Neuron, Volume 99 Supplemental Information A Visual-Cue-Dependent Memory Circuit for Place Navigation Han Qin, Ling Fu, Bo Hu, Xiang Liao, Jian Lu, Wenjing He, Shanshan Liang, Kuan Zhang, Ruijie Li, Jiwei Yao, Junan Yan, Hao Chen, Hongbo Jia, Benedikt Zott, Arthur Konnerth, and Xiaowei Chen
Figure S1. Procedure for AAV injection in the MECII and optical fiber-based Ca 2+ measurements in the MECII-DG projection. Related to Figure 1.
(A) Picture of a glass micropipette. The corresponding volume at each segment is shown on the top. We injected approximately 100 nl of the virus solution into each hemisphere. (B) Diagram showing the injection location in MECII. V1, primary visual cortex; V2L, secondary visual cortex, lateral area; PRh, perirhinal cortex; S, subiculum; Cb, cerebellum. (C) Example of GCaMP5G expression in the MECII. The nuclei were stained with DAPI. (D) Overlay of viral expression areas from 9 mice, indicated in grey. (E) Confocal image of GCaMP5G expression after optical recordings. The fiber track is pointed out by an arrow. (F) Examples of egfp or GCaMP5G or GCaMPf fluorescence in the MECII-DG projection under different behavioral states. (G) Comparison of the amplitudes of egfp, GCaMP5G, and GCaMPf fluorescence (kruskal-wallis test, ***p < 0.001, χ 2 = 20.4, n = 10 trials for each group). (H) Long-term Ca 2+ recordings. These four traces were obtained when the animal was exploring in an open field freely on day 1, 3, 7 and 12 after fiber implantation. (I) Summary of signal amplitudes over 2-week recordings (kruskal-wallis test, p = 0.23, χ 2 = 4.2, n = 7 mice). Box-and-whisker plot: center line, median; box, 25-75 IQR; whiskers, minimum and maximum.
Figure S2. Ca 2+ signals in the MECII-DG projection were decreased by local application of muscimol into the MECII, optics nerve crush as well as execution non-exploration behaviors. Related to Figure 1.
(A) Diagram showing Ca 2+ recording in the MECII DG projection and local muscimol application in the MECII. (B) Example of Ca 2+ signals before, during and after muscimol application. This experiment was performed under anesthesia. (C) Example of Ca 2+ signals and the corresponding animal locomotion before, during and after muscimol application. This experiment was performed in a freely moving mouse. (D) Summary of the muscimol effect (Friedman test, **p < 0.01, χ 2 = 11.14, n = 4 anesthetized mice and 3 freely moving mice). (E) Effects of optic nerve crush on Ca 2+ signals in the MECII DG projection. Left, schematic of procedure for the bilateral crush of optic nerve. Right, example of Ca 2+ recordings before and after optic nerve crush. The Ca 2+ recordings were performed in freely behaving states. (F) Summary of optic nerve crush experiments (Wilcoxon signed-rank test, **p < 0.01, z = 2.45, n = mice) versus sham operated controls (Wilcoxon signed-rank test, p = 0.71, z = - 0.54, n = 5 mice; n.s. denotes no significance). (G) Example of Ca 2+ signals in the MECII-DG projection when running on treadmill, grooming or eating. Left, schematic of treadmill running, grooming or eating (from top to bottom). Right, the corresponding Ca 2+ signals (blue traces) and body movement (black traces). (H) Summary of the amplitudes of Ca 2+ signals (32 trials from 7 mice for each group). The control group indicates the signals when mice were still. Each group was normalized to the corresponding exploration group (Wilcoxon rank sum test, ***p < 0.001, χ 2 = 1.79, n = 7 mice). Box-and-whisker plot: center line, median; box, 25-75 IQR; whiskers, minimum and maximum.
Figure S3. Related to Figure 1. (A) Example of Ca 2+ signals in the MECII-DG projection of a naïve mouse during the water maze task. The vertical green bar indicates the initiation of the performance. (B) Summary of the number of Ca 2+ events at different periods from the onset of task performance (Friedman test, p = 0.11, χ 2 = 7.2, n = 7 mice). Data are represented as mean ± SEM. (C) Two examples of higher amplitude Ca 2+ signals during open field exploration than those during the water maze task in well-trained mice. The red traces indicate Ca 2+ signals during the water maze task, and the blue traces indicate Ca 2+ signals during open field exploration. (D) Overlay of viral expression areas (in grey) from mice. (E) Schematic of virus injection of GCaMP5G in the MECIII and Ca 2+ recording above the axonal projection site in the CA1. (F and G) Post-hoc histological images show
GCaMP5G expression in the MECIII (F) and the axonal projection in CA1 (G). (H to L), Serial parasagittal sections labeled with AAV-Syn-GCaMP5G (green) and stained with an anti-wfs1 (red). (M) The area magnified from the white rectangle in (L). D, dorsal; R, rostral; V, ventral; C, caudal. The nuclei were stained with DAPI (blue).
