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Cell-type-specific drug-inducible protein synthesis inhibition demonstrates that memory consolidation requires rapid neuronal translation

Nature Neuroscience volume 23, pages 281–292 (2020)Cite this article

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Abstract

New protein synthesis is known to be required for the consolidation of memories, yet existing methods of blocking translation lack spatiotemporal precision and cell-type specificity, preventing investigation of cell-specific contributions of protein synthesis. Here we developed a combined knock-in mouse and chemogenetic approach for cell-type-specific drug-inducible protein synthesis inhibition that enables rapid and reversible phosphorylation of eukaryotic initiation factor 2α, leading to inhibition of general translation by 50% in vivo. We use cell-type-specific drug-inducible protein synthesis inhibition to show that targeted protein synthesis inhibition pan-neuronally and in excitatory neurons in the lateral amygdala (LA) impaired long-term memory. This could be recovered with artificial chemogenetic activation of LA neurons, although at the cost of stimulus generalization. Conversely, genetically reducing phosphorylation of eukaryotic initiation factor 2α in excitatory neurons in the LA enhanced memory strength but reduced memory fidelity and behavioral flexibility. Our findings provide evidence for a cell-specific translation program during consolidation of threat memories.

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Fig. 1: Generation of a chemogenetic resource for cell-type-specific protein synthesis inhibition.
Fig. 2: Drug-induced neuronal protein synthesis inhibition.
Fig. 3: Temporally structured protein synthesis is required for LTM consolidation.
Fig. 4: Cell-type-specific protein synthesis inhibition in principal neurons in the LA.
Fig. 5: Bidirectional control of memory strength by phosphorylation of eIF2α S51 in LA principal neurons.
Fig. 6: Artificial reactivation of LA principal neurons compromises the precision of a complex memory.

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Data availability

Sequence information for the targeting vector used to generate the iPKR knock-in mouse line is provided in Supplementary Data 1. Further data that support the findings of this study are available from the corresponding authors upon reasonable request.

References

  1. Tovote, P., Fadok, J. P. & Luthi, A. Neuronal circuits for fear and anxiety. Nat. Rev. Neurosci. 16, 317–31 (2015).

    CAS  PubMed  Google Scholar 

  2. Wheeler, A. L. et al. Identification of a functional connectome for long term fear memory in mice. PLoS Comput. Biol. 9, e1002853 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Clugnet, M. C. & LeDoux, J. E. Synaptic plasticity in fear conditioning circuits: induction of LTP in lateral nucleus of the amygdala by stimulation of the medial geniculate body. J. Neurosci. 10, 2818–2824 (1990).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Maren, S., Ferrario, C. R., Corcoran, K. A., Desmond, T. J. & Frey, K. A. Protein synthesis in the amygdala, but not the auditory thalamus, is required for consolidation of Pavlovian fear conditioning in rats. Eur. J. Neurosci. 18, 3080–3088 (2003).

    PubMed  Google Scholar 

  5. Flexner, L. B., Flexner, J. B., Roberts, R. B. & Delahaba, G. Loss of recent memory in mice as related to regional inhibition of cerebral protein synthesis. Proc. Natl Acad. Sci. USA 52, 1165–1169 (1964).

    CAS  PubMed  Google Scholar 

  6. Davis, H. P. & Squire, L. R. Protein synthesis and memory: a review. Psychol. Bull. 96, 518–559 (1984).

    CAS  PubMed  Google Scholar 

  7. Bourtchouladze, R. et al. Different training procedures recruit either one or two critical periods for contextual memory consolidation, each of which requires protein synthesis and PKA. Learn. Mem. 5, 365–374 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Hoeffer, C. A. et al. Inhibition of the interactions between eukaryotic initiation factors 4E and 4G impairs long-term associative memory consolidation but not reconsolidation. Proc. Natl Acad. Sci. USA 108, 3383–3388 (2011).

    CAS  PubMed  Google Scholar 

  9. Qi, Z. & Gold, P. E. Intrahippocampal infusions of anisomycin produce amnesia: contribution of increased release of norepinephrine, dopamine and acetylcholine. Learn. Mem. 16, 308–314 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Radulovic, J. & Tronson, N. C. Protein synthesis inhibitors, gene superinduction and memory: too little or too much protein? Neurobiol. Learn. Mem. 89, 212–218 (2008).

