The medial entorhinal cortex (MEC) is known to contain spatial encoding neurons that likely contribute to encoding spatial aspects of episodic memories. However, little is known about the role MEC plays in encoding temporal aspects of episodic memories, particularly during immobility. Here using a virtual ‘Door Stop’ task for mice, we show that MEC contains a representation of elapsed time during immobility, with individual time-encoding neurons activated at a specific moment during the immobile interval. This representation consisted of a sequential activation of time-encoding neurons and displayed variations in progression speed that correlated with variations in mouse timing behavior. Time- and space-encoding neurons were preferentially active during immobile and locomotion periods, respectively, were anatomically clustered with respect to each other, and preferentially encoded the same variable across tasks or environments. These results suggest the existence of largely non-overlapping subcircuits in MEC encoding time during immobility or space during locomotion.
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We thank M. Howe for developing the original version of the Door Stop task and thank J. Climer, M. Hasselmo, M. Howe, and J. Issa for useful comments on this manuscript. We thank C. Woolley for use of the freezing microtome and V. Jayaraman, R. Kerr, D. Kim, L. Looger, and K. Svoboda from the GENIE Project (Janelia Farm, Howard Hughes Medical Institute) for GCaMP6. This work was supported by The McKnight Foundation (D.A.D.), The Simons Collaboration on the Global Brain Post-Doctoral Fellowship (J.G.H.), The Chicago Biomedical Consortium with support from the Searle Funds at The Chicago Community Trust (D.A.D.), and the NIH (1R01MH101297; D.A.D.).
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Integrated supplementary information
(a). Epifluorescence image of GCaMP-6f fluorescence through MEC microprism (top) and two-photon imaging FOVs through MEC microprism (bottom). Red dot indicates location of Alexa494 pin marking shown in the histological section. (b). Post-mortem histological sagittal sections from same mouse as in a covering region of MEC labeled with GCaMP6f (green). Morphology shown with DAPi staining (blue) and location of pin marking shown with Alexa594 labeling (red). The presence of lamina dissecans, the relative position of the post-rhinal border to the pin mark and the circular shape of the dentate gyrus shown at the medial-lateral position of the sagittal sections indicates recordings were made in MEC. c. Same as b, but magnified view of GCaMP6f region. Cortical layers marked with red dashed lines. d. Same as c, but only DAPi shown. Repeated 5 times across 5 mice with similar results.
a. Behavioral epochs for Door Stop task. b. Mouse velocity across all correct and incorrect laps for mouse 1 (top) and mouse 2 (bottom). c. Mouse velocity on all correct trials where mouse waited 6-9 seconds before running at the Door Stop for mouse 1 (top) and mouse 2 (bottom). d. Same as b, but with high magnification for velocity. e. Mouse velocity for correct 6-9 second wait trials at Door Stop (grey) and mean across all trials (black) for mouse 1 (top) and mouse 2 (bottom). Purple dashed lines and arrows indicate transition period. f. Mouse velocity for all trials at Door Stop for mouse 1 (top) and mouse 2 (bottom).
Supplementary Figure 3 Analysis of neural activity using lowered velocity thresholds for resting periods, distribution of timing fields across all temporal encoding neurons, examples of spatial cell activity, and correlations between ΔF/F and velocity, all during (invisible door) Door Stop Task.
a. Two examples of mean DF/F versus time across all correct trials in a single session for all neurons in a single FOV during the 6 second door stop wait interval for Mouse 1 (left) and Mouse 2 (right). Data analyzed with upper rest velocity threshold = 5.2 cm/sec (left) and for upper rest velocity threshold = 1.95 cm/sec (right). Mean DF/F normalized to peak for each neuron (each row). Mean DF/F for cells that did not have significant timing fields with lowered velocity threshold were colored visible blue. (left: N = 18 cells from 1 imaging field; right: N = 22 cells from 1 imaging field). b. Distribution of Run (at door)-Rest (at door) Index (see Methods). c. Mean DF/F versus time across all correct trials in the Door Stop task from all time encodings neurons (each row represents single neuron mean DF/F) across all FOVs. Mean DF/F normalized to peak for each neuron (each row). d. Bottom, DF/F versus track location during locomotion for each trial of a single session for 2 individual neurons from the same FOV during Door Stop task running phase. Top, Mean DF/F versus track location across all trials. Purple dashed lines and arrows indicate transition period. e. Mean DF/F versus track location during locomotion across all trials in a single session for all neurons (each row represents single neuron mean DF/F) in a single FOV during the Door Stop task running phase. Mean DF/F normalized to peak for each neuron (each row). f. Histogram of Pearson’s Correlations between mouse velocity and DF/F vs time traces during behavior in the Door Stop task for all active cells (left), temporal encoding and spatial encoding cell (right); transition periods and reward zone excluded.
