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Memory recall and modifications by activating neurons with elevated CREB

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Abstract

Memory is supported by a specific ensemble of neurons distributed in the brain that form a unique memory trace. We previously showed that neurons in the lateral amygdala expressing elevated levels of cAMP response-element binding protein are preferentially recruited into fear memory traces and are necessary for the expression of those memories. However, it is unknown whether artificially activating just these selected neurons in the absence of behavioral cues is sufficient to recall that fear memory. Using an ectopic rat vanilloid receptor TRPV1 and capsaicin system, we found that activating this specific ensemble of neurons was sufficient to recall established fear memory. Furthermore, this neuronal activation induced a reconsolidation-like reorganization process, or strengthening of the fear memory. Thus, our findings establish a direct link between the activation of specific ensemble of neurons in the lateral amygdala and the recall of fear memory and its subsequent modifications.

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Figure 1: CREB and TRPV1 was successfully coexpressed in a subset of lateral amygdala neurons by HSV-mediated gene transfer.
Figure 2: Capsaicin increased the firing rate in the putative ectopic TRPV1-expressing neurons in the lateral amygdala.
Figure 3: Fear memory was recalled by activating lateral amygdala neurons expressing elevated level of CREB.
Figure 4: Fear memory was rendered labile after activating CREB neurons.
Figure 5: Phosphorylation of GluA1at Ser845 was induced by activating neurons expressing elevated level of CREB.
Figure 6: Fear memory was strengthened after multiple reactivations of neurons expressing elevated CREB.

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  • 12 December 2013

    In the version of this article initially published online, the P value in Figure 4a was given as **P < 0.01 instead of *P < 0.05 and the y axis of Figure 1e was labeled “Colocalized cells (%)” instead of “Normalized TRPV1.” The errors have been corrected for the print, PDF and HTML versions of this article.

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Acknowledgements

We thank all our laboratory members for critical discussions and helpful comments on this study and U. Oh (Seoul National University) for rat TRPV1 cDNA plasmid. This work was supported by a National Research Foundation of Korea grant funded by the Korean government (2011-0005755 and 2011-0013173) and the KAIST High Risk High Return Project. J.-H.H. was supported by a TJ Park Science Fellowship, J.K. was supported by the BK21 project of Korean Research Foundation, J.-T.K. was supported by the project of Global Ph.D. Fellowship that National Research Foundation of Korea conducts from 2011, and H.-S.K. was supported by the World Class Institute program of the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (WCI 2009-003).

Author information

Authors and Affiliations

Authors

Contributions

J.-H.H. designed and directed the study. J.K. and J.-H.H. designed the HSV vector and the imaging, western blot and memory reconsolidation experiments. S.A.J. and J.-H.H. designed the memory strengthening experiment. J.-T.K. and J.-H.H. designed the in vivo recording and memory recall experiments. J.K. performed HSV vector construction, virus packaging, imaging, western blot and the memory reconsolidation experiment. J.K. and H.-S.K. performed the memory strengthening experiment. J.-T.K. performed in vivo recording and the memory recall experiment. J.K. and J.-H.H. analyzed the imaging, western blot and memory reconsolidation experiments. J.-T.K. and J.-H.H. analyzed the in vivo recording and memory recall experiments. J.K., H.-S.K. and J.-H.H. analyzed the memory strengthening experiments. J.K., J.-T.K., H.-S.K., S.A.J. and J.-H.H. wrote the manuscript.

Corresponding author

Correspondence to Jin-Hee Han.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 HSV viral vector constructs infected similar percentage of LA neurons.

(a) Representative confocal images showing restricted expression of the transgene in the LA (outlined with a dotted line) by HSV-mediated gene transfer. GFP was used as an expression marker. Scale bar = 200 ÎĽm. (b) Histogram of cell counting result. The percentage number of cell expressing GFP in LA for each group is shown. HSV constructs used in this study infected similar percentages (approximately 15% to 20%) of cells in the LA with no significant differences among groups (n = 9, GFP; n = 11, CREB; n = 9, GFP-TR; n = 19, CREB-TR).

Supplementary Figure 2 Freezing was not induced by drug in mice trained with unpaired conditioning protocol.

