Memory trace interference impairs recall in a mouse model of Alzheimer’s disease

Abstract

In Alzheimer’s disease (AD), hippocampus-dependent memories underlie an extensive decline. The neuronal ensemble encoding a memory, termed engram, is partially recapitulated during memory recall. Artificial activation of an engram can restore memory in a mouse model of early AD, but its fate and the factors that render the engram nonfunctional are yet to be revealed. Here, we used repeated two-photon in vivo imaging to analyze fosGFP transgenic mice (which express enhanced GFP under the Fos promoter) performing a hippocampus-dependent memory task. We found that partial reactivation of the CA1 engram during recall is preserved under AD-like conditions. However, we identified a novelty-like ensemble that interfered with the engram and thus compromised recall. Mimicking a novelty-like ensemble in healthy mice was sufficient to affect memory recall. In turn, reducing the novelty-like signal rescued the recall impairment under AD-like conditions. These findings suggest a novel mechanistic process that contributes to the deterioration of memories in AD.

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Fig. 1: Assessing fosGFP expression in vivo revealed two subpopulations of CA1 neurons under healthy and AD-like conditions.
Fig. 2: Presence of engram cells independent of memory.
Fig. 3: Interference of novelty-like cells with memory engram cells impairs recall.
Fig. 4: Inducing novelty-like activity impaired memory recall, whereas reducing it ameliorated memory impairment.

Data availability

The data that support the findings of this are available from the corresponding authors upon request.

Code availability

The code used for analyzing the data in the current study is available from the corresponding authors upon request.

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Acknowledgements

This work was supported by the DZNE, grants from the Deutsche Forschungsgemeinschaft (SFB 1089 C01 and B06), Centres of excellence in Neurodegeneration (CoEN 3018), the ERA-NET MicroSynDep and the MicroSchiz project. We thank P. Thevenaz and E. Meijering for the development of the ImageJ plugins stackreg and TurboReg. We acknowledge W. Wisden for providing the Addgene plasmids numbers 66794 and 66795. We thank B. Roth for providing the Addgene plasmid number 50475 and the Addgene viral prep number 44361. Addgene plasmid number 26973 was a gift from K. Deisseroth. We thank S. Wiegert, S. Remy, G. Petzold, R. Czajkowski and L. Ewell for helpful discussions on the manuscript, and thank the Light Microscope and Animal Facilities of DZNE for constant support.

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Authors

Contributions

Conceptualization: M.F. and S.P. Methodology: S.P. and L.C.J. Formal analysis: S.P., M.M., F.M. and J.W. Investigation: S.P., L.C.J., J.S., M.M. and E.A.G. Resources: L.Z., S.S., B.S., W.S.J. and D.E. Writing (original draft): M.F. and S.P. Writing (review and editing): M.F., S.P. and E.A.G. Visualization: S.P. Supervision: M.F. Funding acquisition: M.F.

Corresponding authors

Correspondence to Stefanie Poll or Martin Fuhrmann.

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The authors declare no competing interests.

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Peer review information Nature Neuroscience thanks Takashi Kitamura and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 In vivo fosGFP expression is associated with elevated neuronal activity.

a, Scheme of the experimental setting (left) and an exemplary two-photon in vivo image showing jRGECO1a and fosGFP expression (right). b, Fractions of jRGECO1a-expressing cells that are fosGFPpositive (fosGFP+) or -negative (fosGFP). c, Event frequency of fosGFP+ and fosGFP- neurons. Data in b and c from 1953 jRGECO1a-expressing cells in n = 4 mice. d, Experimental timeline to assess fosGFP expression and its underlying neuronal activity, approximated by the Ca2+ event frequency. CE, novel context exposure; CTR, control. ej, Exemplary two-photon images (left) and quantification (right) of UP (e, f), DOWN (g,h) and no change cells (i, j). k, l, Exemplary two-photon image of jRGECO1a-expressing CA1 neurons (k) and respective Ca2+ traces (l) of identified UP, DOWN and no change neurons marked in k. m, n, Ca2+ event frequency of UP, DOWN and no change neurons in the group of mice exposed to a novel context (CE, m) and those under continuous anesthesia (CTR, n). o, Experimental timeline to determine the onset of fosGFP expression upon contextual fear conditioning (cFC). p, Exemplary binary two-photon images (left) and quantification (right) of fosGFP expression onset (ON) and decay (OFF). Data in dn from n = 4 CE and n = 6 CTR mice; c, two-sided Mann-Whitney test; f,h,j, two-sided unpaired t-test; m, n, one-way ANOVA with Holm-Sidak’s multiple comparison test; NS(P > 0.05); *P < 0.05; **P < 0.01; ***P < 0.001 for exact P values see Supplementary Table 1. Data in f, h and j are presented as mean ± SEM; each data point depicts the value of an individual mouse. Data in c, m and n show the median (bold line), 25%- and 75%-quartiles (dashed lines), minimum and maximum values (upper and lower end of violins). Scale bars, 50 µm (a, right), 5 µm (e, g, i), 25 µm (k, right).

