4.13e+03; Tukey’s HSD post hoc, MSN vs PV: P = 9.57e-10; MSN vs CHI: P = 0.772; PV vs CHI: P = 1.65e-04; high speed versus low speed; two-sided sign-test, MSN: sign=1135, n = 1807, ties=6, P = 1.63e-27; CHI: sign=7, n = 11, P = 0.55; PV: sign=5, n = 12, ties=1, P = 0.77). (d) Mean population fluorescence of each cell class as a function of speed. Error bars and center are mean ± s.e.m. Population-wide average fluorescence in PV cells and MSNs generally increased with increasing speed, but not CHIs (Friedman test, MSNs: X 2(6)=3.02e+03, P = 0, n = 7755 neurons; PV’s: X 2(6)=94.1, P = 4.26e-18, n = 79 neurons; CHIs: X 2(6)=9.19, P = 0.16, n = 51 neurons) (e) Mean population fluorescence of each cell class as a function of acceleration. Error bars and center are mean ± s.e.m. Again, population fluorescence in MSN’s and PV’s increased with the magnitude of acceleration, but this trend was not observed in the population of CHI’s. (Friedman test, MSNs: X 2 (7)=3.24e+03, P = 0, n = 7755 neurons; PV’s: X 2(7)=118, P = 1.69e-22, n = 79 neurons; CHI’s: X 2 (7)=3.56, P = 0.83, n = 51 neurons). (f) Animals showed no movement directional preference across imaging sessions, with an equal distribution of directional movement (difference in numbers of left-biased and right-biased sessions between Chat-Cre and PV-Cre mice (CHI: 7 right and 3 left-biased; PV: 12 right and 6 left-biased; two-sided binomial test: 19 right bias, 9 left bias; P = 0.087)). (g) Mean population fluorescence of each cell class as a function of rotation calculated as angular velocity, plots are mean ± s.e.m. (Friedman, main effect of rotation, MSNs: X 2(4)=3.29e+03, P = 0, n = 7755 neurons; PV’s: X 2(4)=1.20e+02, P = 5.59e-25, n = 79 neurons; CHI’s: X 2(4)=1.28e+01, P = 0.012, n = 51 neurons). All three neuron populations exhibited increases in fluorescence with rotational rate, though this trend was more dramatic in the population of PVs. (h) The proportions of MSN, CHI, and PV neurons positively modulated in the final 500ms (−0.5 to 0s) before movement onset relative to the baseline period (−1s to −0.5s). A significantly larger fraction of PVs were positively modulated than the other two cell types (two-sided Fisher tests, PV vs MSN: odds ratio=0.500, P = 0.026; PV vs CHI: odds ratio=0.221, P = 0.019; n MSN=7755 neurons, n PV=79 neurons, n CHI=51 neurons, Bonferroni-corrected for 3 comparisons, data from all 28 sessions and 12 mice). (i) Change in movement speed in PVs, CHIs and MSNs in the 1.5 seconds following a calcium event. Change in positive movement speed following PV events is larger than those following MSN events and the reduction in movement speed is larger for CHIs than those following MSN events (mixed-effects model, ANOVA: F(2,7852)=5.75, P = 0.0032; post hocs, PV vs MSN: t(7852)=−2.76, P = 0.0086; CHI vs PV: t(7852)= 3.26, P = 0.0033; CHI vs MSN: t(7852)=−1.97; P = 0.049; Benjamini-Hochberg corrected, data from all 28 sessions and 12 mice). All analyses were conducted across all 28 recording sessions in all 12 mice. ***=P<0.001, **=P<0.01, *=P<0.05. For all box plot figures, middle lines indicate the median, lower and upper edges of the box indicate quartiles below and above the median, and upper and lower whiskers indicate the points furthest from the median whose value did not exceed 1.5 times the first-to-third quartile range above the third quartile or below the first quartile.

Supplementary Figure 3 Heterogeneous responses of all three cell classes at movement onset.

