Fig. 2: Activation of MEC neurons increases neural responses in CA1-projecting CA3 neurons.

A Schematic drawing shows the laser generator with fiber photometry. B Cartoon depicts the AAVs injection: 250 nl AAV/Retrograde-syn-jGCaMP7s in CA1 (5.0E + 12 vg/ml); 300 nl AAV9-CaMKIIα-ChrimsonR-mCherry for each of two sites in MEC (6.5E + 12 vg/ml); optical fibers implantation in DHP and MEC, respectively. AAV injection (mm) in CA1: AP = −1.80, ML = 1.30, DV = −1.25; In MEC: AP = −5.10, ML = 2.30, DV = −3.25/−2.75; Fiber implantation in CA3 (mm): AP = −1.80, ML = 2.30, DV = −1.80. In MEC: AP = −5.10, ML = 2.30, DV = −2.70. C Fluorescent image after adjusting the look-up table (LUT) showing the AAV expression in DHP and fiber track in CA3. Scale bar: 1000 μm. D Magnified image from Fig. 3C (dashed rectangle) shows an optical fiber monitors the neuronal activities of CA3-SC afferents. Scale bar: 500 µm. E AAV expression and optical fiber track in MEC. Scale bar: 1000 μm (right). Magnified image shows the AAV expression in MEC (left, site 2 in the above figure). Scale bar: 100 μm. F Schematic drawing illustrates the entorhinal–hippocampal circuitry and single light stimulation. G The Ca²⁺ responses in GCaMP 7s-expressing mice (upper) and GFP-expressing mice (lower) before and after the light stimulation. H Averaged Ca²⁺ responses in GCaMP 7s-expressing group (red line) and GFP group (black line) before and after the light stimulation. I Data met normality assumptions (Shapiro–Wilk p > 0.05) but violated variance homogeneity (Levene’s p = 0.021). Statistical comparison of the averaged 5 s (after 0 s) of Ca²⁺ responses in the GCaMP 7s-expressing group (red dots) and GFP group (gray dots) after mice are released into the swimming pool. J Schematic drawing illustrates the entorhinal–hippocampal circuitry and light burst stimulation. K The Ca²⁺ responses in GCaMP 7s-expressing mice (upper) and GFP-expressing mice (lower) before and after the light stimulation. L Averaged Ca²⁺ responses in GCaMP 7s-expressing group (red line) and GFP group (black line) before and after the light stimulation. M Data met normality assumptions (Shapiro–Wilk p > 0.05) but showed severe variance heterogeneity (Levene’s p < 0.001). Statistical comparison of the averaged 5 s of Ca²⁺ responses GCaMP 7s-expressing group (red dots) and GFP group (gray dots) after mice are released into the swimming pool. N Schematic drawing illustrates the entorhinal–hippocampal circuitry and light-TBS stimulation. O The Ca²⁺ responses in GCaMP 7s-expressing mice (upper) and GFP-expressing mice (lower) before and after the light stimulation. P Averaged Ca²⁺ responses in GCaMP 7s-expressing group (red line) and GFP group (black line) before and after the light stimulation. Q Data met normality assumptions (Shapiro–Wilk p > 0.05) but showed severe variance heterogeneity (Levene’s p < 0.001), requiring Welch’s test. Statistical comparison of the averaged 5 s of Ca²⁺ responses GCaMP 7s-expressing group (red dots) and GFP group (gray dots) after mice are released into the swimming pool. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001; ns not significant. Data are reported as mean ± SEM.