Long-Term Characterization of Hippocampal Remapping during Contextual Fear Acquisition and Extinction - PubMed (original) (raw)
Long-Term Characterization of Hippocampal Remapping during Contextual Fear Acquisition and Extinction
Peter J Schuette et al. J Neurosci. 2020.
Abstract
Hippocampal CA1 place cell spatial maps are known to alter their firing properties in response to contextual fear conditioning, a process called "remapping." In the present study, we use chronic calcium imaging to examine remapping during fear retrieval and extinction of an inhibitory avoidance task in mice of both sexes over an extended period of time and with thousands of neurons. We demonstrate that hippocampal ensembles encode space at a finer scale following fear memory acquisition. This effect is strongest near the shock grid. We also characterize the long-term effects of shock on place cell ensemble stability, demonstrating that shock delivery induces several days of high fear and low between-session place field stability, followed by a new, stable spatial representation that appears after fear extinction. Finally, we identify a novel group of CA1 neurons that robustly encode freeze behavior independently from spatial location. Thus, following fear acquisition, hippocampal CA1 place cells sharpen their spatial tuning and dynamically change spatial encoding stability throughout fear learning and extinction.SIGNIFICANCE STATEMENT The hippocampus contains place cells that encode an animal's location. This spatial code updates, or remaps, in response to environmental change. It is known that contextual fear can induce such remapping; in the present study, we use chronic calcium imaging to examine inhibitory avoidance-induced remapping over an extended period of time and with thousands of neurons and demonstrate that hippocampal ensembles encode space at a finer scale following electric shock, an effect which is enhanced by threat proximity. We also identify a novel group of freeze behavior-activated neurons. These results suggest that, more than merely shuffling their spatial code following threat exposure, place cells enhance their spatial coding with the possible benefit of improved threat localization.
Keywords: calcium imaging; contextual fear conditioning; hippocampus; miniaturized microscope; place cell; remapping.
Copyright © 2020 the authors.
Figures
Figure 1.
Experimental design, example data and analysis for full grid assay. A, Illustration of full grid inhibitory avoidance assay and epoch sequence. B, Mean path length (left), freeze duration (middle), and number of stretch-attend postures (right) of mice across recording epochs (±1 SEM) for (top) full grid assay and (bottom) control no shock (No shk) assay (n = 6 full grid; n = 4 control). Statistical comparisons are performed by Wilcoxon rank-sum test for baseline/ext. 1 and baseline/ext. 2 sessions, as well as for matched sessions of the control assay. C, Example paths from day 6 (baseline), day 8 (extinction epoch 1), and day 13 (extinction epoch 2). D, Example injection site, showing lens placement (dotted line) and expression of GCaMP6f in the hippocampal CA1 region. E, Example imaging field of view with cell bodies, color coded by coregistration procedure across the first and last day of recording (days 1 and 13, respectively). F, Example raw calcium traces from a subset of cells recorded during a single session. G, Rows depict the rate maps of all place cell instances in all full grid sessions along the length of the full grid enclosure, sorted by place field center location relative to distance from the left wall (n = 17,051). H, Normalized rate maps of an example place cell, stable across recording epochs; *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 2.
