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A shared neural ensemble links distinct contextual memories encoded close in time

Abstract

Recent studies suggest that a shared neural ensemble may link distinct memories encoded close in time1,2,3,4,5,6,7,8,9,10,11,12. According to the memory allocation hypothesis1,2, learning triggers a temporary increase in neuronal excitability13,14,15 that biases the representation of a subsequent memory to the neuronal ensemble encoding the first memory, such that recall of one memory increases the likelihood of recalling the other memory. Here we show in mice that the overlap between the hippocampal CA1 ensembles activated by two distinct contexts acquired within a day is higher than when they are separated by a week. Several findings indicate that this overlap of neuronal ensembles links two contextual memories. First, fear paired with one context is transferred to a neutral context when the two contexts are acquired within a day but not across a week. Second, the first memory strengthens the second memory within a day but not across a week. Older mice, known to have lower CA1 excitability15,16, do not show the overlap between ensembles, the transfer of fear between contexts, or the strengthening of the second memory. Finally, in aged mice, increasing cellular excitability and activating a common ensemble of CA1 neurons during two distinct context exposures rescued the deficit in linking memories. Taken together, these findings demonstrate that contextual memories encoded close in time are linked by directing storage into overlapping ensembles. Alteration of these processes by ageing could affect the temporal structure of memories, thus impairing efficient recall of related information.

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Figure 1: Calcium imaging CA1 with integrated miniature microscopes while exploring different contexts.
Figure 2: Tagging neural ensembles of contextual memories with the TetTag system.
Figure 3: Memories are contextually linked but distinct.
Figure 4: Age-related deficits in memory linking are rescued by ensemble activation.

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Acknowledgements

We thank B. Khakh for support in the development of the miniaturized microscopes. We thank E. Thai, D. Tarzi, A. Ahuja, K. Lew, E. Lu, E. Stuart, S. Zhang, S. Ghiaee, C. Yang, A. Fariborzi, K. Cheng, N. Rao, A. Chang, C. Grimmick and M. Einstein for help with experiments; N. Rao for assistance with graphical design; and all members of the Silva laboratory for their support. This work was supported by National Institute on Aging R37 AG013622 and the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation to A.J.S.; National Institutes of Health RO1 MH101198, 1U54 HD087101 and VA Merit Award BX00152401A1 to P.G.; National Research Service Award F32 MH97413 and Behavioral Neuroscience Training Grant T32 MH15795 to D.J.C.; Neurobehavioral Genetics Training Grant T32 NS048004 and Neural Microcircuits Training Grant T32 NS058280 to D.A.; Cellular Neurobiology Training Grant T32 NS710133 and Epilepsy Foundation Postdoctoral Research Training Fellowship to T.S.; National Institutes of Health U01 NS094286-01 and David Geffen School of Medicine Dean’s Fund for development of open-source miniaturized microscopes to A.J.S. and P.G.

Author information

Authors and Affiliations

Authors

Contributions

D.J.C., J.S., T.S., D.A. and A.J.S. contributed to the study design. D.A., T.S., D.J.C., P.G. and A.J.S. developed the miniature microscope system. D.A. engineered hardware and software associated with the miniature microscope and wrote the MATLAB analysis suite. T.S., D.J.C., W.S., J.S., S.E.F., J.L. and I.K. performed surgeries. D.J.C., T.S., M.L., W.S. and B.W. conducted calcium imaging and TetTag experiments. M.M. engineered and provided TetTag mice. D.J.C., J.S., T.S., M.L., W.S., B.W., M.V. and M.Z. conducted behavioural experiments. D.J.C., D.A., T.S. and A.L. analysed the data. J.B., D.J.C. and T.S. conducted in vitro physiology experiments. M.T. supported physiology experiments. Y.S. and M.K. made DREADD virus. D.J.C., I.K., B.W. and K.B. managed the mouse colony. D.J.C., T.S., D.A., J.S. and A.J.S. wrote the paper. All authors discussed and commented on the manuscript.

Corresponding authors

Correspondence to Peyman Golshani or Alcino J. Silva.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Stability of fluorescence and overlap.

a, Average normalized mean fluorescence within session. There was no difference between the mean fluorescence across the 3 sessions (one-way repeated measures ANOVA, F2,7 = 0.423, not significant). b, Average normalized mean fluorescence within session. There was no difference between the mean fluorescence across a 10-min session (one-way repeated measures ANOVA, F9,22 = 1.108, not significant). Results show mean ± s.d. c, Higher ensemble overlap with 5 h interval than 7 days. Normalized ensemble overlap is calculated as the ensemble overlap between contexts separated by 5 h divided by the ensemble overlap between contexts separated by 7 days. A normalized overlap value of 1 signifies that there is no difference between the overlap at 5 h and 7 days. The minimum number of calcium events required from each cell for the cell to be considered ‘active’ (inclusion criteria) was systematically increased and the ratio of the ensemble overlap for the different context was calculated. For all inclusion criteria, there is higher ensemble overlap with a 5 h, rather than 7 day, interval (one-sample t-test against 1, (1) t7 = 3.00, P = 0.02, (2) t7 = 2.57, P = 0.04, (3) t7 = 2.42, P = 0.04, (4) t7 = 2.50, P = 0.04, (5) t7 = 2.32, P = 0.05). Results show mean ± s.e.m.

