Extended Data Fig. 5: 6-OHDA injections rapidly ablate SNc dopamine neurons and disrupt motor behaviour without impairing the fidelity of Ca²⁺ event detection.

a, Example coronal brain section from an experimental mouse, immunostained for GCaMP6m (anti-GFP, green) and tyrosine hydroxylase (anti-TH, red). DAPI was used to stain the nuclei (blue). White lines demarcate the position of the implanted microendoscope, and the boundaries of nearby brain areas. AcbC, accumbens core; Cg, cingulate cortex; CPu, caudate/putamen; IC, insular cortex; M1 and M2, motor cortices; Pir, piriform cortex; S1 and S2, somatosensory cortices. Labels are adapted from an anatomical atlas of the mouse brain³⁸. Scale bar, 1 mm. b, Representative midbrain coronal sections acquired 1 day after unilateral infusions of saline (top) or 6-OHDA (middle), or >14 days after 6-OHDA infusion (bottom), and then immunostained for tyrosine hydroxylase (red). DAPI was used to stain the nuclei (blue). Dopamine cell bodies are absent in the SNc of mice that received 6-OHDA, at both 1 and >14 days after 6-OHDA infusion. Scale bar, 500 μm. c, 24 h after infusions into SNc of 6-OHDA but not of saline, mice exhibited disrupted patterns of spontaneous locomotion in an open field arena. P = 4 × 10⁻³ for 6-OHDA and P = 0.8 for saline; n = 7 saline-treated and 14 6-OHDA-treated mice; Wilcoxon signed-rank test for comparisons to the pre-lesion behaviour of each mice. d, The median fluorescence intensity across the entire imaging field (normalized to pre-lesion values on day −5 for each mouse and then averaged across mice) decreased significantly after 6-OHDA lesions in mice of both genotypes. P < 10⁻⁷ comparing median fluorescence intensities (normalized to pre-lesion means) averaged across 5 days before versus 5 days after lesion; Wilcoxon rank-sum test. However, fluorescence intensities stabilized 15–17 days after the 6-OHDA lesion, and there were no further significant changes over time in either mouse line. P > 0.05; n = 5 Drd1a^cre and 7 Adora2a^cre mice; Spearman correlation. Error bars indicate s.e.m. for 5 Drd1a^cre and 7 Adora2a^cre mice. e, The number of active SPNs (mean ± s.e.m.) detected in total on each day of the study, normalized to the mean value (dashed horizontal line) detected before 6-OHDA infusion (days shaded grey). On days on which mice received drug treatments (Fig. 1a), the data shown here are from the initial portions of the recording sessions before drug administration. The number of active cells was stable across the study, except for a single pairwise difference in the number of active iSPNs. Friedman ANOVA; n = 5 Drd1a^cre and n = 7 Adora2a^cre mice; P > 0.05 for Drd1a^cre and P = 0.01 for Adora2a^cre; P = 5 × 10⁻⁴ for post hoc test comparing the number of iSPNs detected 1 day before the lesion and 15 days after the lesion; Fisher’s least significant difference test with a Holm–Bonferroni correction for multiple comparisons. f, Cumulative distributions of peak ΔF/F values for Ca²⁺ events from individual dSPNs (top) and iSPNs (bottom), before and after 6-OHDA lesion. After the lesion, Ca²⁺ event amplitudes were significantly greater in dSPNs (3.7 ± 0.02% (pre-lesion) versus 6.0 ± 0.03% (14 days after)), but smaller in iSPNs (4.4 ± 0.02% (pre-lesion) versus 4.0 ± 0.02% (14 days after)). Data are mean ± s.e.m.; P < 10⁻¹⁰ for both SPN types; Wilcoxon rank-sum test. Data in f and g are from n = 3,332–3,734 dSPNs or iSPNs, from before (day −5) or 14 days (day 14) after 6-OHDA lesion, in n = 12 Drd1a^cre mice and 13 Adora2a^cre mice, respectively. g, Cumulative distributions of the signal detection fidelity (d′) of Ca²⁺ events in individual dSPNs (top) and iSPNs (bottom), before and after 6-OHDA lesion. Owing to the decrease in background fluorescence intensity, Ca²⁺ events in dSPNs became easier to detect after 6-OHDA lesion, as quantified by the increase in d′ values for dSPNs. d′ = 17 ± 0.1 (mean ± s.e.m.; pre-lesion) versus 29 ± 0.2 (14 days after). The changes in d′ values for iSPNs were smaller in magnitude (d′ = 22 ± 0.1 (pre-lesion) versus 19 ± 0.1 (14 days after)), while keeping the optical conditions for Ca²⁺ event detection extremely favourable. Crucially, all changes in Ca²⁺ event detection fidelity values were opposite to those observed for Ca²⁺ event rates in the two cell types (Fig. 3), and thus cannot account for the event rate changes. h, d′ values for Ca²⁺ events from individual dSPNs (left) and iSPNs (right), before and after 6-OHDA lesions, and after drug treatments in mice following 6-OHDA lesion. d′ values increased after 6-OHDA lesions and decreased after the drug treatments in dSPNs. By contrast, d′ values decreased after 6-OHDA lesions and increased after drug treatment in iSPNs. ***P < 10⁻¹⁰ for all conditions in both genotypes; Wilcoxon rank-sum test; values are from n = 1,770–2,027 dSPNs from 5 Drd1a^cre mice and n = 1,719–2,393 iSPNs from 6 Adora2a^cre mice, recorded before (day −5) and after 6-OHDA lesion (day 14), or after the lesion and quinpirole (day 16), SKF81297 (day 18) and l-DOPA (day 20) treatments. As in g, these changes in d′ have an opposite sign to the changes in Ca²⁺ event rates (Fig. 4), and thus cannot account for the latter effects. Throughout the study, d′ values remained extremely high, in that a d′ value >16 corresponds mathematically to a mean rate of <10⁻¹⁰ errors in Ca²⁺ event detection per hour. This nearly vanishing predicted error rate is unattainable experimentally over many hours of recording but underscores the highly favourable conditions for Ca²⁺ imaging.