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. 2020 Mar 13;9:e53129. doi: 10.7554/eLife.53129

Figure 1. Ca2+ activities of the SuM and DG at home cage and during the spatial memory test.

(A) Experimental diagram of the DG and SuM calcium recording at the home cage. (B) Coronal sections showing photometry fiber traces and CaMKII-GCaMP/tdTomato expressed neurons in the DG (left) and SuM (right). Scale bar = 100 µm. (C) A sample of normalized ΔF/F of GCaMP6f signals from the DG and SuM. The traces of GCaMP activity were moderately correlated with R = 0.31, p < 0.0001 at home cage. (D) Sample of GCaMP6f traces of the DG and SuM in 0.1–0.5 Hz spectrum. The traces of GCaMP signals were moderately correlated with R = 0.48, p < 0.0001. (E) Mean correlation of SuM and DG calcium activity in 0.1–0.5 Hz spectrum. n = 10 mice, R = 0.40, p<0.0001. (F) Experimental illustration of in vivo fiber photometry recordings during the NPR test. After 3 days of habituation, mice were recorded for calcium activity during distinct phases (spatial memory encoding versus retrieval) of the NPR test. (G) Time spent exploring the objects during the NPR test. In the test phase, time spent exploring the novel-location object (B) was significantly increased (n = 8 mice, paired t-test, t1,7 = 6.843, **p < 0.01). No difference was found during familiarization (n = 8 mice, paired t-test, t1,7 = 1.593, p = 0.16). (H, L) Scaled color plot of DG and SuM calcium activity while exploring object A (old location) and object B (novel location) during familiarization (H) and test (L) phase, respectively. n = 49, 62, 39, 59 trails. (I, M) Averaged DG and SuM calcium activities during familiarization (I) and test (M). Semi-transparent borders indicate ± SEM. (J, N) Cumulative DG and SuM calcium activities in the NPR test. Time points when mice started to explore the objects were defined as 0 s. Calcium activity was aligned within a 2 s time window at each phase: pre-exploring (−3 ~ −1 s), exploring (−1 ~ 1 s) and post-exploring (1 ~ 3 s). Cumulative activity = ∆F/F × Time (2 s). Calcium activity in both the DG and SuM increased when mice were exploring objects/locations during familiarization (J) and test (N). (K, O) Correlation of the SuM and DG calcium activity during familiarization (K) and test (O). Calcium activity of the SuM and DG during exploration was highly correlated (R = 0.9292, p < 0.0001) during spatial memory retrieval, as compared to a moderate correlation (R = 0.5438, p<0.0108) during spatial memory encoding. (P, Q) Cumulative activity of the DG (P) and SuM (Q) was increased during test, compared to familiarization. DG: Unpaired t-test, t207 = 2.009, *p=0.0459. SuM: Unpaired t-test, t207 = 1.975, *p = 0.0496.

Figure 1.

Figure 1—figure supplement 1. Experimental protocol of fiber photometry recording.

Figure 1—figure supplement 1.

(A) Schematic illustration of the photometry system used for measuring red and green fluorescent sensors in the SuM and DG. CaMKII-GCaMP6f and CAG-tdTomato were delivered to the left DG and SuM. (B) Co-expression of GCaMP6f and tdTomato in DG GCs. Scale bar = 20 µm. (C) Mixed emission spectrum of GCaMP6f and tdTomato. (D) Unmixing coefficients of GCaMP6f and tdTomato by fitting signals to standard emission curves. TdTomato signals were further used as motion correction during recording. (E) Sample traces of simultaneous recording from the SuM and DG post unmixing and normalized process. Those data were used for activity analysis. (F) The color code sample frequency of SuM and DG calcium signals were mainly appeared at 0.1–0.5 Hz. (G) The calcium signals > 0.5 Hz were lower than 0.01 dB/Hz. Due to the low calcium signal associated with >0.5 Hz, we selected 0.1–0.5 Hz frequency range to analyze correlation of calcium activities from the SuM and DG. (H) Heatmap of the correlation coefficient in SuM and DG GCaMP frequency spectrum. (I) Correlation of SuM and DG GCaMP signals in 0.1–0.5 Hz range during home cage recording.
Figure 1—figure supplement 2. Low correlation of GCaMP activity in EC and DG at home cage and during the NPR test.

Figure 1—figure supplement 2.

(A) Experimental diagram of the DG and EC calcium recording. (B) Coronal sections showing photometry fiber traces and CaMKII-GCaMP/tdTomato expressing neurons in the DG (left) and EC (right). Scale bar = 100 µm. (C–D) Samples (C) and correlation (D) of calcium activities of the DG and EC from mice at home cage. n = 4, R = 0.22, p > 0.05. Correlation of SuM and DG calcium activities in 0.1–0.5 Hz range were analyzed. (E) Scaled color plot of DG and EC GCaMP activities in NPR test period, n = 41 trails. (F) Average of DG and EC calcium activities in the test period. Transparent borders indicate ± SEM. During the exploring period, both DG and EC activities were increased. (G) Cumulative activity of the DG and EC during the exploring period (−1 ~ 1 s) in the NPR test. (H) Correlation of averaged DG and SuM calcium activity during the NPR test period. R = 0.21, p > 0.05.