Extended Data Figure 2 Neural ensembles of environments are reliably reactivated at recall of an open field and linear track.

a, Experimental design. Mice were imaged while exploring contexts A and B separated by 7 days and imaged while exploring contexts C and C separated by 7 days. b, There was a higher percentage of cells reactivated when animals explored the same context (C–C) than when animals explored different contexts (A–B) (paired t-test, t3 = 6.305, P = 0.0081, n = 4 mice). c, Mice were trained to run on a 2-m linear track with the miniature microscope for water rewards. Mice were trained 3 days a week for 3 weeks with a delay interval of 2–3 days between each session. Place fields were calculated by deconvolving calcium ΔF/F traces with an exponential to extract approximate spike times. Spikes that remained after crosstalk removal were included for analysis. Animal position was extracted using an automated LED tracking algorithm. A speed threshold (3 cm s−1) was applied to both the animal position and extracted spike timing and the resulting data was spatially binned (6.5-cm bins). Spatial firing rates were calculated by dividing the binned spike counts by the binned occupancy and smoothing with a Gaussian filter (sigma = 6.5 cm). Cells which showed consistent spatial firing modulation on at least three trials, with all other trials showing no bursting activity, were considered as place cells. Normalized spatial firing rates of all matched cells independently meeting the place cell criteria for both days. The data are pooled across 3 mice and include both motion directions. Place fields are ordered by centroid location on session 2. d, A shift of the image registration between sessions results in a decrease in matched place cells. A translational shift both horizontally and vertically was applied to the image registration transformation used in A. Cells were then matched across days and those which met our place cell criteria were kept. The heat map shows the count of matched place cells with a centroid shift of the place field that is less than 33 cm. Optimum matching of cells occurred within a 1-pixel translation of the calculated alignment transformation. e, Distribution of centroid shifts of place fields shown in A compared to the null hypothesis that the cell matching between sessions matches random cells.

Extended Data Figure 3 Five hours after exploration of a context, GFP expression is shut off by doxycycline and excitability is increased.

a, Experimental design. Mice were removed from low levels of dox (40 mg kg−1) and given regular chow for 3 days to open up the GFP tagging window. After receiving administration of high dox (1 g kg−1) for 5 h, mice were injected with 30 mg kg−1 of pentylenetetrazole (PTZ), exposed to a novel context or left in their home cage (HC). An hour later, mice were transcardially perfused and processed for GFP expression. b, There was no difference in GFP expression between the three groups (one-way ANOVA, F2,5 = 0.04, not significant, n = 3, 3, 2 mice), demonstrating that 5 h was enough time for dox (1 g kg−1) to suppress expression of new GFP. c, To test excitability learning-related excitability changes, mice explored a novel context and then were administered high dox to shut off new GFP. Five hours later, mice were euthanized for in vitro slice physiology. d, A two-way repeated measures ANOVA (group × current step) had a significant main effect of group (F2,68 = 4.20, P < 0.05, n = 21, 29, 21 cells). The 5 h GFP+ group had more spikes than the 5 h GFP group (t68 = 2.31, P < 0.05) and home cage GFP (t68 = 2.72, P < 0.05). There was no difference between the 5 h GFP and home cage GFP groups (t68 = 0.61, not significant). Results show mean ± s.e.m.

Extended Data Figure 4 Time course for neuronal overlap and behavioural linking.

a, Design for Ca2+ imaging of neuronal overlap experiment. b, There was a significant difference in overlap across groups (one-way repeated measures ANOVA, F2,12 = 12.43, P = 0.002, n = 7 mice). There was more overlap at 5 h than 2 days (t12 = 3.03, P = 0.01) and 7 days (t12 = 4.72, P = 0.0005). c, Design for transfer of fear experiment. d, There was a significant difference in freezing across groups (one-way ANOVA, F2,43 = 3.55, P = 0.04, n = 20, 14, 12 mice). There was more freezing at 5 h than 2 days (t43 = 2.13, P = 0.04) and 7 days (t43 = 2.31, P = 0.03). e, Design for enhancement experiment. f, There was a significant difference in freezing across groups (one-way ANOVA, F2,45 = 6.38, P = 0.004, n = 22, 14, 12 mice). There was more freezing at 5 h than 2 days (t45 = 2.45, P = 0.02) and 7 days (t45 = 3.32, P = 0.002). Results show mean ± s.e.m.

