Fig. 1: A transgenic cross for optogenetic manipulation of entorhinal cortex layer III and layer resolved ensemble electrophysiology in CA1.

a Representative image of fluorescent in situ hybridization of a sagittal brain slice targeting the transgene GFP (green), fused to the JAWS opsin in the EC LIII-EDGE × JAWS mouse cross. Expression is largely restricted to Entorhinal Cortex Layer III (EC LIII). Counterstain NeuN (blue). Scale bar: 500 μm. Brain-wide GFP label specificity was confirmed this way in all animals (N = 6). b Same as a without the NeuN counterstain. c Percentage of GFP-labeled neurons by brain region. EC LIII entorhinal cortex layer III, EC LII entorhinal cortex layer II, Pir piriform area, PPir postpiriform transition area, EC LV entorhinal cortex layer V, PirA piriform-amygdala area (N = 6 animals), highlighting highly similar expression patterns across all six animals used for EC LIII terminal inhibition. Data is presented as mean values ± SEM. d–h Representative high magnification images of dorsal MEC showing NeuN (blue), GFP (green) and PCP4 (violet) staining. Cropped images from brain-wide image shown in (a, b). Entorhinal cortex layer specificity was confirmed this way in all animals (N = 6). Scale bar: 100 μm. i Simplified schematic of the Hybrid Drive, with optic fiber. j Schematic of the hippocampus and entorhinal cortex showing tetrode bundle, silicon probe, and optic fiber location in CA1 and specific expression of the JAWS opsin to a subset of EC LIII pyramidal cells projecting to CA1. s.r. stratum radiatum, s.l.m. stratum lacunosum moleculare. k 3D reconstruction from histology of tetrode (purple dots) and silicon probe (black shank) tracts. The hippocampal formation is depicted in green. D dorsal, V ventral, C caudal, R rostral. Tracts were assigned to the Allen Mouse Brain Atlas and visualized in 3D using MeshView (https://www.nesys.uio.no/MeshView/). l Representative Nissl staining showing tetrode (black arrows) and silicon probe (balck bar) tracts from the Hybrid Drive implant. Tetrode, silicon probe, and optic fiber positions were confirmed using Nissl staining in all animals (N = 6). Scale bar: 100 μm. s.o. stratum oriens, s.pyr. stratum pyramidale, s.r. stratum radiatum, s.l.m. stratum lacunosum moleculare. m Example of suppressed firing in a CA1 cell following repeated laser stimulation (red, 250 ms). Shaded areas are the 95% confidence intervals of the mean. n Schematics of the linear track paradigm. Red, laser on periods.