    CAS  PubMed  Google Scholar 

  11. Banko, J. L. et al. The translation repressor 4E-BP2 is critical for eIF4F complex formation, synaptic plasticity, and memory in the hippocampus. J. Neurosci. 25, 9581–9590 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Costa-Mattioli, M. et al. eIF2alpha phosphorylation bidirectionally regulates the switch from short- to long-term synaptic plasticity and memory. Cell 129, 195–206 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Jiang, Z. et al. eIF2alpha phosphorylation-dependent translation in CA1 pyramidal cells impairs hippocampal memory consolidation without affecting general translation. J. Neurosci. 30, 2582–2594 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Heumeller, M. et al. A genetically encodable cell type-specific protein synthesis inhibitor. Nat. Methods 16, 699–702 (2019).

    Google Scholar 

  15. Klann, E. & Dever, T. E. Biochemical mechanisms for translational regulation in synaptic plasticity. Nat. Rev. Neurosci. 5, 931–942 (2004).

    CAS  PubMed  Google Scholar 

  16. Sonenberg, N. & Hinnebusch, A. G. Regulation of translation initiation in eukaryotes: mechanisms and biological targets. Cell 136, 731–745 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Takei, N., Kawamura, M., Hara, K., Yonezawa, K. & Nawa, H. Brain-derived neurotrophic factor enhances neuronal translation by activating multiple initiation processes: comparison with the effects of insulin. J. Biol. Chem. 276, 42818–42825 (2001).

    CAS  PubMed  Google Scholar 

  18. Flores, M. V., Strawbridge, J., Ciaramella, G. & Corbau, R. HCV-NS3 inhibitors: determination of their kinetic parameters and mechanism. Biochim. Biophys. Acta 1794, 1441–1448 (2009).

    CAS  PubMed  Google Scholar 

  19. Gentile, I. et al. Asunaprevir, a protease inhibitor for the treatment of hepatitis C infection. Ther. Clin. Risk Manag. 10, 493–504 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Saelens, X., Kalai, M. & Vandenabeele, P. Translation inhibition in apoptosis. J. Biol. Chem. 276, 41620–41628 (2001).

    CAS  PubMed  Google Scholar 

  21. Butko, M. T. et al. Fluorescent and photo-oxidizing TimeSTAMP tags track protein fates in light and electron microscopy. Nat. Neurosci. 15, 1742–1751 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Donnelly, M. L. et al. The ‘cleavage’ activities of foot-and-mouth disease virus 2A site directed mutants and natually occuring ‘2A-like’ sequences. J. Gen. Virol. 82(Pt 5), 1027–1041 (2001).

    CAS  PubMed  Google Scholar 

  23. Klinakis, A. et al. Igf1r as a therapeutic target in a mouse model of basal-like breast cancer. Proc. Natl Acad. Sci. USA 106, 2359–2364 (2009).

    CAS  PubMed  Google Scholar 

  24. Doyle, J. P. et al. Application of a translational profiling approach for the comparative analysis of CNS cell types. Cell 135, 749–762 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Tronche, F. et al. Disruption of the glucocorticoid receptor gene in the nervous system results in reduced anxiety. Nat. Genet. 23, 99–103 (1999).

    CAS  PubMed  Google Scholar 

  26. Dieterich, D. C. et al. Labeling, detection and identification of newly synthesized proteomes with bioorthogonal non-canonical amino-acid tagging. Nat. Protoc. 2, 532–540 (2007).

    PubMed  Google Scholar 

  27. Schmidt, E. K., Clavarino, G., Ceppi, M. & Pierre, P. SUnSET, a nonradioactive method to monitor protein synthesis. Nat. Methods 6, 275–277 (2009).

    CAS  PubMed  Google Scholar 

  28. Huang, Y.-Y., Martin, K. C. & Kandel, E. R. Both protein kinase A and mitogen-activated protein kinase are required in the amygdala for the macromolecular synthesis-dependent late phase of long-term potentiation. J. Neurosci. 20, 6317–6325 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Johnson, L. R. et al. A recurrent network in the lateral amygdala: a mechanism for coincidence detection. Front. Neural Circuits 2, 3 (2008).