Extended Data Fig. 2 Characterization of fosGFP expressing neurons.

a, Coronal mouse brain section showing the analyzed part (framed) of dorsal CA1. b, Quantification of endogenous c-Fos-positive (c-Fos+) and fosGFP-positive (fosGFP+) neurons in wild-type and fosGFP mice, respectively, and APP/PS1 and fosGFP-APP/PS1 mice; data from n = 9 wild-type, n = 6 fosGFP, n = 5 APP/PS1 and n = 9 fosGFP-APP/PS1 mice. c, Representative confocal images of c-Fos+ and fosGFP+ neurons in CA1 of fosGFP and fosGFP-APP/PS1 mice; for each mouse, five brain slices were stained and analyzed (see d), with similar results. d, Fraction of endogenous c-Fos+ nuclei that were simultaneously positive for fosGFP; data from n = 3 fosGFP and n = 3 fosGFP-APP/PS1 mice. eg, Method to convert 8-bit raw data into binary images. Exemplary measurement of background fluorescence intensity (BG) in a fosGFP raw data image with manually placed circular masks (red, 7.45 μm) (e). Exemplary fosGFP raw data (f, left) and corresponding binary image (f, right). Circular masks (white) were manually placed above putatively fosGFP expressing nuclei to measure their fluorescence intensity (f, left). Nuclei with a fosGFP fluorescence intensity above or below threshold (TH) were defined as fosGFP+ or fosGFP-, respectively (g). h, Images showing the density of fosGFP+ neurons around MeXO4-stained Aß-plaques (yellow) and randomly placed virtual Aß-plaques in fosGFP-APP/PS1 and fosGFP mice, respectively; 250 ×250 μm excerpts of a 1.9 ×1.9 mm field of view; images were acquired once for each mouse; other excerpts show similar results. i, j, Density of fosGFP+ neurons in CA1 irrespective of Aß plaque distance (i), and in the proximity (<50 µm, near) or distant (>50 µm, far) to MeXO4-stained Aß-plaques (j) comparing fosGFP-APP/PS1 and fosGFP mice, respectively. k, Exemplary images of fosGFP-expressing neurons’ fluorescence intensities in CA1 of fosGFP and fosGFP-APP/PS1 mice; 250 ×250 μm excerpts of a 1.9 ×1.9 mm field of view; image was acquired once, other excerpts show similar results; white circles (radius = 50 µm) in h and k depict the area close to a MeXO4-positive Aβ-plaque (in fosGFP-APP/PS1 mice) or a virtual plaque (in fosGFP mice), respectively. l, m, Fluorescence intensity of fosGFP-expressing neurons in fosGFP and fosGFP-APP/PS1 mice regardless of Aß plaque distance (l), and in proximity (<50 µm, near) or distant (>50 µm, far) to MeXO4 stained Aß plaques (m); data in hm from n = 8 fosGFP and n = 6 fosGFP-APP/PS1 mice; statistics were done over mice, except for l, m; data in l are from 2873 (fosGFP) and 2092 (fosGFP-APP/PS1) cells; data in m are from 1373 (fosGFP – near), 674 (fosGFP-APP/PS1-near), 1500 (fosGFP – far) and 1418 (fosGFP-APP/PS1 – far) cells; d, i, two-sided unpaired t-test; j, two-way ANOVA with Holm-Sidak’s correction for multiple comparisons; l, two-sided Mann-Whitney test; m, Kruskal-Wallis test with Dunn’s correction for multiple comparisons; *P < 0.05, **P < 0.01, ***P < 0.001, for exact P values see Supplementary Table 1. Data in b, d, i, j are presented as mean ± SEM; each data point depicts the value of an individual mouse. Data in l and m show the median (bold line), 25%- and 75%-quartiles (dashed lines), minimum and maximum values (upper and lower end of violins). Scale bars, 40 µm (c), 20 µm (e,f), 50 µm (h, k).

