Extended Data Fig. 1: Characterizations of striatal SPN Ca²⁺ activity patterns in live brain slices and recorded in behaving mice using a chronic microendoscopy preparation.

a, We performed whole-cell patch-clamp recordings of SPNs in acute brain slices, using dual epifluorescence and infrared Nomarski (IR-DIC) microscopy to guide the recordings. We selectively recorded from fluorescent dSPNs and iSPNs in brain slices from Drd1a^cre and Adora2a^cre mice that had been injected into the striatum with AAV2/9-CAG-FLEX-GCaMP6m-WPRE.SV40. The numerical aperture used for the fluorescence recordings was matched to that used for Ca²⁺ imaging studies in freely behaving mice (Methods). b, Illustrative traces (top) of neural membrane potential (V_m) showing the changes that result upon stepwise injections of electrical current (bottom). The traces exhibit typical waveforms for SPNs. c, To visualize Ca²⁺ transients evoked by different numbers and frequencies of action potentials in iSPNs and dSPNs in brain slice recordings, we elicited spiking with 1-nA pulses (1-ms duration; 1 to 10 pulses, delivered at 5, 10, 20, 50 and 100 Hz). Top, an example of a V_m trace during 1–10 pulses injected at 10 Hz. Middle, example traces of current pulse injections delivered at 10 Hz. Bottom, a representative trace of somatic Ca²⁺ transients in response to the same 10-Hz current injections. d, Example Ca²⁺ activity traces for each current injection pattern tested in brain slices. As expected, electrical stimulation at higher frequencies evoked larger amplitude Ca²⁺ transients, with more sharply rising Ca²⁺ waveforms than those evoked at lower stimulation frequencies. Arrows mark current injections. e, f, Mean ± s.e.m. values for Ca²⁺ transient amplitudes (e) and area under the Ca²⁺ transient waveform (AUC; f) as a function of the number of action potentials evoked, for dSPNs and iSPNs in brain slices. Values for each cell are normalized to those evoked at the highest stimulation intensity (10 action potentials, 100 Hz). Ca²⁺ event amplitudes rose linearly with the number of action potentials (R² > 0.99 for 5–20 Hz; R² > 0.97 for all frequencies). There were no significant differences in event amplitude or AUC between dSPNs and iSPNs, at any stimulus intensity. n = 9 dSPNs and 8 iSPNs; two-way, mixed-design ANOVA. Exact P values can be found in the Supplementary Information for all extended data figures. g, h, Mean ± s.e.m. values for half-rise (g) and half-decay (h) times (t_1/2), measured for the Ca²⁺ events evoked in dSPNs and iSPNs in brain slices, plotted as a function of the number of action potentials. Consistent with their more prolonged stimulus durations, lower stimulation frequencies yielded greater increases in the transient rise time. Ca²⁺ transient decay times were nearly independent of action potential frequency or number. i, Mean ± s.e.m. values of the AUC of stimulus-evoked Ca²⁺ events in dSPNs and iSPNs in brain slices, under control conditions or perfused with SKF81297 (1 μM) or quinpirole (10 μM), respectively. Neither drug significantly affected somatic Ca²⁺ event AUCs in either SPN type, regardless of stimulus intensity. n = 6 dSPNs and 6 iSPNs; two-way, repeated-measures ANOVA. The individual data points are plotted alongside the mean values. j, Two example time-lapse fluorescence image series, acquired over the course of experiments in a Drd1a^cre mouse (top) and an Adora2a^cre mouse (bottom). Scale bars are 125 μm and apply uniformly to all panels within each row. Insets show example Ca²⁺ transients in the absence and presence of dopamine receptor agonist SKF81297 or quinpirole. Scale bars are uniformly sized for all insets. k, Surgical implantation of the microendoscope did not affect the ability of mice to improve their motor performance on the accelerating rotarod assay, as shown by their increased latencies to fall off the rod, which parallel the performance improvements of control mice without an implanted microendoscope. P = 0.8; two-way, repeated-measures ANOVA; n = 13 control and 20 implanted mice. Inset, Microendoscope implantation did not alter levels of spontaneous movement in an open field arena. P = 0.7; n = 5 control and 6 implanted mice; Wilcoxon rank-sum test. Data points from individual mice are shown as open circles. l–n, Box-and-whisker plots of the rates (l), amplitudes (m) and full-width at half-maximum (FWHM) durations (n) of Ca²⁺ events observed in live normal (untreated) mice in individual dSPNs and iSPNs, before (Pre-lesion) and after (Post 14-d) dopaminergic lesions, as the mice were resting (left) or moving (right). In resting mice, Ca²⁺ event rates decreased in dSPNs and increased in iSPNs after the lesion (l) as characterized in Fig. 3. When the mice were moving, Ca²⁺ event rates in iSPNs were similar before and >14 days after the lesion, whereas the rates in dSPNs were depressed after the lesion. Each box-and-whisker plot is based on n = 3,325–3,703 dSPNs or iSPNs, from 12 Drd1a^cre and 13 Adora2a^cre mice, respectively, tracked before or 14 days after the 6-OHDA lesion. Horizontal lines denote median values, boxes cover the middle two quartiles and whiskers span 1.5× the interquartile range. Data denoted as ‘Pre-lesion’ were taken on day −5. Data denoted as ‘Post 14-d’ were taken on day 14.