Extended Data Fig. 3: Cell type-specific tracing for the PF→STN circuit, and PV⁺ STN neurons are critical for motor learning, are excitatory in nature, and lack local STN connectivity.

a-c, Monosynaptic retrograde RV tracing from VGLUT2⁺ or PV⁺ STN neurons. Images show starter cells in STN (a), FISH co-staining of GFP with VGLUT2 or PV (b), and corresponding PF labeling (c, lower magnification image relative to Fig. 2h) (n = 7 mice per group). d-e, Expression of a Cre-dependent hM4Di-mCh virus in STN (d), FISH co-staining of mCh with VGLUT2 or PV (e) in Vglut2-Cre or PV-Cre mice, related to experiments in Fig. 2j, k. f, Inhibition of STN VGLUT2^- neurons during rotarod using a Cre-Off virus (DO-NpHR-eYFP) injected in STN of Vglut2-Cre mice. eYFP mice received a control Cre-Off virus (DO-eYFP) in place of the NpHR virus (n = 7 mice per group). FISH co-staining of VGLUT2 with eYFP (used a red fluorophore to visualize the eYFP probe). g, Inhibition of STN PV⁺ neurons that receive PF inputs during rotarod using an anterograde AAV expressing Cre injected in PF, and a Cre-On/Flp-On virus (COn/FOn-NpHR3.3-eYFP) injected in STN of PV-Flp mice. eYFP mice received a control Cre-On/Flp-On virus (COn/FOn-eYFP) in place of the NpHR3.3 virus (n = 7 mice per group). FISH co-staining of PV with eYFP (used a red fluorophore to visualize the eYFP probe). h-i, FISH staining of GFP-labeled VGLUT2⁺ neurons and endogenous VGLUT2 expression (h) or GFP-labeled PV⁺ neurons and endogenous PV expression (i) showed a high degree of overlap for each of these mouse lines. GFP labeling was achieved by injecting a Cre-dependent AAV in STN of Vglut2-Cre or PV-Cre mice. j-k, Half width (j) and spontaneous firing (k) measured in ex vivo recordings from STN VGLUT2⁺ and PV⁺ neurons. To visualize VGLUT2⁺ and PV⁺ neurons, a Cre-dependent mCh virus was injected in STN of Vglut2-Cre or PV-Cre mice (Half width, VGLUT2⁺: n = 18 neurons (6, 5, 7), PV⁺: n = 12 neurons (4, 4, 4) from 3 mice each; Spontaneous firing, VGLUT2⁺: n = 9 neurons (3, 3, 3), PV⁺: n = 9 neurons (3, 3, 3) from 3 mice each). l, Input resistance (R_in), membrane time constant (tau), membrane capacitance (C_m), and action potential amplitude of STN VGLUT2⁺ and PV⁺ neurons. To visualize VGLUT2⁺ and PV⁺ neurons, a Cre-dependent mCh virus was injected in STN of Vglut2-Cre or PV-Cre mice (R_in, tau, and C_m, VGLUT2⁺: n = 21 neurons (8, 8, 5), PV⁺: n = 13 neurons (5, 5, 3) from 3 mice each; Amplitude, VGLUT2⁺: n = 18 neurons (6, 5, 7), PV⁺: n = 12 neurons (4, 4, 4) from 3 mice each). m, VGLUT2, PV FISH staining in wild type STN sections. Plot (right) shows the proportion of VGLUT2⁺ or VGLUT3⁺ neurons among the total number of PV⁺ STN neurons (n = 5 VGLUT2⁺, n = 4 VGLUT3⁺ mice). n, Representative traces of evoked IPSCs (n = 10 neurons (3, 4, 3) from 3 mice) and evoked EPSCs (n = 10 neurons (2, 4, 4) from 3 mice) recorded in STN neurons during activation of ChR2-eYFP-expressing PV⁺ STN neurons. PV-Cre mice were injected with a Cre-dependent ChR2-eYFP virus in STN. Data are presented as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001. NS, not significant. Two-tailed unpaired t test (c, j-m) and two-tailed paired t test (f, g). P < 0.0001 (c), Cre-Off eYFP P = 0.001, Cre-Off NpHR P = 0.12 (f), eYFP P = 0.019, NpHR3.3 P = 0.85 (g), P = 0.0012 (j), P = 0.62 (k), R_in P = 0.14, Tau P = 0.11, C_m P = 0.26, Amplitude P = 0.51 (l), P = 0.0001 (m)

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