Fig. 2

Comparison of EF1α-DIO-EGFP expression pattern driven by M1-Cre and by the non-CA2-specific Cre virus in dorsal CA2 of WT mouse and neuron type identification in the infected CA2. A Schematics of stereotactic virus injection at dorsal CA2 of WT mouse. B1–B4 Representative confocal serial sections of hippocampus infected with M1-EGFP (green) and stained with RGS14 (red) (n = 6 mice). B5 Magnified image of M1-EGFP (green) collocated with RGS14 (red) in CA2 region (n = 9 mice). C1–C4 Representative confocal serial sections of hippocampus infected with CMV-EGFP (green) and stained with RGS14 (red) (n = 5 mice). C5 Magnified image of CMV-EGFP (green) and RGS14 (red) (n = 9 mice). Image showing the expression range of CMV-EGFP exceeds the CA2 region, there were some EGFP and RGS14 overlap. D1–D4 Representative confocal serial sections of hippocampus infected with AAV9/hSyn-Cre & AAV9/EF1α-DIO-EGFP (green) in dCA2 and stained with RGS14 (red) (n = 4 mice). D5 Magnified image of hSyn driven EGFP (green) and RGS14 (red). Image showing no hSyn-EGFP cells and RGS14 overlapping, and EGFP expressed within CA2. E1-E4 High magnification confocal images of CA2b displaying M1-EGFP unstained with RGS14 (dotted circles) and overlapped with RGS14. F1 confocal image of dCA2 infected with M1-EGFP, showing M1-EGFP (green) and NeuN (red) colocalization (n = 3 mice). F2 image showing no Parvalbumin (red) and M1-EGFP (green) colocalization (n = 3 mice). F3 image showing no GAD67 (red) and M1-EGFP (green) colocalization in pyramidal cell layer (Py) but a little overlap in Oriens layer (Or) (n = 4 mice). F4 image showing no M1-EGFP (green) and Calretinin (red) colocalization (n = 4 mice). F5 image showing dCA2 infected with M1-EGFP stained with Calbindin (red). G Proportion of different cells in the hippocampus infected with M1-EGFP (AAV9/M1-Cre & AAV9/EF1α-DIO-EGFP or AAV5) (n = 6 mice) or CMV-EGFP (n = 4 mice) or AAV9/hSyn-Cre & AAV9/EF1α-DIO-EGFP (hSyn-EGFP) (n = 3 mice) on confocal coronal section. Black dash lines represent RGS14 stained CA2 region (Red bars). RGS14⁺ cells in RGS14 stained region were defined as total CA2 cells and is normalized as 100%. Green bars: the proportion of infected EGFP⁺ cells compared to RGS14⁺ cells; Yellow bars: the proportion of EGFP⁺RGS14⁺ cells to the total CA2 cells, Blue bars: the proportion of EGFP⁺RGS14⁻ cells to the total CA2 cells within RGS14 stained region. All means were calculated from no less than 3 slices for each biological sample. H Scatter chart displaying the number of EGFP⁺ neuron outside-RGS14-region in each treatment. Data are presented as mean ± SEM. I Quantitative analysis showing the percentage of M1-EGFP cells stained with various markers in (F1–F4). All means calculated from no less than 5 brain slices of each biological sample. J Quantitative analysis of M1-EGFP cells receiving Calbindin-positive mossy fibers (MF⁺) and not receiving mossy fibers (MF^–) in F5. All sections above were stained with DAPI (blue). Scale bars: 100 μm (B5, D5, F3, F5), 200 μm (B1–B4, C1–C4, D1–D4, C5), 20 μm (E1–E4), 50 μm (F1, F2, F4)