Fear acquisition decreases mean rate, place field size and increases information content. A, Example rate maps of place cell with a decrease in mean rate from baseline to extinction (Ext). B, Example rate maps of place cell with a decrease in field size from baseline to Ext. C, Correlation of change in field size and change in mean rate from baseline to Ext 1 and 2 (n = 1620 Ext 1, n = 1535 Ext 2; baseline/Ext 1 Spearman r = −0.03, p > 0.05; baseline/Ext 2 Spearman r = 0.00, p > 0.05). D, Correlation of the change in spatial information content and place field size from baseline (n = 1620 Ext 1, n = 1535 Ext 2; baseline/Ext 1 Spearman r = 0.12, p < 0.001; baseline/Ext 2 Spearman r = 0.14, p < 0.001). For E–G, statistical comparisons are performed by Wilcoxon signed-rank test for paired cells at baseline/Ext 1 and baseline/Ext 2 (see Materials and Methods). E, Changes in place cell firing properties from baseline across epochs of the full grid assay. Bars show the changes in firing properties (±1 SEM) of pooled place cells that coregister between baseline and Ext epochs 1 and 2 (n = 1620 baseline/Ext 1; n = 1535 baseline/Ext 2). F, Change in spatial information content (±1 SEM) from baseline to extinction 1 and 2 epochs, recalculated for only place cells that did not show a concurrent decrease in place field size (n = 793 baseline/Ext 1; n = 748 baseline/Ext 2). G, Changes in firing properties of hippocampal CA1 place cells across epochs of control (no shock) assay. Bars show the changes in firing properties (±1 SEM) of pooled cells that coregister between no-shock days 4 and 6 and days 8 and 10 and 11 and 13 (n = 302, days 4–10/days 8–10; n = 304, days 4–10/days 11–13); **p < 0.01, ***p < 0.001.
Figure 3.
Effects of behavioral sampling on place cell firing properties. A, Bars depict the same place cell firing properties (±1 SEM) described in Figure 2_E_, but resampled such that, for each session, there is an equal number of samples from the left and right side of the enclosure (n = 1620 baseline/Ext 1; n = 1535 baseline/Ext 2). B, top, Lines depict example tracks from preshock and postshock sessions. Bottom, Bars show the fraction of time the mouse spent at binned positions along the length of the enclosure, with the corresponding bimodality coefficient in red. C, The correlation of the bimodality coefficient with mean event rate, place field size and information content for all preshock sessions (n = 36 preshock sessions); ***p < 0.001.
Figure 4.
Place cell firing properties calculated using samples for which the speed was higher than 2 cm/s. A, Changes in place cell firing properties from baseline across epochs of the full grid assay. Bars show the changes in firing properties (±1 SEM) of pooled place cells that coregister between baseline and Ext epochs 1 and 2 (n = 1620 baseline/Ext 1; n = 1535 baseline/Ext 2). Statistical comparisons are performed by Wilcoxon signed-rank test for paired cells at baseline/Ext 1 and baseline/Ext 2. B, top, Changes in firing properties from baseline across epochs of local grid assay. Bars show the changes in firing properties from baseline of pooled place cells (±1 SEM) that coregister between baseline (BL) and extinction (Ext) epochs 1, 2, or 3. Statistical comparisons are performed by Wilcoxon signed-rank test for paired cells at baseline/Ext 1, baseline/Ext 2, and baseline/Ext 3. Bottom, The change in firing properties for place cells with place field centers on the left (L) or right (R) side of the enclosure across epochs. Bars show the change in firing properties (±1 SEM) from baseline of pooled place cells, L and R, that coregister between baseline and Ext epochs 1, 2, or 3 (n = 1630 baseline/Ext 1; n = 1591 baseline/Ext 2; n = 1576 baseline/Ext 3). Statistical comparisons made by Wilcoxon rank-sum test for change in left and right sides (n = 24 per epoch); *p < 0.05, ***p < 0.001.
Figure 5.
