Using immunohistochemistry, we confirmed that EYFP reporter expression was present in both S1 and M1. YFP-positive cells in these areas were morphologically identified as pyramidal neurons, and were in layers II-III, V-VI. To confirm this we further analyzed the tissue using markers for inhibitory neurons such as parvalbumin, somatostatin and calbindin as well as GAD67/65 and examined cell bodies and axonal terminals at the areas around the site of infusion. We found no evidence for co-localization between EGFP and any of the inter-neuronal markers therefore excluding the possibility for significant opsin presence in cells other than pyramidal neurons, and confirmed that optical stimulation was selectively activating excitatory pyramidal neurons in M1 and S1. (A) Low magnification image of the coronal section processed anti-GFP antibody showing the medio-lateral aspect of YFP expression in the somatosensory cortex of monkey J (areas 1, 2, 3). The black arrowhead indicates the location of the injector needle track; the adjacent tissue (black frame) is microscopically enlarged in B to show laminar distribution of the YFP-positive cells. In monkey G (Yazdan-Shahmorad et al., 2016), we saw some cortical thinning post-mortem. In contrast, the histology for monkey J, presented here does not show change in the cortical thickness. (B) Densely YFP-positive cells are located predominantly in layers II-III and V-VI, and also show typical pyramidal morphology (cells in white frames are further enlarged in panels C-E). White arrowheads on bottom panels C-E point to typical pyramidal cells in layers II-III (C); layer V (D); and layer VI (E). Scale bars: A, 2 mm; B, 200 μm; C-E, 100 μm. (F) Stained parvalbumin neurons (red) and YFP-expressing cells (green), no overlap indicated high specificity. Scale bar: 30 μm.