(a, b) All movement modulated neurons (n = 465 out of 546 neurons) identified in a representative recording session from a Chat-Cre mouse, sorted by rank-sum z-statistic (a; lowest: top, highest: bottom) and aligned to movement onset (n = 459 MSN and 6 CHIs). (b) Population fluorescence of the neurons classified as either positively (n = 356), negatively (n = 109), or non-modulated (n = 81) by movement onset from the neurons shown in (a). Shaded regions and center are mean ± s.e.m. (c) Population pie charts across all animals (n = 12 mice) showing proportion of neurons modulated by movement onset, separated by polarity (+ or –) and cell type. Across all animals, the proportion of cells not modulated by movement onset was similar across cell types: 24.2% for MSNs, 19.6% for CHIs, and 12.7% for PVs. MSNs and PV neurons generally were positively modulated by movement (MSN’s: 54.4% positive vs. 21.4% negative, binomial test P = 1.15e-250, n = 4216 positive vs 1664 negative; PV’s: 73.4% positive vs. 13.9% negative, binomial test P = 7.08e-09, n = 58 positive vs 11 negative. Each test was Bonferroni-corrected for 3 total comparisons within each cell genotype: all binomial tests were two-sided). However, nearly equal numbers of CHIs were positively or negatively modulated by movement (43.1% positive vs. 37.3% negative, P = 1, Bonferroni-corrected), consistent with the observation that a substantial portion of CHIs signal reductions in movement (Fig. 2f,h). Also see Supplementary Fig. 7 for individual analysis of CHI populations.

Supplementary Figure 4 PV activity separated by movement modulation type.

(a) As described in the main text, PVs almost uniformly exhibited increases in fluorescence associated with motion onset. A small population did not, characterized fully here. Mean population fluorescence for positively (red) and negatively (black) modulated PVs, aligned to movement onset. (b) Quantification of the pre-onset movement and post-onset movement windows shown above. Both positively and negatively modulated PV neurons showed a significant change in GCaMP6f fluorescence at movement onset, but in opposite directions (change from pre-onset period; sign-test, Positive-PVs: sign=0, n = 58 neurons from 6 mice, P = 6.94e-18; Negative-PVs: sign=11, n = 11 neurons, P = 9.77e-04). (c) Mean population fluorescence for positively (red) and negatively (black) modulated PVs, aligned to peak velocity. (d) Quantification between the pre-peak velocity and post-peak velocity windows shown above. Only positive-PV neurons showed a significant change in fluorescence between these two time windows (change from pre-peak period; sign-test, positive-PVs: sign=14, n = 58 neurons, P = 1.00e-04; negative-PVs: sign=4, n = 11 neurons, P = 0.55). (e) Mean population fluorescence for positively (red) and negatively (black) modulated PVs, aligned to movement offset. (f) Quantification between the pre-offset and post-offset window shown above. The positive-PV population showed a significant decrease in GCaMP6f fluorescence at movement offset, but there was no change in the negative-PV population (change from pre-offset period; sign-test, positively modulated PVs: sign=45, n = 58 neurons, P = 3.01e-05; negatively modulated PVs: sign=5, n = 11 neurons, P = 1). (g) Anatomical map from a representative PV-Chrimson mouse showing 230 MSNs and the proportion of neurons whose activity is significantly correlated with optogenetic stimulation periods (25 MSNs, in red). PV cells are shown in black. The effect of optogenetic stimulation was not uniform across the entire population of MSNs, and did not generally increase or decrease activity in MSNs (bootstrap test, comparing proportion correlated to 1: P = 0; n sig=37, n total=493 neurons in 4 mice) or an all inactive zero-correlation state (bootstrap test, one-sided, comparing proportion correlated to 0: P = 0; n sig=37, n total=493 neurons in 4 mice). (h) Normalized change in movement speed following calcium events of positively or negatively modulated PVs. Speed tended to remain elevated following an event in the positive-PV population, similar to that observed in the PV population when considered as a whole. However, there was no significant difference in speed across time bins following and event in either cell type: Mixed-effects model; ANOVA for cell type-time bin interaction: F(4,330)=2.14, P = 0.075. n = 11 negatively modulated neurons and 57 positively modulated neurons (1 neuron did not produce a measured calcium event). (i) Linear model predictions of population fluorescence (left) and movement speed (right) based on the activity of positively modulated PVs (red) or negatively modulated PVs (black). Cross-validated correlation values for positively and negatively modulated PV populations predicting MSN fluorescence or speed. Positive PV cells were better predictors of MSN activity than negative-PVs (n = 7 sessions, sign-test, sign=0, P = 0.016), but positive-PVs were not statistically better predictors of speed than negative-PVs (n = 7 sessions, sign-test, sign=1, P = 0.13). Unless otherwise specified, all figures here utilized data from all 10 sessions from all 6 PV-Cre mice. In this figure, all error bars and centers indicate mean ± s.e.m., and all shaded regions indicate the mean ± s.e.m. All sign-tests were two-sided.

