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Memory retrieval by activating engram cells in mouse models of early Alzheimer’s disease

Nature volume 531, pages 508–512 (2016)Cite this article

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Abstract

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive memory decline and subsequent loss of broader cognitive functions1. Memory decline in the early stages of AD is mostly limited to episodic memory, for which the hippocampus has a crucial role2. However, it has been uncertain whether the observed amnesia in the early stages of AD is due to disrupted encoding and consolidation of episodic information, or an impairment in the retrieval of stored memory information. Here we show that in transgenic mouse models of early AD, direct optogenetic activation of hippocampal memory engram cells results in memory retrieval despite the fact that these mice are amnesic in long-term memory tests when natural recall cues are used, revealing a retrieval, rather than a storage impairment. Before amyloid plaque deposition, the amnesia in these mice is age-dependent3,4,5, which correlates with a progressive reduction in spine density of hippocampal dentate gyrus engram cells. We show that optogenetic induction of long-term potentiation at perforant path synapses of dentate gyrus engram cells restores both spine density and long-term memory. We also demonstrate that an ablation of dentate gyrus engram cells containing restored spine density prevents the rescue of long-term memory. Thus, selective rescue of spine density in engram cells may lead to an effective strategy for treating memory loss in the early stages of AD.

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Figure 1: Optogenetic activation of memory engrams restores fear memory in early AD mice.
Figure 2: Neural correlates of amnesia in early AD mice.
Figure 3: Reversal of engram-specific spine deficits rescues memory in early AD mice.
Figure 4: Recovery of multiple types of HPC-dependent memories from amnesia in early AD.

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  • 23 March 2016

    The PDF was replaced to correct the presentation of Extended Data Figures 3 and 7.

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Acknowledgements

We thank X. Liu for the c-Fos-tTA construct; S. Huang, T. Okuyama and T. Kitamura for help with experiments; W. Yu, S. LeBlanc and X. Zhou for technical assistance; L. Brenner for proofreading; and all members of the Tonegawa laboratory for their support. We thank M. Luo for sharing the DTR coding sequence. This work was supported by the RIKEN Brain Science Institute, the Howard Hughes Medical Institute, and the JPB Foundation (to S.T.).

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Authors and Affiliations

  1. Department of Biology and Department of Brain and Cognitive Sciences, RIKEN-MIT Center for Neural Circuit Genetics at the Picower Institute for Learning and Memory, Massachusetts Institute of Technology, Cambridge, 02139, Massachusetts, USA

    Dheeraj S. Roy, Autumn Arons, Teryn I. Mitchell, Michele Pignatelli, Tomás J. Ryan & Susumu Tonegawa

  2. Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, 02139, Massachusetts, USA

    Autumn Arons, Tomás J. Ryan & Susumu Tonegawa

Authors
  1. Dheeraj S. Roy
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  2. Autumn Arons
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  3. Teryn I. Mitchell
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  4. Michele Pignatelli
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  5. Tomás J. Ryan
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  6. Susumu Tonegawa
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Contributions

D.S.R. and S.T. contributed to the study design. D.S.R., A.A., T.I.M., M.P. and T.J.R. contributed to the data collection and interpretation. D.S.R. cloned all constructs. D.S.R. and A.A. conducted the surgeries, behaviour experiments and histological analyses. D.S.R. and S.T. wrote the paper. All authors discussed and commented on the manuscript.

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Correspondence to Susumu Tonegawa.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Characterization of 7-month-old early AD mice.

a–d, Images showing hippocampal Aβ+ plaques lacking in control mice (a, b) and 7-month-old AD mice (c), which showed an age-dependent increase in 9-month-old AD mice (d). e, f, Images showing neuronal nuclei (NeuN) staining of DG granule cells in control (e) and 7-month-old AD (f) mice. g, NeuN+ fluorescence intensity of the granule cell layer from control and AD sections shown in e, f (n = 8 mice per group). h, i, Heat maps showing exploratory behaviour in an open field arena from control (h) and 7-month-old AD (i) mice. j, k, Distance travelled (j) and velocity (k) did not differ between control and AD groups (n = 9 mice per group). l, m, Images showing adult newborn neurons (DCX+) in DG sections from control mice (l) that are double positive for NeuN (m). n, Percentage of NeuN+ cells among DCX+ cells (n = 3 mice). o, p, Images showing DCX+ neurons in DG sections from control (o) and AD (p) groups (n = 4 mice per group). q, DCX+ cell counts from control and AD mice. Data are presented as mean ± s.e.m.

