Fig. 1

Outer layer of Vb neurons in medial entorhinal cortex project to hippocampal dentate gyrus in mice. (A) Injection of CTB488 and CTB555 into dorsal (top) and ventral DG (bottom), respectively. CTB488 (green). CTB555 (red). DAPI (blue). Scale bar, 500 μm (top), 1 mm (bottom). (B) Parasagittal sections of the MEC labeled with CTB488 (green), CTB555 (red) and immunostained with Ctip2 (blue). Arrowheads indicate CTB488⁺ cells and CTB555⁺cells in Vb, individually. Scale bar, 500 μm. (C–D) Magnified images of B, top square (C) and bottom square (D), respectively. Yellow arrowheads indicate CTB488 and CTB555 double positive cells. White arrowheads indicate CTB555 single positive cells. Scale bar, 100 μm. (E–H) Immunohistochemistry for CTB555⁺ cells using GABAergic interneuron makers, respectively (left). Merged images of left panel and CTB555⁺ cell, individually (right). (E) GAD67, (F) Parvalbumin, (G–H) Somatostatin. Example image of somatostatin negative CTB555⁺ cell (G) and somatostatin positive CTB555⁺ cell (H). Scale bar, 10 μm. (I) Injection of CTB555 (red) into vDG (top), CTB488 (green) into BLA (middle), and CTB647 (blue) into NAc (bottom), respectively. Scale bar, 1 mm. (J) Parasagittal sections of the MEC labeled with CTB488, CTB555, CTB647. Scale bar, 500 μm. (K) Magnified images of J, respectively. Scale bar, 200 μm. (L) Injection of CTB555 into AD/AV. Scale bar, 1 mm. (M) Parasagittal sections of the MEC labeled with CTB555 (top left, red) and immunostained with PCP4 (top right, green), NeuN (bottom left, blue) and merged image (bottom right). Scale bar, 200 μm. (N) Magnified image of square area in M. Scale bar, 100 μm. (O) Distribution of vDG-projecting CTB⁺ cells (top, n = 7 mice) and AD/AV-projecting CTB⁺ cells (bottom, n = 6 mice) in MEC Vb which represented by 50 bins through the outer to the inner layer of Vb. (P) Parasagittal section of the MEC Vb labeled with CTB488 (green) and CTB555 (red) and immunestained with PCP4 (blue), which CTB488 and CTB555 were injected into AD/AV and vDG, respectively. Scale bar, 50 μm. (Q) Summary for projection of MEC layer Vb neurons into hippocampus