Fig. 3

Molecular and cellular features of M1⁺RGS14⁻ neuron in CA2 Py layer. A1–A2 Images of CA1 pyramidal neurons (A1) and CA3 pyramidal neurons (A2) infected with AAV9/EGFP. Dotted lines in A1 and A2 enclosed stratum pyramidale. A3 Image of CA2 RGS14 positive and negative neurons infected with AAV9/M1-EGFP. Dotted lines enclosed RGS14 stained CA2 region (red). White arrows show M1⁺RGS14⁻ (M1 for Map3k15) neuron, white arrowheads with black edge show M1⁺RGS14⁺ neurons. B1-B4 Images of CA2b pyramidal neurons infected with AAV9/M1-EGFP, (B1, B2 the blue arrowheads with black edge indicate RGS14-failed expressed M1⁺ neurons). B3 B4 High magnification images shows CA2b M1⁺RGS14⁻ overlapped with CaMKII-α in B2 (the blue arrowhead with black edge). C1–C4 IHC images showing M1-EGFP expressed neurons stained with PCP4 (C1) and RGS14 (C2). Superposed image of M1-EGFP and PCP4 (C3). Superposed image of PCP4 and RGS14 (C4). Dotted lines enclose CA2. White symbol in C3 show M1⁺PCP4⁻ neurons (green) (arrowheads) and M1⁺PCP4⁺ neurons (yellow) (arrows). The dotted lines on Or divide the four zones of CA2. Yellow five-pointed stars in C4 show RGS14⁺PCP4⁻. The standard for distinguishing Z1–Z4 is that Z1, Z2 each occupy 20% of the RGS14 staining area, Z3, Z4 each occupy 30%. Z1, Z2 occupy a small amount of space outside the cropped area. High magnification IHC images showing M1-EGFP expressed neurons stained with RGS14 (D3) and failed stained with PCP4 (arrow head), and arrow displays M1-EGFP stained with PCP4 (D1, D2). E1 IHC image showing M1⁺RGS14⁻ neurons located in RGS14 stained region. Dotted lines enclosed RGS14 stained CA2 region (red). E2–E5 Magnified images of the delineating area in F showing overlay between CaMKII-α (magenta) and M1⁺RGS14⁻. Dotted circles show M1⁺RGS14⁻ neurons. F1–F4 IHC images showing M1-EGFP stained with RGS14 (F1) and CaMKII-α (F2). Superposed image of M1-EGFP and RGS14 (F3). Merged image of M1-EGFP and CaMKII-α (F4). Dotted circles indicate RGS14⁻EGFP⁺ neurons in F3 and overlay with stained CaMKII-α in F4. Scale bars: 200 μm (A1–A3), 50 μm (B1, B2, C1–C4, E1, F1–F4), 20 μm (E1–E4, D1–D4), 2 μm (B3, B4)