Extended Data Figure 2 Labelling and engram activation of early AD mice on DOX.

a, Mice are taken off DOX for 24 h in the home cage (HC) and subsequently trained in CFC. DG sections (n = 3 mice per group) revealed 2.05% ChR2–eYFP labelling in the home cage, consistent with the previously established engram tagging strategy11. b, Mice were injected with a virus cocktail of AAV9-c-Fos-tTA and AAV9-TRE-ChR2-eYFP. After 1 day off DOX, kainic acid was used to induce seizures. Image showing efficient labelling throughout the DG. c, ChR2–eYFP cell counts from DG sections shown in b (n = 3 mice). d, Behavioural schedule for optogenetic activation of DG engram cells. e, Memory recall 1 day after training (test 1) showed less freezing of AD mice compared with control mice (n = 8 mice per group). f, Engram activation with blue light stimulation (left). Average freezing for the two light-off and light-on epochs (right). Statistical comparisons are performed using unpaired t-tests; **P < 0.01. Data are presented as mean ± s.e.m.

Extended Data Figure 3 Chronic DG engram activation in early AD mice did not rescue long-term memory.

a, Behavioural schedule for repeated DG engram activation experiment. Ctx, context. b, AD mice in which a DG memory engram was reactivated twice a day for 2 days (AD + ChR2) showed increased STM freezing levels compared with memory recall before engram reactivation (ChR2-STM test, n = 9 mice per group). c, Memory recall 1 day after repeated DG engram activations (ChR2-LTM test). NS, not significant. Statistical comparisons are performed using unpaired t-tests; *P < 0.05, **P < 0.01. Data are presented as mean ± s.e.m.

Extended Data Figure 4 Engram activation restores fear memory in triple-transgenic and PS1/APP/tau models of early AD.

a, Triple-transgenic mouse line obtained by mating c-Fos-tTA transgenic mice11,28 with double-transgenic APP/PS1 AD mice10. These mice combined with a DOX-sensitive AAV virus permits memory engram labelling in early AD. b, Triple-transgenic mice were injected with AAV9-TRE-ChR2-eYFP and implanted with an optic fibre targeting the DG. c, Image showing DG engram cells of triple-transgenic mice 24 h after CFC. d, ChR2–eYFP cell counts from control and triple-transgenic AD mice (n = 5 mice per group). e, Behavioural schedule for engram activation. f, Memory recall 1 day after training (test 1) showed less freezing of triple-transgenic AD mice compared with control mice (n = 10 mice per group). g, Engram activation with blue light stimulation (left). Average freezing for the two light-off and light-on epochs (right). h, Triple-transgenic AD model (3×Tg-AD) as previously reported18. A cocktail of AAV9-c-Fos-tTA and AAV9-TRE-ChR2-eYFP viruses were used to label memory engrams in 3×Tg-AD mice. i, Image showing memory engram cells in the DG of 3×Tg-AD mice 24 h after CFC. j, ChR2–eYFP cell counts from DG sections of control and 3×Tg-AD mice (n = 4 mice per group). k, Behavioural schedule for engram activation. l, Memory recall 1 day after training (test 1) showed less freezing of 3×Tg-AD mice compared with control mice (n = 9 mice per group). m, Engram activation with blue light stimulation (left). Average freezing for the two light-off and light-on epochs (right). Statistical comparisons are performed using unpaired t-tests; *P < 0.05, **P < 0.01. Data are presented as mean ± s.e.m.

Extended Data Figure 5 Dendritic spines of engram cells in 7-month-old early AD mice.

a, Average dendritic spine density of DG engram cells showed an age-dependent decrease in 7-month-old APP/PS1 AD mice (n = 7,032 spines) as compared to 5-month-old AD mice (n = 4,577 spines, n = 4 mice per group). Dashed line represents spine density of control mice (1.21). b, Left, average dendritic spine density of CA3 engram cells in control (n = 5,123 spines) and AD mice (n = 6,019 spines, n = 3 mice per group). Right, average dendritic spine density of CA1 engram cells in control (n = 9,120 spines) and AD mice (n = 7,988 spines, n = 5 mice per group). NS, not significant. Statistical comparisons are performed using unpaired t-tests; **P < 0.01. Data are presented as mean ± s.e.m.

