Genetic strategy for CRISPR-Cas9-mediated generation of knock-in lines.

a, Schematic of generation of D1-Cre mice. (Top) Drd1 gene with (middle) targeting vector replacing the stop codon with a T2A sequence and Cre recombinase resulting in (bottom) a modified locus expressing Cre under the control of the endogenous Drd1 promoter. b, PCR-based genotyping strategy for D1-Cre mice using 5’ and 3’ primer sets. Positive bands denote presence of T2A-Cre insert in F1 mice, which are not present in wildtype (WT) or negative (H2O) controls. c, d, Genetic schematic (c) and PCR-genotyping strategy (d) for the generation of A2a-Cre mice, with insertion of a T2A-Cre sequence replacing the Adora2a stop codon. e, f, Genetic schematic (e) and PCR-genotyping strategy (f) for the generation of D1-FlpO mice, with insertion of a T2A-FlpO sequence replacing the Drd1 stop codon. g, h, Genetic schematic (g) and PCR-genotyping strategy (h) for the generation of A2a-FlpO mice, with insertion of a T2A-FlpO cassette replacing the Adora2a stop codon. i, j, Genetic schematic (i) and PCR-genotyping strategy (j) for the generation of DAT-FlpO mice, with insertion of a T2A-FlpO sequence replacing the Slc6a3 stop codon. M: DNA mass ladder.

Screening primers

Whole-brain histological validation of D1 and D2 knock-in lines.

a-e, Representative images of mouse brain sections from D1-Cre;Ai9 mice showing Cre-dependent tdTomato expression. f-j, Representative images of mouse brain sections from D1-FlpO;RCE:FRT mice showing FlpO-dependent EGFP expression. k-o, Representative images of mouse brain sections from A2a-Cre;Ai9 mice showing Cre-dependent tdTomato expression. p-t, Representative images of mouse brain sections from A2a-FlpO;RCE:FRT mice showing FlpO-dependent EGFP expression. Sagittal sections (a, f, k, p): ML +1.90. Coronal sections (b, g, l, q): AP +1.40. Coronal sections (c, h, m, r): AP +0.60. Coronal sections (d, i, n, s): AP -1.60. Coronal sections (e, j, o, t): AP -3.20. Scale-bar: 1000µm.

Histological validation of DAT-FlpO knock-in line.

a, Representative coronal section from a DAT-FlpO;Ai65 mouse. TH antibody immunoreactivity (labeling midbrain dopamine neurons) significantly overlapped with FlpO-dependent tdTomato expression driven by the knock-in DAT-FlpO line. b, Viral expression of a FlpO-dependent YFP construct into the midbrain of DAT-FlpO mice confirms the restricted expression of YFP to midbrain VTA and SNc regions.

Electrophysiological validation of D1-Cre and A2a-Cre knock-in lines.

a,b, Left: representative histological staining of neurobiotin-filled SPNs from whole-cell recordings from D1-Cre;Ai9 mice (a) and A2a-Cre;Ai9 mice (b). Right: DIC images of striatal SPNs, with tdTomato expression driven in D1 SPNs (a) or D2 SPNs (b). c,d, Representative whole-cell recording traces of SPNs with current injections evoking voltage responses and action potential firing. e, f-I curves quantifying SPN firing in response to injected current values. f, I-V curves quantifying SPN voltage changes in response to injected current values. g, Resting membrane potentials across all recorded SPNs (D1-Cre tdTom+ -84.34 ± 1.17 mV, n = 21 cells / 5 mice; D1-Cre tdTom--83.95 ± 1.40 mV, n = 21 cells / 5 mice; A2a-Cre tdTom+ -80.28 ± 1.94 mV, n = 16 cells / 5 mice; A2a-Cre tdTom--82.24 ± 1.05 mV, n = 20 cells / 5 mice). h, Rheobase currents for all recorded SPNs across D1-Cre and A2a-Cre animals (D1-Cre tdTom+ 310.48 ± 10.83 pA; D1-Cre tdTom-172.38 ± 10.37 pA; A2a-Cre tdTom+ 176.25 ± 9.70 pA; A2a-Cre tdTom-258.00 ± 13.37 pA). Significantly decreased rheobase currents in putative D2 SPNs (D1-Cre tdTom+ vs tdTom-: p < 0.0001; A2a-Cre tdTom+ vs tdTom-: p = 0.0142).

Electrophysiological validation of D1-FlpO and A2a-FlpO knock-in lines.

a,b, Left: representative histological staining of neurobiotin-filled SPNs from whole-cell recordings from D1-FlpO;Ai65F mice (a) and A2a-FlpO;Ai65F mice (b). Right: DIC images of striatal SPNs, with tdTomato expression driven in D1 SPNs (a) or D2 SPNs (b). c,d, Representative whole-cell recording traces of SPNs with current injections evoking voltage responses and action potential firing. e, f-I curves quantifying SPN firing in response to injected current values. f, I-V curves quantifying SPN voltage changes in response to injected current values. g, Resting membrane potentials across all recorded SPNs (D1-FlpO tdTom+ -81.87 ± 1.30 mV, n = 21 cells / 5 mice; D1-FlpO tdTom--79.01 ± 1.77 mV, n = 19 cells / 5 mice; A2a-FlpO tdTom+ -80.60 ± 1.82 mV, n = 16 cells / 5 mice; A2a-FlpO tdTom--82.32 ± 1.05 mV, n = 22 cells / 5 mice). h, Rheobase currents for all recorded SPNs across D1-FlpO and A2a-FlpO animals (D1-FlpO tdTom+ 291.43 ± 9.72 pA; D1-FlpO tdTom-183.16 ± 8.27 pA; A2a-FlpO tdTom+ 183.75 ± 9.53 pA; A2a-FlpO tdTom-294.55 ± 7.92 pA). Significantly decreased rheobase currents in putative D2 SPNs (D1-FlpO tdTom+ vs tdTom-: p < 0.0001; A2a-FlpO tdTom+ vs tdTom-: p < 0.0001).

Electrophysiological validation of DAT-FlpO knock-in line.

a, Left: histological section of DAT-FlpO;Ai65F brain containing VTA and SNc. Middle: post-recording histology of dopamine neuron filled with neurobiotin during whole-cell records. Right: DIC image (top) and epifluorescence image of tdTomato dopamine neurons (bottom) targeted for recordings (pipette solution containing alexa dye). b, Representative voltage traces of VTA (left) and SNc (right) neurons in response to current injections. c, f-I curves quantifying dopamine neuron firing in response to injected current values. d, I-V curves quantifying dopamine neuron voltage changes in response to injected current values (red line indicated linear fit). e, Input resistances across recorded dopamine neurons, based on linear fits of I-V curves (SNc: 360.37 ± 43.76 MΩ, n = 6 cells; VTA: 544.97 ± 77.39 MΩ, n = 9 cells). f, Directly measured input resistances (SNc: 344.90 ± 46.11 MΩ; VTA: 497.60 ± 73.32 MΩ). g, Measured resting membrane potentials across all dopamine neurons (SNc: -55.03 ± 1.43 mV; VTA: -56.18 ± 1.70 mV). h, Measured rheobase currents across all dopamine neurons (SNc: 20.00 ± 5.16 pA; VTA: 42.22 ± 17.14 pA).