Remapping effects are more prominent near the shock grid. A, Illustration of local grid inhibitory avoidance assay and epoch sequence. B, Bars show percent time spent on the shock grid, path length, the number of stretch-attend postures, and freeze duration (±1 SEM) for recording days 4–16 (n = 8). C, Changes in firing properties of hippocampal CA1 place cells across epochs of local grid assay. Bars show the changes in firing properties from baseline of pooled place cells (±1 SEM) that coregister between baseline (BL) and extinction (Ext) epochs 1, 2, or 3 (n = 1630 baseline/Ext 1; n = 1591 baseline/Ext 2; n = 1576 baseline/Ext 3). Statistical comparisons are performed by Wilcoxon signed-rank test for paired cells at baseline/Ext 1, baseline/Ext 2, and baseline/Ext 3. D, top, Distribution of place field centers for all mice in example baseline and extinction 1 sessions. The number of place fields in the left and right side are annotated inside the histogram. Note that, in extinction 1, there is an increase in the number of fields on the right side, which contains the shock grid. Middle, Bars show the percent change in the number of place field centers (±1 SEM) from baseline on the left and right sides of the enclosure for Ext epochs 1–3 in shocked mice. Statistical comparisons made by Wilcoxon signed-rank for change in left and right sides (n = 8 per epoch). Bottom, Same as middle but for control mice (n = 4 per epoch). For E–G, the change in firing properties for place cells with place field centers on the left (L) or right (R) side of the enclosure across epochs. Bars show the change in firing properties (±1 SEM) from baseline of pooled place cells, L and R, that coregister between baseline and Ext epochs 1, 2, or 3. E, top, Example place fields on the left and right sides of the enclosure that, respectively, increase and decrease mean rate. F, top, Example place fields on left and right sides of the enclosure that decrease place field size. G, top, Example place fields on the left and right sides of the enclosure that increase information content. Local grid: n = 701 baseline/Ext 1 left; n = 886 baseline/Ext 1 right; n = 657 baseline/Ext 2 left; n = 899 baseline/Ext 2 right; n = 704 baseline/Ext 3 left; n = 866 baseline/Ext 3 right; control: n = 150 baseline/Ext 1 left; n = 171 baseline/Ext 1 right; n = 166 baseline/Ext 2 left; n = 158 baseline/Ext 2 right); *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 6.
Place field stability measures are affected by the inhibitory avoidance assay. A, Correlation matrix showing Pearson r values of the rate maps of coregistered place cells in the local grid and (B) control (no-shock) assays across days. For each session correlation pair, only rate map locations with more than five samples in both sessions were used. C, Bars show the mean correlation (±1 SEM) of coregistered cells within neighboring sessions of each epoch, local grid, and control. Statistical comparisons performed by Wilcoxon rank-sum test between days 4 and 6 and all subsequent epochs (n = 546, mean cell count per local grid epoch; n = 97, mean cell count per control epoch). D, Bars show the amount of peak shift, defined as the distance (cm) between maximum rate map bins for coregistered neurons in neighboring sessions (n = 1383, mean cell count per local grid epoch; n = 295, mean cell count per control epoch). Only sessions in which > 90% of the enclosure length was explored were used in this analysis. E, F, Bars show the mean percent of place fields gained (E) or lost (F) in the first session of each extinction epoch, relative to baseline session 6. Statistical comparisons performed by Wilcoxon rank-sum between days 8 and 13 for local grid and control (n = 16 local grid sessions, n = 8 control sessions). G, Bars show the fraction of place cells to coregister for contiguous sessions within each epoch, local grid, and control. Statistical comparisons are performed by Wilcoxon rank-sum between days 4 and 6 and all subsequent epochs (n = 24 local grid sessions per epoch, n = 12 control sessions per epoch). H, Bars show the fraction of place cells to coregister within each epoch, separately for the left and right side of the local grid enclosure. Statistical comparisons are performed by Wilcoxon rank-sum between baseline and all subsequent epochs (n = 24 local grid left and right sessions per epoch); *p < 0.05, **p < 0.01, ***p < 0.001, n.s., not significant.
Figure 7.