Extended Data Figure 5 Calcium imaging during retrieval.

a, Design for Ca2+ imaging of neuronal overlap at retrieval. Order of contexts during retrieval was counterbalanced. b, There was higher overlap of the neuronal ensemble at 5 h than 7 days (paired t-test, t7 = 2.55, P = 0.04, n = 8 mice). Results show mean ± s.e.m.

Extended Data Figure 6 Replication of memory linking experiments in young (3–6 months old) C57Bl/6NIA mice.

a, Design for transfer of fear experiment. b, There was a significant difference in freezing across the groups (one-way ANOVA, F2,20 = 9.49, P = 0.001, n = 8, 7, 8 mice). There was no difference between freezing levels in context C or B (t20 = 0.99, not significant). Animals had less freezing in context D than C (t20 = 4.19, P = 0.0004) and B (t20 = 3.06, P = 0.006). c, Design for enhancement experiment. d, There was a significant difference in freezing (one-way ANOVA, F2,46 = 4.071, P = 0.023, n = 16, 17, 16 mice). The 5 h group had more freezing than the home cage (HC) group (t46 = 2.72, P = 0.0278) and 7 day group (t46 = 2.612, P = 0.012). There was no difference between home cage or 7 day groups (t46 = 0.335, not significant). Results show mean ± s.e.m.

Extended Data Figure 7 Exploring the same context twice enhances memory regardless of time.

a, Experimental design. b, There was a significant difference in freezing (one-way ANOVA, F3,44 = 2.92, P = 0.04, n = 10, 11, 13, 14 mice). Consistent with the prior experiment, there was more freezing in the 5 h BC than the 7 day BC group (t44 = 2.19, P < 0.05). The 7 day BC group also had more freezing than the 5 h CC (t44 = 2.35, P < 0.05) and 7 day CC (t44 = 2.48, P < 0.05) groups, however there were no difference between the 5 h CC and 7 day CC (t44 = 0.06, not significant) and 5 h CC and 5 h BC (t44 = 0.31, not significant) groups. Results show mean ± s.e.m.

Extended Data Figure 8 NMDA receptor activity is required for overlap of neural ensembles and behavioural enhancement.

a, Design for Ca2+ imaging of neuronal overlap with MK-801 or saline. b, There was no difference in the number of cells active during exploration of the first context between saline-injected (SAL) and MK-801 groups (unpaired t-test, t6 = 0.58, not significant, n = 4, 4). c, There was lower overlap of the neuronal ensemble in the MK-801 group than in the SAL group (paired t-test, t3 = 3.45, P = 0.04, n = 4 mice). d, Design for behavioural enhancement experiment. e, There was lower freezing in the MK-801 than in the SAL group (unpaired t-test, t22 = 2.65, P = 0.015, n = 12, 12 mice). f, Design for behavioural control experiment. g, There was no difference in freezing between SAL and MK-801 groups (unpaired t-test, t22 = 0.22, not significant, n = 12, 12 mice). Results show mean ± s.e.m.

Extended Data Figure 9 Control experiments for aged mice.

a, Design for experiment of recall for single contextual experience. b, There was no difference in reactivation of cells between young and old mice during recall (unpaired t-test, t6 = 0.59, not significant, n = 4, 4 mice). c, Design for experiment with single context pre-exposure in young and old mice. d, There was no difference in freezing behaviour to exposures of a single context (unpaired t-test, t29 = 0.24, not significant, n = 16, 15 mice). e, Design for replication of TetTag experiment in old mice. f, There was no difference in the levels of overlapping ensembles between the 5 h and 7 day groups (unpaired t-test, t6 = 0.06, not significant, n = 3, 5 mice). Results show mean ± s.e.m.

Extended Data Figure 10 CNO activates cells with DREADD receptors and does not increase anxiety in aged mice.

a, Mice infected with DREADD virus in CA1 were injected with saline (SAL) or clozapine-N-oxide (CNO) and then euthanized 90 min post-injection for immunofluorescence staining. b, There was no difference in the percentage of DREADD-positive cells (labelled with GFP) between SAL and CNO groups (unpaired t-test, t7 = 0.01, not significant, n = 3, 6 mice). c, DREADD-positive cells (labelled with GFP) had more ZIF when injected with CNO than SAL (unpaired t-test, t7 = 5.08, P = 002). d, Representative examples of ZIF, DREADD, DAPI as well as merged images of CA1. e, Design for elevated plus maze experiment in aged mice with DREADD virus. f, A two-way ANOVA showed no main effect of injection (F1,9 = 0.75, not significant, n = 6, 5 mice) and a significant main effect of arms (F1,9 = 71.03, P < 0.0001). There was no significant interaction between injection and arms (F1,9 = 0.003, not significant). Results show mean ± s.e.m.

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Cai, D., Aharoni, D., Shuman, T. et al. A shared neural ensemble links distinct contextual memories encoded close in time. Nature 534, 115–118 (2016). https://doi.org/10.1038/nature17955

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