Fig. 1. The density of dSPN terminals in the GPe account for more than half the density of SNr terminals.

A Strategy for anterograde tracing of direct pathway striatal projection neuron (dSPN) axons/terminals using the presynapse-targeted tracer Synaptophysin-GCaMP6s. B Synaptophysin-GCaMP6s is largely absent from axons (blue arrow) and enriched in terminals (white arrow) (representative images from N = 5 mice). C Synaptophysin-targeted dSPN terminals (cyan) colocalize with the presynaptic GABA marker VGAT (red), appearing white (blue arrows). D The density of dSPN Synaptophyin-GCaMP6s+ (antibody amplified for GFP) terminals in the globus pallidus externus (GPe) (83%) reaches more than half the density in the entopeduncular nucleus (EP) (123%) and substantia nigra reticulata (SNr) (134%) (ANOVA: region p < 0.001; Tukey post-hocs: **p < 0.01) (N = 5 mice). Data are mean ± SEM. E Confirmation that dSPN terminals in the GPe arise from axons projecting to the SNr (representative images from N = 3 mice). Left: Injection of retrograde herpes-simplex virus (HSV) expressing a flexed YFP into the GPe and red retrobeads (retrobeads-647) into the SNr of Drd1-cre mice. Right: YFP+ cell bodies colocalized with retrobeads+ cells in the DMS, identifying dSPNs projecting to both GPe and SNr (white arrows). Note that retrobeads had a puncta-like labeling pattern, while YFP staining either had a puncta-like pattern or covered the whole soma. White or red puncta in YFP-positive soma indicate colocalization. There were also retrobeads-, YFP+ cell bodies, identifying neurons projecting only to the GPe (blue arrow). Out of 159 YFP+ cells counted, 147 were also retrobeads+ (92.5%), 12 were retrobeads- (7.5%). Out of 148 retrobeads+ cells counted, all but 1 was YFP+ (99.3%). Exact p-values are given in Supplementary Dataset S2. See also Supplementary Figs. S2, S3.