The zona incerta (ZI) supports diverse behaviors including binge feeding, sleep-wake cycles, nociception, and hunting. Diverse ZI functions can be attributed to its heterogeneous neurochemical characterization, cytoarchitecture, and efferent connections. The ZI is predominantly GABAergic, but we recently identified a subset of medial ZI GABA cells that are marked by the enzyme tyrosine hydroxylase (TH) and produce dopamine (DA). While the role of GABA within the ZI is well studied, less is known about the functions of ZI DA cells. To identify potential roles of ZI DA cells, we further phenotyped them and mapped their efferent fiber projections. We showed that wild-type TH-immunoreactive (-ir) ZI cells did not express somatostatin or calretinin immunoreactivity. We next validated a Th-cre
L10-Egfp mouse line and found that medial Egfp ZI cells were more likely to be TH-ir. We therefore delivered a Cre-dependent virus into the medial ZI of Th-cre or Th-cre
L10-Egfp mice and selected two injection cases for full brain mapping, namely, cases with the lowest and highest colocalization between TH-ir and virally transduced, DsRed-labeled cells, to identify common target sites. Overall, DsRed-labeled fibers were distributed brainwide and were most prominent within the motor-related midbrain (MBmot), notably the periaqueductal gray area and superior colliculus. We also observed numerous DsRed-labeled fibers within the polymodal association cortex-related thalamus (DORpm), like paraventricular thalamic nucleus and nucleus of reunions, that processes external and internal sensory input. Overall, ZI DA cells displayed a similar fiber profile to ZI GABA cells and may integrate sensory input to coordinate motor output at their target sites.