    PubMed  PubMed Central  Google Scholar 

  30. Costa-Mattioli, M. et al. Translational control of hippocampal synaptic plasticity and memory by the eIF2alpha kinase GCN2. Nature 436, 1166–1173 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Zhu, P. J. et al. Suppression of PKR promotes network excitability and enhanced cognition by interferon mediated disinhibition. Cell 147, 1384–1396 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Stern, E., Chinnakkaruppan, A., David, O., Sonenberg, N. & Rosenblum, K. Blocking the eIF2alpha kinase (PKR) enhances positive and negative forms of cortex dependent taste memory. J. Neurosci. 33, 2517–2525 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Igaz, L. M., Vianna, M. R., Medina, J. H. & Izquierdo, I. Two time periods of hippocampal mRNA synthesis are required for memory consolidation of fear motivated learning. J. Neurosci. 22, 6781–6789 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Wilensky, A. E., Schafe, G. E. & LeDoux, J. E. Functional inactivation of the amygdala before but not after auditory fear conditioning prevents memory formation. J. Neurosci. 19, RC48 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Quirk, G. J., Repa, C. & LeDoux, J. E. Fear conditioning enhances short-latency auditory responses of lateral amygdala neurons: parallel recordings in the freely behaving rat. Neuron 15, 1029–1039 (1995).

    CAS  PubMed  Google Scholar 

  36. Schafe, G. E. & Ledoux, J. E. Memory consolidation of auditory pavlovian fear conditioning requires protein synthesis and protein kinase A in the amygdala. J. Neurosci. 20, RC96 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Rogan, M. T., Staubli, U. V. & LeDoux, J. E. Fear conditioning induces associative long-term potentiation in the amygdala. Nature 390, 604–607 (1997).

    CAS  PubMed  Google Scholar 

  38. Sidrauski, C., McGeachy, A. M., Ingolia, N. T. & Walter, P. The small molecule ISRIB reverses the effects of eIF2α phosporylation on translation and stress granule assembly. eLife 4, e05033 (2015).

    PubMed Central  Google Scholar 

  39. Pasini, S., Corona, C., Liu, J., Greene, L. A. & Shelanski, M. L. Specific downregulation of hippocampal ATF4 reveals a necessary role in synaptic plasticity and memory. Cell Rep. 11, 183–191 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Ma, Y. & Hendershot, L. M. Delineation of a negative feedback regulatory loop that controls protein translation during endoplasmic reticulum stress. J. Biol. Chem. 278, 34864–34873 (2003).

    CAS  PubMed  Google Scholar 

  41. Lin, C.-J. et al. Targeting synthetic lethal interactions between Myc and the eIF4F complex impedes tumorigenesis. Cell Rep. 1, 325–333 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Back, S. H. et al. Translation attenuation through eIF2α phosphorylation prevents oxidative stress and maintains the differentiated state in beta cells. Cell Metab. 10, 13–26 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Shrestha, P. & Klann, E. Lost memories found. Nature 531, 450–451 (2016).

    CAS  PubMed  Google Scholar 

  44. Ambruster, B. N., Li, X., Pausch, M. H., Herlitze, S. & Roth, B. L. Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand. Proc. Natl Acad. Sci. USA 104, 5163–5168 (2007).

    Google Scholar 

  45. Li, S. et al. Promoting axon regeneration in the adult CNS by modulation of the melanopsin/GPCR signaling. Proc. Natl Acad. Sci. USA 113, 1937–1942 (2016).

    CAS  PubMed  Google Scholar 

  46. Thompson, K. J. et al. DREADD agonist 21 (C21) is an effective agonist for muscarinic-based DREADDs in vitro and in vivo. ACS Pharmacol. Transl. Sci. 1, 61–72 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Batista, G. et al. Translational control of auditory imprinting and structural plasticity by eIF2α. eLife 2016, e17197 (2016).

    Google Scholar 

  48. Kalai, M. et al. The caspase-generated fragment of PKR cooperate to activate full-length PKR and inhibit translation. Cell Death Differ. 14, 1050–1059 (2007).

    CAS  PubMed  Google Scholar 

  49. Abdou, K. et al. Synapse-specific representation of the identity of overlapping memory engrams. Science 360, 1227–1231 (2018).

    CAS  PubMed  Google Scholar 

  50. Ryan, T. J., Roy, D. S., Pignatelli, M., Arons, A. & Tonegawa, S. Engram cells retain memory under retrograde amnesia. Science 348, 1007–1013 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Trinh, M. A. et al. The eIF2 α kinase PERK limits the expression of hippocampal metabotropic glutamate receptor-dependent long-term depression. Learn. Mem. 21, 298–304 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Santini, E. et al. Exaggerated translation causes synaptic and behavioural aberrations associated with autism. Nature 493, 411–416 (2013).

