(A–C) Cre-dependent, AAV anterograde tracing of striato-nigral projections from VS and DS using D1-Cre mice (A) shows segregated terminal fields in SNr (B), in which VS innervates the medial SNr. DS and VS innervate EPN and LH, respectively (C). N = 3, including CTb-based tracing. Scale bars: 500 µm (A) and 200 µm (B, C). (D–F) The same strategy as in panels (A–C) for mapping DMS versus DLS efferents. N = 3, including CTb-based tracing. Scale bars: 500 µm (D) and 200 µm (E, F). (G–I) Dual Cre-dependent AAV anterograde tracing of nigro-thalamic projections using PV-Cre mice. Conjugated GFP and tdTomato are separately expressed in the lateral and medial SNr (G), which then target distinct thalamic nuclei (H, I). Insets emphasize the medial SNr projections to VA and to the caudal VM. Scale bars: 200 µm (G) and 500 µm (H,I). (J–L) Thalamo-cortical neurons projecting to mPFC, M2 and M1 were identified using three fluorophores of CTb. Insets in (K, L) indicate VA and caudal VM thalamus that contain M1- and M2-projecting thalamic neurons, which correspond to the insets in (H) and (I) where medial SNr sends axon terminals. Scale bars, 500 µm. (M, N) Schematic illustration showing that medial SNr, which receives VS and DMS inputs, projects to VA and caudal VM thalamus, which in turn project to M1 and M2, as the mechanism for limbic-to-motor connectivity through the direct pathway. (O, P) Ex vivo electrophysiology is used to determine the functional strength of inputs from DLS or VS onto SNr neurons projecting to motor thalamus. Injections of red fluorescent protein retrobeads in VA-VL and AAV.hsyn.ChR2.eYFP in the striatum resulted in striato-nigral labeled axons and retrogradely labeled retrobeads+ cells in the SNr (left). Images indicate recording pipettes attached to retrobeads+ cells (middle). Example traces recorded from lateral (O) and medial SNr neurons (P) under optical stimulation (blue bars above traces, right). Under the glutamate receptor antagonists (CNQX and DL-APV), IPSCs are visible immediately after stimulation, which were abolished by application of picrotoxin (PTX) that blocks GABAa receptors. Scale bars: 20 µm. (Q, R) Mean amplitude (medial, 0.902 ± 0.235; lateral, 0.842 ± 0.206; unpaired t-test, t11 = 0.177, p=0.862) and paired pulse ratio (medial, 0.697 ± 0.099; lateral, 0.778 ± 0.196; unpaired t-test, t11 = 0.410, p=0.690) for all recorded neurons in medial (n = 8 from seven mice) and lateral SNr (n = 5 from two mice). There is a neuron in medial SNr that did not respond to optical stimulation, and we excluded it from the analysis. Note that this neuron was located outside of a ChR2.YFP positive area. Data presented as mean ± SEM. Abbreviations: cp, cerebral peduncle; ic, internal capsule; mt, mammillothalamic tract; fr, fasciculus retroflexus; MD, mediodorsal; CM, centromedial; PC, paracentral; CL, centrolateral; LP, lateral posterior; PO, posterior; VPM, ventroposterior medial thalamus; PrL, prelimbic cortex; Cg, cingulate cortex.