Extended Data Fig. 3 Impaired learning and short-term memory in APP/PS1 mice.

a, Experimental protocol for fear conditioning in context A: two minutes of exploration (pre-shock) are followed by three 0.5 mA foot shocks spaced by one minute intervals. Mouse was removed from the chamber one minute after the last foot shock (see also methods section). b, Freezing behavior of wild-type and APP/PS1 mice during conditioning. c, Experimental paradigm for testing short term memory (STM). d, Freezing behavior of wild-type and APP/PS1 mice during STM test, one hour after conditioning. e, f, Fraction of c-Fos-positive (c-Fos+) per DAPI-positiv (DAPI+) neurons (e) and density of DAPI+ neurons upon STM test (f). a, b, Data from n = 17 wild-type and n = 18 APP/PS1 mice; cf, data from n = 4 wild-type and n = 4 APP/PS1 transgenic mice; b, two-way ANOVA with Holm-Sidak’s correction for multiple comparisons; d, f, two-sided unpaired t-test; e, two-sided Mann-Whitney test; **P < 0.01, for exact P values see Supplementary Table 1. Data are presented as mean ± SEM; each data point depicts the value of an individual mouse.

Extended Data Fig. 4 ON cells upon memory recall.

Inter-group comparison of the fold change of ON cells upon memory recall (d3–4 in A-A/B), corresponding to Fig. 1m–p. Data according to Fig. 1, n = 7 fosGFP A-A (G1), n = 6 fosGFP-APP/PS1 A-A (G2), n = 6 fosGFP A-B (G3) and n = 3 fosGFP-APP/PS1 A-B (G4) mice; data include BL and A-A/B imaging, comprising measurements from 4322 (G1), 5099 (G2), 3755 (G3) and 2195 (G4) cells, respectively; statistics were done over mice; two-way ANOVA with Holm-Sidak’s multiple comparisons test; NS (P > 0.05), for exact P values see Supplementary Table 1. Data are presented as mean ± SEM; each data point depicts the value of an individual mouse.

Extended Data Fig. 5 Optogenetic CA1 memory trace activation induces recall in APP/PS1 mice.

a, AAV-c-Fos-tTA and AAV-PTRE-tight-hChR2-EYFP were injected bilaterally into the hippocampus of APP/PS1 mice and wild-type littermates (left) before optical fibers were implanted to target CA1 (right). b, Experimental timeline. c, Representative confocal images showing hChR2-EYFP expression in wild-type (upper) and APP/PS1 mice (lower), respectively; for each mouse, four brain slices were stained and analyzed (see i, j), with similar results. d, e, Freezing during the stimulation protocol in context C, before (control, black) and after tagging (hChR2, green) of the same set of APP/PS1 (d, left) and wild-type mice (e, left). Average freezing during intervals without (off) and with (on) light stimulation in hChR2-tagged APP/PS1 (d, right) and wild-type (e, right) mice, respectively. f, g, Freezing during test I (f) and test II (g) comparing wild-type and APP/PS1 mice. h, Comparison of net freezing during light stimulation (on - off, average freezing during light on periods minus average freezing during light off periods) between wild-type and APP/PS1 mice. i, j, Density of hChR2-positive (hChR2+) cells regardless of Aß plaque distance (i), and in proximity (<50 µm, near) or distant (>50 µm, far) to MeXO4 stained Aß plaques in wild-type and APP/PS1 mice (j). Data from n = 6 wild-type and n = 8 APP/PS1 mice; d (right), e (right), two-sided paired t-test; fj, two-sided unpaired t-test; ****P < 0.0001, ***P < 0.001, for exact P values see Supplementary Table 1. Data are presented as mean ± SEM; each data point depicts the value of an individual mouse.

Extended Data Fig. 6 Mimicking novelty−like activity in CA1 impaired recall performance.

a, b, Experimental approach. c, d, Overview (left) and zoom (right) of exemplary confocal images of coronal brain sections showing CA1 targeted expression of hM3D(Gq)-mCherry and c-Fos in vehicle- (c) and CNO-treated mice (d); for each mouse, four brain slices were stained and analyzed (see e), with similar results. e, Density of c-Fos-positive (c-Fos+) neurons in vehicle- and CNO-treated mice; the value of each mouse represents an average of four analyzed brain slices. f, Freezing behavior of vehicle- and CNO-treated mice. Data from n = 5 CNO-treated and n = 4 saline-treated mice; e, f, two-sided unpaired t-test; ****P < 0.0001, *P < 0.05, for exact P values see Supplementary Table 1. Data are presented as mean ± SEM; each data point depicts the value of an individual mouse. Scale bars, 500 µm (c, left), 50 µm (c, right).