Supplementary Figure 4 Temporal and spatial information carried by individual cells during the immobile timing phase and navigation phase of the (invisible door) Door Stop task.
a. Heatmap histogram of number of cells carrying various combinations of spatial and temporal information for all active cells (cells with a mean DF/F > 0.7% during either locomotion periods along the track or immobile periods waiting at the door, which equates roughly to at least 5 calcium transients during one of these behavioral periods). No test for the statistical significance of a cell’s spatial or temporal information was included here (see b for plot including only cells with significant information). b. Same as a, but only including cells with significant spatial or temporal (or both) information (based on Bootstrap test, see Methods). Spatial and temporal information tests for each cell were performed separately and independently, such that a cell could carry significant spatial, temporal, both or neither information (neither not included in histogram). c. Angular histogram of data shown in b; number of cells plotted as a function of angle (θ, see red angle in inset) from 0 to π/2 (binned in increments of π/16, see black wedge in inset). Real data (blue) and randomized data (black: mean and STD across all shuffles; grey: individual shuffle distributions). d. Scatter plot of data presented in b; each circle represents a single cell. Cells with only significant temporal information (red), cells with only significant spatial information (blue) and cells with both significant temporal information and significant spatial information (grey). Histograms of temporal information subset (top) and spatial information subset (right). e. Scatter plot of data presented in b; each circle represents a single cell. Cells sorted by k-means cluster analysis with 2 clusters.
Supplementary Figure 5 Sequence progression across time-encoding cells correlates with animal wait time in (invisible door) Door Stop task.
a. Examples of normalized DF/F sequence for all cells from an individual trial for error waits (mean error wait = 5.1 ± 0.5 sec; left), short waits (mean short wait = 6.5 ± 0.3 sec; middle) and long waits (mean long wait = 8.0 ± 0.7 sec; right). Cells were ordered according to each cell’s mean center of mass across all short wait trials. Red, pink and green lines are linear fits of error (red, left), short (pink, middle) and long (green, right) wait sequences (N = 7 cells from 1 imaging field). b. Plot of slope (from linear fits of cell activations per second) as a function of animal wait time for all individual trials (each circle represents a single trial, as seen in a). Cells were ordered according to each cell’s mean COM across all correct (6-9.5 sec) trials. N=86 wait trials from 4 imaging fields in 3 mice.
Supplementary Figure 6 Encoding properties of temporally and spatially selective cells across different environments and behavioral tasks.