Supplementary Figure 5 Examples of neuron activity at movement onset.

(a, b) GCaMP6f fluorescence recorded from individual representative MSNs, aligned at all movement onset events in a recording session. Two different recording sessions from two different animals are depicted in (a) and (b). Mean results across all trials is shown below. (c) GCaMP6f fluorescence recorded from a PV cell, aligned at movement onset for all identified onset events from a typical recording session in a PV-Cre animal, and the mean result at the bottom, showing the pre-movement onset increase in PV activity. (d) GCaMP6f fluorescence recorded from a CHI, aligned at movement onset for all identified onset events from a single recording session in a Chat-Cre animal, and the mean result at the bottom showing a decrease in activity at motion onset.

Supplementary Figure 6 Movement-triggered GCaMP6f fluorescence in cells recorded from a representative PV-Cre mouse and a representative Chat-Cre mouse.

(a) Colormap presents fluorescence intensity across all MSNs from a representative Chat-Cre animal, sorted by the timing of peak fluorescence intensity during the 5 second windows centered at movement onset (top). Mean population average of movement triggered fluorescence from MSNs (n = 473 neurons; blue) and identified CHIs (n = 4 neurons; green) from this recording session (bottom). (b) Same as (a) but for a representative PV-Cre animal (top), and across all MSN (n = 247 neurons; blue) and PV neurons (n = 7 neurons; orange) from that session (bottom). Note the rise in the PV population prior to motion onset. All shaded regions and centers in lower plots are mean ± s.e.m.

Supplementary Figure 7 Heterogeneity in motion coding within the population of CHIs.

(a) Mean population fluorescence for positively (red) and negatively (black) modulated CHIs, aligned to movement onset. (b) Quantification between the pre-onset movement and post-onset movement windows shown above. Both positively and negatively modulated CHI neurons showed a significant change in GCaMP6f fluorescence at movement onset, but in opposite directions (change from pre-onset period; sign-test, Positive-CHIs: sign=0, n = 22 neurons from 6 mice, P = 4.77e-07; Negative-CHIs: sign=19, n = 19 neurons, P = 3.81e-06). (c) Mean population fluorescence for positively (red) and negatively (black) modulated CHIs, aligned to peak velocity. (d) Quantification between the pre-peak velocity and post-peak velocity windows shown above. Neither positively nor negatively modulated CHI neurons showed a statistically significant change in fluorescence between these two time windows (change from pre-peak period; sign-test, Positive-CHIs: sign=10, n = 22 neurons, P = 0.83; Negative-CHIs: sign=6, n = 19 neurons, P = 0.167), although the population of Negative-CHIs closely resembles the relationship seen in the whole population. (e) Mean population fluorescence for positively (red) and negatively (black) modulated CHIs, aligned to movement offset. (f) Quantification between the pre-offset and post-offset window shown above. Each population showed a trend towards a change in GCaMP6f fluorescence at movement offset; specifically a decrease in the positive-CHI population, and an increase in the negative-CHI population (change from pre-offset period; sign-test, positively modulated CHIs: sign=16, n = 22 neurons, P = 0.052; negatively modulated CHIs: sign=5, n = 19 neurons, P = 0.063). (g) Anatomical map showing MSNs (544, blue dots), significantly correlated with a positively modulated CHI (red lines), and those correlated with a negatively modulated CHI (black lines). The MSNs correlated with these two types of CHIs were largely non-overlapping (n = 8 sessions, one-sided permutation-test, 5 with p = 0, P = 0.0082, P = 0.0026, P = 0.0028). Only 9 MSNs (yellow) were correlated with both a positively and negatively modulated CHI in the example shown. (h) Normalized change in movement speed following a calcium event in positively modulated CHIs versus negatively modulated CHIs. The change in speed following an event in either population was similar, and there was no significant interaction between the two CHI types across the subsequent two seconds: Mixed-effects model; ANOVA for cell type-time bin interaction: F(4,190)=0.593, P = 0.668. n = 18 negatively-modulated neurons and n = 22 positively-modulated neurons (1 negatively modulated neuron did not produce a measured calcium event). (i) Linear model predictions of population fluorescence (left) and movement speed (right) based on the activity of positively modulated CHIs (red) or negatively modulated CHIs (black). Cross-validated correlation values for positively and negatively modulated CHI populations predicting MSN fluorescence (n = 8 sessions, sign-test, sign=6, P = 0.29), and speed (n = 8 sessions, sign-test, sign=4, P = 1) indicated that the two populations did not differ from one another with respect to their predictive power. Unless otherwise specified, all figures here utilized data from all 10 sessions from 6 Chat-Cre mice. All error bars and centers indicate mean ± s.e.m., and all shaded regions indicate the mean ± s.e.m. All sign-tests were two-sided.

