In contrast, very sparse labeling was found in the caudal half, the parietal, visual, auditory, and entorhinal cortices. In SNc-targeted cases, the most dense labeling was found in the primary and secondary motor cortices (M1 and M2)

(Figures 5B, 5E, 5H, and S4). Somatosensory cortex (S1) has moderate labeling, but, due to its large size, it provides the largest number of inputs among cortical areas (Figure 3). VTA dopamine neurons receive fewer cortical inputs than SNc dopamine neurons, but the lateral orbitofrontal cortex (LO) is the major sources of cortical inputs to VTA dopamine neurons (Figures 3, 5A, and 5G). Areas encompassing the medial prefrontal cortex (PrL, IL, DP, and MO) and the cingulate cortex (Cg1 and Cg2) have moderate labeling. These results demonstrate

that dopamine neurons in the VTA and SNc receive significant numbers of cortical inputs from overlapping but distinct areas. AZD2281 NVP-AUY922 cost At more caudal regions, the intermediate layer of the superior colliculus (SC) and supraoculomotor (ventrolateral) periaqueductal gray (PAG) contained large numbers of labeled neurons in both VTA- and SNc-targeted cases (Figure S6C). The dorsal raphe (DR) contained the densest population of labeled neurons both for VTA- and SNc-targeted cases, with slightly stronger projections to VTA (Figure S6D; also see Figure 3). The pedunculotegmental nucleus (PTg) and cuneiform nucleus (CnF) preferentially project to SNc dopamine neurons, whereas laterodorsal tegmental nucleus (LDTg) preferentially projects to VTA dopamine neurons (Figure S6D). The parabrachial nucleus (PB), both ipsilateral and contralateral to the injection side, projects to both VTA and SNc dopamine neurons (Figure S6E). We also found that cerebellar nuclei project to dopamine neurons (Figure S6F). The aforementioned results are, to a large degree, consistent with previous results using conventional tracers (Geisler and Zahm, 2005) but differ in some critical ways. For example, some areas such as the septum and mHb were not labeled heavily in our experiment, found despite being strongly labeled in previous

experiments involving injection of a retrograde tracer (Fluoro-gold) in VTA (Geisler and Zahm, 2005). Furthermore, even in the areas that were labeled both in our and in other previous experiments, our methods resulted in labeling of more specific subsets of neurons (see below). To test whether these differences are due to the greater specificity of our labeling methods, we performed a control experiment using rabies virus that was not pseudotyped with EnvA but still lacks RG (SADΔG-GFP) (therefore, this virus can infect mammalian cells but cannot spread transsynaptically). In these experiments, injection of the virus into VTA resulted in a significant number of retrogradely labeled neurons in the septum and mHb (Figures S3A, S3B, S3D, and S3E).