Extended Data Fig. 7 Presence of doxycycline prevents DREADD expression.

a, b, Experimental approach. c, Density of c-Fos-positive (c-Fos+) neurons after memory retrieval; the value of each mouse represents an average of four analyzed brain slices. d, Freezing behavior of non-treated and non-labeled mice during recall on d8 (test). e, Overview (left) and zoom (right) of an exemplary confocal image of a coronal brain sections showing CA1 lacking expression of hM3D(Gq)-mCherry; for each mouse, four brain slices were stained and analyzed (see c), with similar results. Data from n = 5 wild-type mice. Data are presented as mean ± SEM; each data point depicts the value of an individual mouse. Scale bars, 500 µm (e, left), 50 µm (e, right).

Extended Data Fig. 8 Reduced endogenous c-Fos expression in hM4D(Gi)-positive CA1 pyramidal neurons.

a,b, Scheme of the experimental setting (a) and timeline (b) for reducing noise-like activity (ensemble of neurons active in context B) during memory recall of the conditioned context A (see Fig. 3g–k). c, d, Intensity distribution of endogenous c-Fos measured in the nuclei of hM4D(Gi)-mCherry-negative (mCherry-) and hM4D(Gi)-mCherry-positive (mCherry+) cells in both APP/PS1 (c) and wild-type mice (d). e, Quantification of hM4D(Gi)-mCherry expression in wild-type and APP/PS1 mice. Data from n = 5 wild-type and n = 3 APP/PS1 mice; c, d, two-sided Mann-Whitney test; e, two-sided unpaired t-test; NS (P > 0.05); ****P < 0.0001, for statistical details and exact P values see Supplementary Table 1. Data in c and d show the median (bold line), 25%- and 75%-quartiles (dashed lines), minimum and maximum values (upper and lower end of violins). Data in e are presented as mean ± SEM; each data point depicts the value of an individual mouse.

Extended Data Fig. 9 CNO alone has no effect on exploratory behavior or memory recall.

a, Travelled distance in context B (left) and freezing in the conditioned context A (right) comparing vehicle- and CNO-treated wild-type mice expressing hM4D(Gi). Data in a from n = 4 vehicle- and n = 8 CNO-treated wild-type mice. b, Travelled distances in context B comparing wild-type and APP/PS1 mice expressing hM4D(Gi) (left) or lacking DREADD expression (right); data in b (left) from n = 8 wild-type and n = 10 APP/PS1 mice; data in b (right) from n = 4 wild-type and n = 4 APP/PS1 mice. c, Experimental timeline (left). Travelled distance in context B (middle) and freezing in the conditioned context A (right) comparing vehicle- and CNO-treated wild-type mice. Data in c from n = 6 vehicle-treated and n = 7 CNO-treated wild-type mice. a (left), two-sided Mann-Whitney test; a (right), b, c, two-sided unpaired t-test; NS (P > 0.05), for exact P values see Supplementary Table 1. Data are presented as mean ± SEM; each data point depicts the value of an individual mouse.

Extended Data Fig. 10 AAV injection and DREADD expression alone have no effect on memory recall.

a, b, Experimental setup (a) and timeline (b) to determine the influence of either hM4D(Gi) expression or sham injection (just AAV-c-Fos-tTA) on memory recall. c, Exemplary confocal images showing hM4D(Gi)-mCherry expression in groups A, B and C; for each mouse, four brain slices were stained, with similar results. d, Freezing behavior of groups A, B and C during memory test. Data from n = 6 group A, n = 6 group B and n = 6 group C mice. d, one-way ANOVA with Holm-Sidaks multiple comparison test; NS (P > 0.05), for exact P values see Supplementary Table 1. Data are presented as mean ± SEM; each data point depicts the value of an individual mouse. Scale bars, 500 µm (c, left), 50 µm (c, right).

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Poll, S., Mittag, M., Musacchio, F. et al. Memory trace interference impairs recall in a mouse model of Alzheimer’s disease. Nat Neurosci (2020). https://doi.org/10.1038/s41593-020-0652-4

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