Figure S4. Histological evidence of the locations of electrophysiological recordings in MEC and analysis of spatially-modulated cells. Related to Figure 3. (A) Post-hoc histological image showing the locations of tetrodes in all 11 recorded mice. Yellow arrows indicate the positions of electrode tracks. Red dots indicate the recording positions. (B) Example of electrophysiological signals in MECII when the mouse was performing the radial arm maze task. The raw trace (black trace), the filtered trace (high-pass filtering at a cut-off frequency of 250 Hz), the subtracted trace (subtraction of
reference), and the detected spikes are shown. (C) Three consecutive trials from one well-trained mouse showing three intermittent firing cells. These three cells exhibited stable firing activities at three particular regions across multiple trials and were defined as spatial cells. The average firing rates during navigation between the start to the target are shown in different colors on the bottom. (D) Summary of the fractions of spatial and non-spatial cells (n = 149 intermittent firing cells from 10 mice).
Figure S5. Swimming, eating, running and delayed fear conditioning behaviors were not affected by optogenetic inhibition of the MECII-DG projection activity. Related to Figure 4. (A) Three consecutive trials of Ca 2+ signals in the MECII-DG projection of a control mouse with or without 594 nm light stimulation. The mouse expressed only GCaMP5 but no
enphr. (B) Summary of the amplitudes of Ca 2+ signals with or without 594 nm light stimulation. The amplitude of Ca 2+ signals was normalized to the mean value before light stimulation (Friedman test, p = 0.17, χ 2 = 3.5, n = 1 trials). (C) Summary of swimming speed in the enphr group (Friedman test, p = 0.50, χ 2 = 1.4, n = 10) and the sham-control group (Friedman test, p = 0.5, χ 2 = 0.33, n = ) during consecutive light-off, light-on and light-off trials. (D) Schematic for 594 nm light stimulation during eating behavior. A five-second light pulse was delivered every 20 sec. 27 trials were administered over 9 min. (E) Summary of the failure rate of eating stop during light stimulation. This failure rate was calculated by normalizing the number of eating events during light-on periods to that immediately prior to each light-on period (Wilcoxon ranksum test, p = 0.34, z = 0.95, n = mice; n.s. denotes no significance). (F) Schematic for optogenetic experiments in the open field with 594 nm light stimulation (top) and representative exploring paths during the light-off (left, gray) and light-on (right, orange) trials. Six trials of light off/on periods were performed consecutively. (G) The running velocity was calculated during each trial, and no significant differences were observed between the light-off and light-on trials in the enphr group (Wilcoxon signed-rank test, p = 0.21, z = 1.2, n = ) or in the sham-control group (Wilcoxon signed-rank test, p = 0.53, z = 0.3, n = ). Data are represented as mean ± SEM. (H) Experimental protocol of delayed fear conditioning (DFC). At the training session, the unconditioned stimulus (US, electrical shock, 1 s, 0. ma intensity) was applied immediately following the conditioned stimulus (CS, pure tone at 94 Hz, 9.9 s, 70 db SPL). This pairing was repeated five times, with an interval of 210 sec. 24 hours after conditioning, the fear levels of mice were tested in a novel chamber. 10 CS stimuli were applied at an interval of 210 sec. The light stimulation was delivered together with every two CS presentations. (I) Summary of freezing levels in light-on and light-off trials (CS versus baseline: Friedman test, **p < 0.01, χ 2 = 11.19, n = 7 mice; light-on trials versus light-off trials: Wilcoxon signed-rank test, p = 0.17, n = 7 mice; n.s. denotes no significance). (J) Post hoc histology of enphr expression in bilateral CA1 areas from a mouse after optogenetic inhibition experiments Box-and-whisker plot: center line, median; box, 25-75 IQR; whiskers, minimum and maximum.
Tetrode location (medial to lateral) (mm) Tetrode location (ratio to the dorsoventral extent of MEC) Sporadic firing cells Intermittent firing cells Sustained firing cells Total MECII cells in well-trained state Sporadic firing cells Intermitten t firing cells Sustained firing cells Total MECII cells in naive state Table S1. Distribution of electrophysiologically-recorded MEC neurons across animals and their locations. Related to Figure 3. Mouse ID 1 2 3 4 5 7 9 10 11 Total 9 5 12 7 14 14 11 23 115 9 4/ 5 1/ 1/ 12 7 7/ 14 14 / 11 1/ 23 52/115 4/ 9 4/ 5 7/ 12 7/ 7 7/ 14 9/ 14 3/ 11 7/ 23 3/115 2/ 9 3/ 1 11 --- 1/ 34 2/ 24 33 3/ 2 1 1/ 12/15 7/ 9 11/ 1 / 1 11 --- 27/ 34 1/ 24 2/ 33 22/ 2 13/ 1 149/15 9 2/ 1 1/ 11 --- / 34 4/ 24 33 1/ 2 3/ 1 2/ 24/15 17 13 17 22 19 21 7 15 2 20 --- 3.2 3.2 3.2 2.9 3.1 2. 2.9 3.1 2. 2.9 3.1 2. 2. 2. ---