Supplementary Figure 2: Histology for hippocampus-implanted mice.

iWT mice shown on right, iCre-KO mice shown on left. Mouse ID is shown at the top of each brain slice. All scale bars are 1 mm. a) Nissl stained sagittal sections of implant site. Black arrow marks the recording location of CA1 cells. While in some cases tetrodes were lowered below CA1, only cells from CA1 were included in analysis. The morphology of the hippocampus of mouse #106 was distorted during brain extraction, however the site of the lesion was still evident. (b) Overlaid images of DAPI and GFP for the slice containing the tetrode track. Importantly, GFP was not detected in the hippocampus in these slices. Images of DAPI and GFP were taken separately and then combined in Photoshop. The intensities of the blue and green channels were adjusted separately to allow for clear visualization of the GFP expression. White arrowheads denote the end of the tetrode track. (c-d). GFP expression in the left (c) and right/implanted (d) hemispheres. White lines denote the boundary between MEC and neighboring brain structures. In two mice (iCre-KO, #99 and #118) GFP was not detectable in the left/non-implanted hemisphere. GFP expression was greatest in and largely restricted to the MEC. In a few mice a small amount of GFP leaked into the neighboring subiculum. Any GFP observed in hippocampus was further quantified using Image-Pro Plus software. Two iCre-KO mice had minor GFP expression in CA1 (#99: infected area <<1 % right CA1, ~15 cells; #144: infected area <2 % left CA1, <<1 % right CA1). GFP was also detected in the CA1 of two iWT mouse (#100: infected area = ~1 % of right CA1; #119: infected area <4 % left CA1). Two iCre-KO mice had GFP expression in a small portion of dentate gyrus (#87: infected area = ~7 % of bilateral dentate gyrus; #118: infected area <1 % of left dentate gyrus), where HCN1 expression is very low^1,2. (e) There was no difference in the mean or maximum field size of place cells recorded in iCre-KO mice with or without minor spread to hippocampus [HPC] (n = 59 place cells from 4 mice with perfect virus restriction, n = 67 place cells from 4 mice with minor virus spread; mean ± SD; mean field size without spread = 455.28 ± 353.99 cm², with spread = 424.13 ± 323.36 cm², two-tailed WRS Z = 0.32, p = 0.75; max field size without spread: 556.99 ± 377.99 cm², with spread: 495.71 ± 329.28 cm², two-tailed WRS Z = 0.79, p = 0.43). Box depicts the first and third quartiles. The median is depicted by a solid line. Whiskers indicate the range, except for data falling above the third quartile or below the first quartile by at least 1.5 times the interquartile range (plotted separately). (f) Exclusion of all mice with any hippocampal spread did not affect the primary place cell findings (virus perfectly restricted to MEC: n = 64 cells from 9 iWT mice, 59 cells from 4 iCre-KO mice). (g) boxplot of mean and maximum place field sizes (mean ± SD; mean place field size, iWT = 259.76 ± 186.94 cm², iCre-KO = 455.28 ± 353.99 cm², two-tailed WRS Z = −3.66, p = 2.5e-4; max place field size, iWT = 342.19 ± 200.11 cm², iCre-KO = 556.99 ± 377.99 cm², two-tailed WRS Z = −3.50, p = 4.6e-4). Box depicts the first and third quartiles. The median is depicted by a solid line. Whiskers indicate the range, except for data falling above the third quartile or below the first quartile by at least 1.5 times the interquartile range (plotted separately). (h) Scatterplot of distance from the nearest boundary versus max field size (F(3,123) = 7.50, p = 0.0071, ANCOVA). Blue and red lines depict the line of best linear fit for iWT and iCre-KO place cells, respectively. (i) Boxplot showing the difference in size between fields located in the center (> 14 cm; n = 11 iWT cells, 16 iCre-KO cells) versus the boundaries (< 14 cm; n = 53 iWT cells, 43 iCre-KO) of the environment, which was greater for iCre-KO (red) place cells compared to iWT (blue) (two-tailed t-test on bootstrapped distributions of the mean difference, t(19998) = 141.21, p << 0.0001). Box depicts the first and third quartiles. The median is depicted by a solid line. Whiskers indicate the range, except for data falling above the third quartile or below the first quartile by at least 1.5 times the interquartile range (plotted separately). ***p<0.0001.