Behavior procedure and histogram showing the percentage of freezing induced by drug. All mice were injected with CREB-TR vector. During training, shock (0.5 mA) was delivered first and 1 minute later tone was presented for 30 seconds. One group of mice was infused with capsaicin (n = 8) for drug-induced freezing test and the other group of mice infused with vehicle (n = 9) as a control.

Supplementary Figure 3 Activating a subset of random neurons in LA by drug did not induce freezing in mice with strong fear memory or elevated CREB.

(a) Mice in GFP-TR group (Fig. 3) were divided into two subgroups, Strong (>50% freezing, n = 3) and Weak (<50% freezing, n = 5), according to their freezing level in the auditory CS retention test. Although CS-induced freezing was much greater in the Strong group (82.4% ± 4.8%) than in the Weak group (27.5% ± 6.8%), mice in the Strong group did not exhibit significant drug-induced freezing and there was no significant difference in capsaicin-induced freezing between Strong (5.3% ± 2.6%) and Weak (5.0% ± 1.7%) groups. The freezing during 10–15 minutes period (Fig. 3b) was used for analysis. (b) Behavior procedure and histogram showing the percentage of freezing induced by drug (5-minute bins). In this experiment, GFP-TR vector was first injected and 2 days later CREB alone vector injected again within the LA but at slightly different site. During testing, one group of mice was administered capsaicin (n = 5) and the other group of mice vehicle (n = 4) as a control.

Supplementary Figure 4 Reconsolidation process was intact in viral vector injected mice.

(a) Behavior experimental procedure. Mice were tested before (Test 1) and 24 hours after (Test 2) anisomycin or vehicle infusion. During test session, an auditory CS was presented and CS-induced freezing was monitored. (b) Histograms showing behavioral results. Mice were injected with either CREB-TR (n = 12 for VEH and n = 7 for ANI) or GFP-TR (n = 4 for VEH and n = 8 for ANI) viral vectors and trained for auditory fear conditioning. **P > 0.01, ***P > 0.001.

Supplementary Figure 5 Anisomycin treatment after control vehicle injection instead of capsaicin had no amnesic effect on auditory fear memory.

(a) Behavior experimental procedure. Twenty-four hours after training, vehicle was administered, followed by anisomycin or vehicle treatment. (b) Histogram showing behavioral results. Mice were injected with either CREB-TR (n = 12 for VEH and n = 9 for ANI) or GFP-TR (n = 4 for VEH and n = 4 for ANI) viral vectors and trained for auditory fear conditioning.

Supplementary Figure 6 No differences in the expression level of tGluA1 and pGluA1 across different groups (CREB-TR, GFP-TR and Post CREB-TR) with no drug or control vehicle injection.

(a) Experimental procedures for western blot analysis. Top: CREB-TR and GFP-TR groups. Bottom: Post CREB-TR group. Quantification of tGluA1 and pGluA1 protein expression in LA of mice with no injection (b) or vehicle injection (d) by western blots. Representative blot images (b, d) and histogram (c, e) showing normalized level of tGluA1 and pGluA1 protein expression (right) are shown. For the comparison, the expression level of tGluA1 and pGluA1 protein was normalized to control β-actin (n = 5 for all conditions). Full immunoblots are shown in Supplementary Figure 8.

Supplementary Figure 7 Fear memory was strengthened naturally by multiple reactivations of memory trace by CS under the conditions parallel to drug-induced reactivations.

(a) Behavior experimental procedure. (b) Histogram showing behavior results. The percentage of freezing before (pre CS) and after (CS) tone presentation during testing is shown. Mice were injected with CREB-TR viral vector and trained for auditory fear conditioning with relatively weak shock (0.4 mA). One group of mice (n = 7, 3 CS) were exposed to 10-minute CS tone at the homecage once per day over 3 days for memory reactivations. In control group (n = 10, No CS), all procedures were same as except no CS was presented during reactivation period. Baseline freezing (pre CS) was determined by measuring freezing for 2 minutes right before tone CS presentation. *P > 0.05.

Supplementary Figure 8 Full-length immunoblots of Fig. 1, 5 and sFig. 6

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Kim, J., Kwon, JT., Kim, HS. et al. Memory recall and modifications by activating neurons with elevated CREB. Nat Neurosci 17, 65–72 (2014). https://doi.org/10.1038/nn.3592

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