Freeze cell stability and place coding. A, A GLM was used to identify cells that are significantly modulated by freezing (see Materials and Methods). Example recording from an extinction session of (top) individual freeze and nonfreeze cells and (bottom) the corresponding mouse speed. B, Example recording of mean z-scored freeze and nonfreeze cell activity, normalized by maximum value. C, Bars depict the Spearman correlation r value (±1 SEM) between freeze and nonfreeze cell activity (mean z-scored dF/F of all freeze or nonfreeze cells) for each session. Statistical comparison performed by Wilcoxon rank-sum test between baseline and grouped extinction epochs (n = 24 baseline sessions, n = 72 extinction sessions). D, Mean z-scored freeze aligned activity for all freeze cells at each behavioral instance, ±1 SEM (n = 41,411 freeze aligned dF/F traces). E, Bars show the mean z-scored dF/F activity during walking, stretch, low speed (<3 cm/s) and freeze behavior (n = 2078). F, A GLM was again used to identify freeze cells, but with the latter half of freeze bouts excluded (“training data” = modeled freeze bouts; “testing data” = excluded freeze bouts). To assure that no single calcium event appeared in both training and testing sets, only freeze bouts from the testing set that were separated by at least 10 s from those of the training set were used. For each freeze and nonfreeze cell classified in this manner, we quantified the percent of freeze bouts that were accompanied by calcium events per session (±3 s from behavior onset). This was done separately for training and testing sets. Bars show this mean percent measure for (±1 SEM) freeze and nonfreeze cells, across both training and testing sets. G, Trace of the mean freeze cell dF/F (±1 SEM) for freeze bouts from the “testing data” (n = 13,756 freeze aligned dF/F traces; for details, see F and Materials and Methods). H, Data from an example cell categorized as both freeze and place encoding. Figures show (top) the rate map for an example session and (bottom) activity for the same session with freeze behavior highlighted in green and times in which the mouse traversed the place field in yellow. I, Pie charts show (top left) the percentage of all local grid cells categorized as freeze cells per session, (top right) the percentage of all local grid cells categorized as place cells per session, (bottom left) the percentage of these freeze cells categorized as place cells per session, and (bottom right) the percentage of place cells categorized as freeze cells per session (n = 16); *p < 0.05, ***p < 0.001.
Figure 8.
Firing properties of freeze cells that were also categorized as place cells across epochs of the local grid assay. Bars show the change in (A) mean event rate (B), place field size, and (C) spatial information content of pooled place cells (±1 SEM) that are categorized as freeze cells in baseline (BL) and coregister between BL and extinction (Ext) epochs 1, 2, or 3 (n = 194 baseline/Ext 1; n = 183 baseline/Ext 2; n= 181 baseline/Ext 3). Statistical comparisons are performed by Wilcoxon signed-rank test for paired cells at baseline/Ext 1, baseline/Ext 2, and baseline/Ext 3; *p < 0.05.
Figure 9.
Freeze cells encode freeze behavior rather than freeze location. A, top, Example traces of freeze cells in the local grid assay that were also categorized as place cells and (middle) mouse speed. Bottom, Color-coded ovals depict place field centers corresponding to traces (top) of matching color. B, Rows depict the rate maps of all freeze-activated place cells in all local grid sessions along the length of the enclosure, sorted by place field center location (n = 1264). C, Bar depicts the mean distance (±1 SEM) of freeze-activated place cell field centers from freeze location for all local grid recording sessions (n = 5268 freeze-activated place cell instances). D, top, Example traces of freeze cells activated during freeze bouts at multiple locations within the enclosure and (middle) mouse speed. Color-coded bouts correspond to the enclosure quadrant (bottom) in which they occurred. E, For sessions in which freeze bouts occurred in more than one quadrant of the enclosure (D, bottom), 90% of all freeze cells were active during freeze bouts across multiple quadrants (n = 309 freeze cell instances; see Materials and Methods).
Figure 10.
Freeze cell activity is distinct from pSWR activity. A, Example recording of (top) freeze cells and cells that contribute to pSWR activity, (middle) the corresponding mouse speed, and (bottom) the number of active neurons per sample, with the 3-SD threshold shown for pSWR categorization. B, Bars compare dF/F (±1 SEM) of all cells (left) and the number of coactive neurons (right) during all freeze bouts and pSWR events (n = 1616 freeze bouts, n = 8378 pSWR events). C, Pie chart depicts the fraction of pSWR events that occurred during freeze bouts (3.53 ± 0.99%), periods of low speed locomotion (<4 cm/s; 8.52 ± 3.54%), high speed (>4 cm/s; 1.96 ± 1.27%), and non-locomotive, nonfreeze samples (85.99 ± 4.18%; n = 8); ***p < 0.001.
References
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