Extended Data Figure 6 High-fidelity responses of oChIEF+ cells and dendritic spines of DG engram cells after in vitro optical LTP.

a, EC cells were injected with a virus cocktail containing AAV9-TRE-oChIEF-tdTomato for activity-dependent labelling. b, Image showing a biocytin-filled oChIEF+ stellate cell in the EC. c, 100 Hz (2-ms pulse width) stimulation of an oChIEF+ cell across 20 consecutive trials. Spiking responses exhibit high fidelity. d, Average dendritic spine density of biocytin-filled DG cells showed an increase after optical LTP induction in vitro (n = 1,452 spines, n = 6 cells). Statistical comparisons are performed using unpaired t-tests; *P < 0.05. Data are presented as mean ± s.e.m.

Extended Data Figure 7 Behavioural rescue and spine restoration by optical LTP is protein-synthesis dependent.

a, Modified behavioural schedule for long-term rescue of memory recall in AD mice in the presence of saline or anisomycin (left). Memory recall 2 days after LTP induction followed by drug administration showed less freezing of AD mice treated with anisomycin (AD + 100 Hz + Aniso) compared with saline-treated AD mice (AD + 100 Hz + saline, n = 9 mice per group; right). Dashed line represents freezing level of control mice (48.53). Ctx, context. b, Average dendritic spine density in early AD mice treated with anisomycin after LTP induction (n = 4,810 spines) was decreased compared with saline-treated AD mice (n = 6,242 spines, n = 4 mice per group). Dashed line represents spine density of control mice (1.21). Statistical comparisons are performed using unpaired t-tests; *P < 0.05. Data are presented as mean ± s.e.m.

Extended Data Figure 8 Rescued early AD mouse behaviour in a neutral context and control mouse behaviour after in vivo optical LTP.

a, After the long-term rescue of memory recall in AD mice (test 2; Fig. 3m), animals were placed in an untrained neutral context to measure generalization (n = 10 mice per group). Rescued AD mice (AD + 100 Hz) did not display freezing behaviour. b, Left, average dendritic spine density of DG engram cells from control mice remained unchanged after optical LTP induction in vivo (control + 100 Hz, n = 4,211 spines, n = 3 mice; control data from Fig. 2c). Right, the behavioural rescue protocol applied to early AD mice (Fig. 3m) was tested in age-matched control mice (n = 9 mice per group). Similar freezing levels were observed after optical LTP (test 2) as compared to memory recall before the 100 Hz protocol (test 1). NS, not significant. Statistical comparisons are performed using unpaired t-tests. Data are presented as mean ± s.e.m.

Extended Data Figure 9 Optical LTP using a CaMKII-oChIEF virus did not rescue memory in early AD mice.

a, AAV virus expressing oChIEF-tdTomato under a CaMKII promoter. b, CaMKII-oChIEF virus injected into MEC and LEC. c, d, Images showing tdTomato labelling in a large portion of excitatory MEC neurons (c) as well as the PP terminals in the DG (d). e, In vivo optical LTP protocol23. f, Behavioural schedule for long-term rescue of memory recall in AD mice (left). In contrast to the engram-specific strategy, long-term memory could not be rescued by stimulating a large portion of excitatory PP terminals in the DG (right; n = 9 mice per group). NS, not significant. Statistical comparisons are performed using unpaired t-tests. Data are presented as mean ± s.e.m.

Extended Data Figure 10 Normal DG mossy cell density after engram cell ablation.

a–d, Images showing DG engram cells after saline treatment (a) and the corresponding calretinin positive (CR+) mossy cell axons (b). DTR–eYFP engram cell labelling after DT treatment (c) and the respective CR+ mossy cell axons (d). e, CR+ fluorescence intensity of mossy cell axons from saline- and DT-treated DG sections shown in a–d (n = 8 mice per group). Data are presented as mean ± s.e.m.

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Roy, D., Arons, A., Mitchell, T. et al. Memory retrieval by activating engram cells in mouse models of early Alzheimer’s disease. Nature 531, 508–512 (2016). https://doi.org/10.1038/nature17172

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