Extended Data Fig. 4: Simultaneous recordings of dSPNs and iSPNs show that the two cell types encode movement with indistinguishable spatiotemporal patterns of activation.

a, To image dSPN and iSPN Ca²⁺ activity concurrently, we prepared mice that expressed GCaMP6m in both cell types but tdTomato only in dSPNs (Methods). Mice were head-fixed on a running wheel beneath the objective lens of a two-photon microscope. A piezoelectric actuator moved the axial position of the objective to allow volumetric imaging across four planes at different depths of the tissue (15-μm axial spacing). The mice were free to run or rest on the wheel (Supplementary Video 3). We tracked the motion of the wheel using a rotary encoder (500 encoder pulses per revolution) that provided a read-out of the instantaneous locomotor speed. We computed the mean speed of the mouse at a time-resolution matching that of the two-photon volume acquisition rate (6 Hz, or 166 ms per time bin). We identified periods of movement by marking all time bins in the mean speed trace with values >0.2 cm s⁻¹. To identify instances of motion onset, we selected all time bins for which speeds were <0.2 cm s⁻¹ for at least 1 s in the immediately prior time bins, and >0.2 cm s⁻¹ for at least 1 s in the immediately subsequent time bins. To identify instances of motion offset, we used the opposite criterion. b, Histological section of dorsomedial striatum expressing tdTomato (red) in Drd1a^cre positive cells and immunostained for GFP (green) to visualize GCaMP6m expression. Closed arrowheads point to three example dSPNs that expressed both GCaMP6m and tdTomato. Open arrowheads point to three putative iSPNs that expressed GCaMP6m but not tdTomato. Scale bar, 50 μm. c, Representative cell maps from each of the four imaging planes in an example mouse with 118 detected dSPNs (blue) and 183 detected iSPNs (red). Scale bars, 100 μm. d, Representative traces of Ca²⁺ activity from 10 dSPNs (blue) and 10 iSPNs (red) from the same mouse as in c. Grey shading here and in e, g, h denotes periods classified as movement on the running wheel. e, Locomotor speed on the running wheel (top) and Ca²⁺ activity traces of individual dSPNs (middle) and iSPNs (bottom), during part of an imaging session in an example mouse. Note the clear correlation between locomotion and Ca²⁺ activity in both cell types. f, Mean cumulative distribution functions of Ca²⁺ event rates in dSPNs (n = 699) and iSPNs (n = 1020) were nearly identical, during periods of rest (left) and running (right). g, Mean Ca²⁺ event rates in dSPNs and iSPNs as a function of mouse locomotor speed. Events rates are shown normalized to their mean levels when the mice were resting (speed <0.2 cm s⁻¹). Grey shading denotes speeds at which we classified the mouse as moving (>0.2 cm s⁻¹). h, Mean locomotor speed (bottom) and the fraction of SPNs that are activated (top), plotted as a function of time relative to motion onset (left) and offset (right). We determined the onset time of neural activity as the time at which the mean percentage of active cells was ≥3 s.d. of the percentage of active cells during the baseline periods of −3 to −1 s relative to motion onset. Using this criterion, dSPNs and iSPNs respectively activated −666 ± 97 ms and −733 ± 51 ms before motion onset (mean ± s.e.m; n = 5 mice; Methods), which were statistically indistinguishable. P = 0.9; Wilcoxon rank-sum test. i, Mean locomotor speed as a function of time relative to the occurrences of Ca²⁺ events in dSPNs and iSPNs. For each cell, we normalized these traces to the mean speed 20 s before Ca²⁺ excitation, then averaged the traces across all cells of each type and all mice. j, Mean pairwise coactivity (Methods) for dSPN–dSPN, iSPN–iSPN and dSPN–iSPN cell pairs, computed as a function of the distances between the pairs of cells, normalized to coactivity values in temporally shuffled datasets (1,000 distinct shuffles) in which time-correlated activity patterns were scrambled. Cyan shading indicates proximal (20–100 μm) cell pairs. Data are from periods of movement on the running wheel. k, Pairwise coactivity values (mean ± s.e.m.) were significantly greater for proximal cell pairs than those in temporally shuffled datasets (*P < 0.05; Wilcoxon rank-sum test; n = 5 mice), and did not depend on the SPN types. n.s. denotes P > 0.05; Wilcoxon rank-sum test. Data points from individual mice are shown as open circles. l, We created GLMs to make time-dependent predictions of mouse locomotor speed based on the ΔF(t)/F₀ activity traces of the cells determined by two-photon Ca²⁺ imaging. We used the GLM libraries in MATLAB (Mathworks), using a Gaussian noise model and taking the identity function as the linking function. We used 70% of the time bins in the set of ΔF(t)/F₀ traces for training the GLM, and 30% for testing it. To study the accuracy of the speed predictions as a function of the number of cells (n) included in the GLM, for each value of n, we randomly chose n cells from the total available, constructed the GLM and computed the Pearson’s correlation coefficient between the actual and predicted speed traces. For each n value, we repeated this procedure with 10 different randomly chosen subsets of cells and then computed the mean correlation coefficient of the real and predicted speeds across the 10 different sub-samplings. In an example mouse, traces of the actual running speed (grey) were well fit by models based on the activity of either dSPNs (blue) or iSPNs (red). m, Mean Pearson correlation coefficients for an example mouse between the actual and predicted running speeds, for GLMs based on either dSPNs or iSPNs (10 sets of randomly chosen cells for each abscissa value, out of 301 total cells). Inset shows the correlation coefficients (mean ± s.e.m.) from n = 5 mice, computed using either 115 dPSNs or 115 iSPNs from each mouse. Data points from individual mice are shown as open circles and were statistically indistinguishable across the two cell types. P = 0.13; Wilcoxon signed-rank test. f–j, Colour shading denotes s.e.m. for n = 5 Drd1a^cre × Ai14 mice.