“…From all colabeled neurons, the greatest numbers were found in layer ii (78.9%, mean = 101.1 neurons), followed by layers iii and i, respectively (iii: 16.9%, mean = 21.7 neurons; i: 4.2%, mean = 5.5 neurons; F ( 2,7 )=206.47, p <0.0001; Fig 1D ). Importantly, layer ii contains the densest collection of projection neurons (superficial pyramidal neurons and semilunar cells) extending fibers into the OT (Bekkers and Suzuki, 2013; Haberly, 2001; Mazo et al, 2017; Neville and Haberly, 2004; Shipley and Ennis, 1996). While this AAV is, by design, specific for transducing excitatory neurons, we nevertheless confirmed that the neurons infected with AAV and thus expressing ChR2 are indeed glutamatergic by performing anti-VGlut1 immunohistochemistry.…”