Supplementary Figure 8 Activity in different striatal cell classes is selectively associated with changes in rotational rate versus movement direction.

(a) Change in movement direction (assessed as zero crossings of the x-axis on the spherical treadmill) aligned to calcium events in PV and CHIs. Zero crossings were compared between periods when a calcium event occurred to all other periods when calcium events were absent. PV calcium events were associated with an increase in changes in movement direction, but not CHI calcium events (two-sided sign-test, sign=51, n = 78 neurons in 6 PV-Cre mice, P = 0.0088; sign=29, n = 50 neurons in 6 Chat-Cre mice, P = 0.32). (b) Optogenetic stimulation of PV neurons led to an increase in changes in movement direction, but not optogenetic stimulation of CHIs (two-sided Wilcoxon rank-sum test, PV-Crimson: w=1.55e+04, P = 0.0058, n laser-on=116 periods, n laser-off=124 periods, in 4 mice; Chat-Crimson mice: w=1.83e+04, P = 0.19, n laser-on=130 periods, n laser-off=138 periods, in 4 mice). *=P<0.05, **=P<0.01, ***=P<0.001. Changes in movement direction were compared between laser on and laser off periods in PV-Chrimson and Chat-Chrimson mice. Thus PV activation was associated with a general change in movement, including changes in rotational behavior coincident with increased velocity. (c) Normalized change in rotational rate aligned to calcium events in MSNs, CHIs and PVs. Mixed model analysis did not reveal significant interactions between time bins and cell types (Mixed-effects model, F(8,39260) =2.11, P = 0.061). Only MSN events were followed by changes in rotation that were significantly different from pre-event levels, specifically at the 0.5-1 second, 1-1.5 second, and 1.5-2.0 second time windows (t(38630)=8.31, P = 6.11e-16; t(38630)=11.3, P = 1.52e-28; t(38630)=5.66, P = 6.23e-08; Benjamini-Hochberg correction for 12 cell-time comparisons. n MSN=7727, n CHI=50, and n PV=78. A total of 30 cells out of 7855 did not produce an event that was included in this analysis, see methods for details; MSN, n = 28 cells; PV, n = 1 cell; CHI, n = 1 cell). Error bars indicate mean ± s.e.m. For all box plot figures, middle lines indicate the median, lower and upper edges of the box indicate quartiles below and above the median, and upper and lower whiskers indicate the points furthest from the median whose value did not exceed 1.5 times the first-to-third quartile range above the third quartile or below the first quartile.

Supplementary Figure 9 Heterogeneous responses of all three cell classes to different aspects of movement.