a-b. (Visible door) Door Stop task environment switch. a. Views of “visible Door Stop” tracks that mice navigated in during environment switch paradigm (Fig. 4). Views from beginning of tracks. b. Examples of cells with timing fields and spatial fields that either remained coding for time or space across environments, switched coding time or space across environments, or were selective for time or space only in one environment. Bottom, DF/F versus time (left) or position (right) for 5 individual neurons during the (visible door) door stop wait interval and running periods across the tracks in environment 1 (E1) and environment 2 (E2). Scale bars indicate 100% DF/F. Top, Mean DF/F versus time (left) and position (right) across all correct wait trials and runs along the track. c-f. Linear track task timing cells. c. Views of linear tracks that mice navigated in during environment switch paradigms (Figs. 4 and 5, Supplementary Fig. 7). No door stop component was included in this task. Mice spontaneously stopped during linear track navigation. d. Histogram of instantaneous velocity from all mice (n=6) navigating in virtual linear track task (top). Histogram of duration of spontaneous rest periods from all mice navigating in virtual linear track task (Bottom). e. Bottom, DF/F versus time for each rest period of a single session for 4 individual neurons from the same FOV. Top, Mean DF/F versus time across all rest periods (red) and mean locomotion velocity (black) across same periods. f. Mean DF/F versus time across all rest periods from a single session (each row represents single neuron mean DF/F) in a single FOV (top). Mean DF/F normalized to peak for each neuron (each row). Mean mouse velocity during rest periods (bottom). g-j. Classical conditioning to linear track switch g. Schematic of classical trace conditioning paradigm (top) and view of linear virtual track environment (below). CS (auditory click) was presented 6 seconds before reward delivery. Head-fixed mice performed classical trace conditioning task in complete darkness (treadmill was fixed in place and could not rotate) and were subsequently switched into the virtual linear track navigation task (VR screens were turned on and treadmill was free to rotate, no door stop, see c-f above); this switch paradigm was used in Fig. 4. h. Examples of cells with timing fields during the trace conditioning period and during rest periods on the virtual linear track navigation task. Bottom, DF/F versus time (left, classical conditioning; middle, linear track) and position (right, linear track) for 2 individual neurons. Scale bars indicate 100% DF/F. Top, Mean DF/F versus time (left, classical condition; middle, linear track) and position (right). i. Mean DF/F versus time across all trace periods in a single session for all neurons (each row represents single neuron mean DF/F) in a single FOV during the 5.5 second trace period in the classical conditioning task. Mean DF/F normalized to peak for each neuron (each row). j. Top, Mean normalized mouse lick frequency versus time during trace period, across all trace periods during example single session in the classical trace conditioning task, demonstrating anticipatory licking. Bottom, normalized mouse lick frequency versus time during trace period shown for all trace periods in the classical trace conditioning task. k-m. Characterization of cell encoding properties across days in (invisible door) Door stop task. k. Bottom, DF/F versus time for each correct trial of a single session for the same individual neuron with timing fields during the 6 second door stop wait interval on day 1 (left) and day 3 (right). Top, Mean DF/F versus time across all correct trials for the same cell on day 1 (left) and day 3 (right). l. Same as b, but for an example cell with spatial fields. m. Fraction of cells with timing or spatial fields that encoded the same variable (orange) or switched variables (blue) across days (t=inf, df = 25, P<.0001, Student’s Paired T-Test). N = 26 cells across 4 imaging fields. *** indicate P<0.0001. n. Histogram (left) and mean (right) of absolute value of RRI difference for each cell across environments (including all track environment switches: invisible door, visible door, linear track, but not including classic conditioning to linear track switches) for real (blue) and shuffled data (brown). Cells included are from top and bottom quartiles of RRI distribution in environment 1. (N = 236 cells across 10 imaging fields; P<0.001 for Shuffle Test). Box plot: Red lines indicate median, the edges of the box are the 25th and 75th percentiles, the whiskers depict range of data (excluding outliers), and the outliers are plotted individually indicated by red “+” markers. *** indicate P<0.001 for Shuffle Test.
Supplementary Figure 7 The spatial representation formed by populations of spatial cells in MEC is present from the first moments of new experiences.
a. Views of linear tracks (no door stop) mice navigated in during environment switch paradigm. b. Bottom, DF/F versus position for each track traversal (trial) of a single session for 2 individual neurons during first session in novel linear track. Trial 1 was the first time the mice ever ran down the track in the novel environment (light blue trace). Top, Mean DF/F versus position across all run periods (dark blue); DF/F trace from first run period is also shown (light blue). c. Pearson’s Correlation between the calcium transients during each run period and the mean spatial field over all periods (y-axis) as a function of number of run periods in novel environment (x-axis); grey = mean across all cells in a single FOV in a single session; black = mean ± SEM across all cells in all sessions. N = 5 imaging fields across 3 mice. d. Mean fraction of trials with transients occurring within the significant spatial field across all cells for the first half of run trials in the session versus the second half of run trials in the session. (n = 5 imaging fields from 3 mice; P = 0.625, two-sided Paired Wilcoxon Signed Rank Test). e. Cumulative distribution of the trial number at which a transient in the significant spatial field first occurred in the novel session across all spatial encoding cells.
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Heys, J.G., Dombeck, D.A. Evidence for a subcircuit in medial entorhinal cortex representing elapsed time during immobility. Nat Neurosci 21, 1574–1582 (2018). https://doi.org/10.1038/s41593-018-0252-8
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