(a) Summed population calcium activity, and the corresponding movement speed and rotation from a representative recording session from a PV-Cre mouse. Movement onsets and rotation onsets, highlighted with arrows, often co-occurred reflecting a general increase in motor output on the spherical treadmill. (b) 600 identified neurons from the animal depicted in (a) revealing the distribution of cells differentially modulated by speed, rotation, or both, and are color coded and plotted anatomically. Similar to recent reports, quantitative analysis from the full population (n = 7709 neurons) revealed that MSNs close together (<100µm) were likely to be related to the same aspects of movement (Effect of distance by neuron category; two-sided Wilcoxon rank-sum, speed sensitive, w=4.66e+06, n speed=1139 MSNs, n other=6570 MSNs, P = 1.25e-04; rotation: w=5.31e+06, n rotation=1222MSNs, n other=6487 MSNs, P = 4.30e-17; conjunctive: w=1.95e+07, n conjunctive=4706 MSNs, n other=3003 MSNs, P = 1.83e-48). (c) Population pie charts showing proportion of neurons modulated by speed alone (red), rotation alone (purple) or both (black), sorted by cell type. All three cell types are more likely to be both speed and rotation sensitive (conjunctive) than either alone. The number of conjunctive neurons differed significantly from speed alone (binomial test, all two-sided): MSN’s, P = 0, n conjunctive=4736 neurons, n speed=1144 neurons; CHI’s, P = 3.08e-07, n conjunctive=37 neurons, n speed=4 neurons; PV’s, P = 2.27e-08, n conjunctive=58 neurons, n speed=11 neurons; or rotation alone: MSN’s, P = 0, n conjunctive=4736 neurons, n rotation=1230 neurons; CHI’s, P = 8.52e-07, n conjunctive=37 neurons, n rotation=2 neurons; PV’s, P = 2.71e-11, n conjunctive=58 neurons, n rotation=6 neurons; Bonferroni-corrected p-values. Total number of MSNs: 7755 (1144 speed-related, 1230 rotation-related, 4736 conjunctive); total number of PVs: 79 (11 speed-related, 6 rotation-related, and 58 mixed); total number of CHIs: 51 (4 speed-related, 2 rotation-related, and 37 mix-related) from 6 PV-Cre mice and 6 Chat-Cre mice. *=P<0.05, **=P<0.01, ***=P<0.001.

Supplementary Figure 10 Deconvolution of calcium signals.

(a) Color map showing GCaMP6f activity from 375 identified neurons shown in Fig. 2a. (b) Calcium traces of (a), deconvolved into a binary “spike-like” signal used for pairwise correlation analysis.

Supplementary Figure 11 Population activity during optogenetic stimulation in Chat-Cre and PV-Cre mice.

(a) PV population fluorescence (orange) and MSN population fluorescence (blue) during optogenetic simulation of PVs in PV-Chrimson mice. Plotted are normalized fluorescence across 100 laser pulses delivered at ~15 Hz. PV population fluorescence, but not MSN fluorescence, rose significantly at laser onset in the first second of laser illumination (two-sided sign-test, sign=2, n = 26 PVs, P = 1.05e-05; sign=239, n = 493 MSNs in 4 animals, P = 0.53). Interestingly, MSN population fluorescence showed a small increase ~1 second after laser onset, coinciding with the onset of optogenetically evoked movement in PV-Crimson mice (Fig. 2h,i). (b) Same as (a) but during optogenetic stimulation of CHIs in Chat-Chrimson animals. Both CHI and MSN population fluorescence rose at laser onset, and was significantly elevated in the first second of laser illumination (MSN events: two-sided sign-test, sign=279, n = 740 MSN neurons in 4 animals, P = 2.86e-11, CHI events: sign-test, sign=3, n = 21 CHIs in 4 animals, P = 0.0015). (c) PV population fluorescence (orange) and MSN population fluorescence (blue) during same laser illumination protocol (100 laser pulses delivered at ~15 Hz) in control PV-Cre mice expressing only tdTomato but not Chrimson (n = 4). (d) Same as (c) but for control Chat-Cre animals expressing tdTomato but not Chrimson (n = 4). Optogenetic stimulation was without effect on fluorescence in either population in the absence of Chrimson. (e) Normalized change in calcium event probability in PV cells (orange) and MSNs (blue) during laser stimulation in PV-Chrimson mice (n = 4 PV-Chrimson mice). (f) Same as (e) but for calcium event probability in CHI cells (green) and MSN cells (blue) in Chat-Chrimson mice (n = 4 Chat-Chrimson mice). Plots are mean ± s.e.m., see Fig. 7 for further quantification of the effects of stimulation.

Supplementary Figure 12 Regression coefficients for mixed-effects model predicting linear velocity.

(a) Regression coefficients for the fluorescence-cell-genotype interaction term from a large “multivariate” mixed-effects model. Fluorescence-PV and fluorescence-CHI coefficients are plotted with respect to MSNs (dashed line), the reference cell type. Mix-effect regression model considered individual neurons' fluorescence, fluorescence-cell-genotype interactions, and linear velocity at two different lags as predictors. The horizontal line (orange and green) represents the actual coefficient values from the large multivariate model encompassing all sessions from all mice for each cell type. PVs have the largest interaction term and CHIs have the smallest interaction term, indicating that high fluorescence values from PVs more positively predict velocity relative to either of the other two cell types while CHIs are the poorest positive predictors of velocity. (Mixed-effects model, PV:fluorescence vs MSN:fluorescence, ### = t(4.69e+07)=7.04, P = 2.95e-12; CHI:fluorescence vs MSN:fluorescence, ### = t(4.69e+07)=4.38, P = 1.20e-05; CHI:fluorescence vs PV:fluorescence, *** = t(4.69e+07)=7.85, P = 1.21e-14). The individual dots correspond to regression coefficients from all sessions from all animals with at least one interneuron (17 PV sessions from 6 PV mice and 10 CHI sessions from 6 CHI mice).

Supplementary information

Supplementary Figures 1–12.

Reporting Summary

41593_2019_341_MOESM3_ESM.mp4

Supplementary Video 1 Striatal neuron activity coincident with instantaneous mouse voluntary movement. Wide-field calcium video showing activity in the dorsal striatum from a representative mouse while running on a spherical treadmill. GCaMP6f fluorescence is shown on the left (pseudo-color), and right (grayscale) as a mirror image. Integrated mouse movement interpolated from the optical sensors is shown in the center. Video is 35 seconds long, and played at 1X speed. Neural activity on the left has been color coded to highlight dynamic changes. Green neurons highlight cells that exhibited activity during the session, and form the anatomical map under the imaging window. Active neurons are pseudo-colored red, while the rising phase of calcium fluorescence is pseudo-colored blue. Thus, blue occurs briefly before red. Mouse direction and speed is updated across frames in the center with future and past paths highlighted. Path is projected 60 frames (~3 seconds) in either direction and path color indicates future or past rate of movement. Yellow path denotes periods of acceleration and purple path denotes periods of deceleration.

41593_2019_341_MOESM4_ESM.mp4

Supplementary Video 2 PV neuron activity relative to MSN activity and movement speed. Video shows calcium activity in several MSNs and a single PV neuron, in a typical recording session in a PV-Cre mouse. Video is 15 seconds long, and played at 1X speed. The PV neuron, positive for tdTomato, is outlined in red. All other neurons are non-tdTomato labeled cells and are putative MSNs. The speed of the mouse in cm/s is shown in the gray bar in the right margin. Note that the PV neuron shows activity through movement and in advance of the MSN cell activity that is coincident with a change in velocity.

Supplementary Video 3

CHI neuron activity relative to MSN activity and movement speed. Video shows calcium activity in several MSNs and a single CHI, in a typical recording session in a Chat-Cre mouse. Video is 15 seconds long, and played at 1X speed. The CHI, positive for tdTomato, is outlined in red. All other neurons are non-tdTomato labeled cells and are putative MSNs. The speed of the mouse in cm/s is shown in the gray bar in the right margin. Note that MSN activity increases coincident with a change in velocity, and CHI activity occurs just prior to a reduction in speed.

41593_2019_341_MOESM6_ESM.mp4

Supplementary Video 4 Change in movement direction upon optogenetic stimulation of PV cells in a PV-Cre mouse expressing Chrimson. Video from a representative PV-Chrimson expressing mouse during optogenetic activation of PV neurons while the mouse is running on a spherical treadmill. Video is 12 seconds long, and played at 1X. Note the increase in side to side movement or “change of direction” events coinciding with laser onset. Sessions contained 13-23 stimulation trials and the optogenetic experiment was repeated 2X in each PV-Chrimson (n = 4) or PV-tdT control (n = 4) animal across different days. See Figure S8b for population quantifications from all sessions.

41593_2019_341_MOESM7_ESM.mp4

Supplementary Video 5 Decrease in movement speed upon optogenetic stimulation of CHI cells in a Chat-Cre mouse expressing Chrimson. Video from a representative Chat-Chrimson expressing mouse during optogenetic activation of CHI neurons while the mouse is running on a spherical treadmill. Video is 12 seconds long, and played at 1X. Note the marked decrease in movement speed that occurs during stimulation. Sessions contained 13-23 stimulation trials and the optogenetic experiment was repeated 2X in each Chat-Chrimson (n = 4) or Chat-tdT control (n = 4) animal across different days. See Figure 3b for population quantifications from all sessions.

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Gritton, H.J., Howe, W.M., Romano, M.F. et al. Unique contributions of parvalbumin and cholinergic interneurons in organizing striatal networks during movement. Nat Neurosci 22, 586–597 (2019). https://doi.org/10.1038/s41593-019-0341-3

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