    CAS  PubMed  Google Scholar 

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Acknowledgements

We thank M. Marmacz for expert technical assistance, C. Rice (The Rockefeller University) for providing the plasmid for HCV polyprotein, M. Ryan (University of St Andrews) for the 2A plasmid, A. Klinakis (Biomedical Research Foundation Academy of Athens), A. Domingos and J. Friedman (The Rockefeller University) for the plasmid containing the STOP sequence and the Eef1a1 targeting plasmid, P. Vandenabeele (Ghent University) for the PKRk plasmid, R. Kaufman (Sanford Burnham Prebys Medical Discovery Institute) for the Eif2s1 (S51A); CAG Prfl.Eif2s1.fl.GFP mouse line and J. Pelletier (McGill University) for the Col1a1TRE GFP.shmir4E.389 mouse line. We thank the Allen Brain Institute for providing AAV.CAG Pr.DIO.tTA. We thank all members of the Klann laboratory for critical feedback and discussions. This study was supported by National Institute of Health Grants (nos. NS034007 and NS047384) to E.K., a Howard Hughes Medical Investigator grant to N.H. and a Brain and Behavior Research Foundation NARSAD Young Investigator grant to P.S.

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Author notes

  1. These authors contributed equally: Prerana Shrestha, Pinar Ayata.

Authors and Affiliations

  1. Center for Neural Science, New York University, New York, NY, USA

    Prerana Shrestha, Pedro Herrero-Vidal, Francesco Longo, Alexandra Gastone, Joseph E. LeDoux & Eric Klann

  2. HHMI Laboratory of Molecular Biology, The Rockefeller University, New York, NY, USA

    Pinar Ayata & Nathaniel Heintz

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Contributions

P.S., P.A. and N.H. conceptualized the iPKR system. P.S. and E.K. created the conceptual design of all in vivo work. P.A. designed and characterized the iPKR system in vitro and generated the iPKR mouse model under the supervision of N.H. P.S. carried out behavior training, and collected and analyzed in vivo and ex vivo data. P.H.-V. carried out behavior training. F.L. carried out and analyzed slice electrophysiology. A.G. helped with mouse behavior training. J.E.L. provided critical advice on behavioral design. P.S. and E.K. wrote the paper. All authors read and commented on the paper.

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Correspondence to Nathaniel Heintz or Eric Klann.

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Extended data

Extended Data Fig. 1 Characterization of iPKR system.

a) The toxicity of NS3/4 inhibitor ASV was determined by central i.c.v. administration of varying doses of the drug. Representative Western blots (top) shows the levels of cFos, phospho-eIF2α, total eIF2α, and control β-Actin band in response to the administration of ASV at the doses : 0, 10 nM, 100 nM (1 μM in 2 μl saline). Bar graph with individual data points shows quantification of cFOS (left) and phospho/total eIF2α (right) normalized to β-Actin. n=3 independent Western blots, 3 mice per group. One-way ANOVA. b) Schematic of the engineering approach for chemogenetic protein synthesis inhibitor plasmid construct consisting of NS3/4 protease, EGFP and iPKR kinase domain separated by 2A ribosome skipping sites under CMV promoter. Control plasmids harbored iPKR without NS3/4 protease or unmodified PKR kinase domain (PKRk). c) Metabolic S35 labeling of de novo translation in vitro showed significantly decreased translation in the presence of PKRk and iPKR (**p<0.01) but the translation block was lifted by the co-expression of NS3/4 protease that degrades iPKR (**p<0.01). n = 2 biological replicate lysates per group; One way ANOVA followed by Bonferroni’s post-hoc test. F(5,6) = 19.01, **p=0.0013. d) iPKR expression is correspondingly regulated by NS3/4 protease (**p<0.01), whereas unmodified PKRk levels were unaltered by NS3/4 protease. n = 2 per group; One way ANOVA followed by Bonferroni’s post-hoc test. F(5,6) = 22.21, ***p=0.0008. Data are presented as ± SEM.

Source data

Extended Data Fig. 2 Generation of iPKR mouse.

a) Schematic showing the subcloning and targeting strategy of the multicistronic cassette containing loxP-flanked STOP cassette, NS3/4 protease, EGFP-L10, and iPKR kinase domain, which were separated by 2A ribosome skipping sites. The entire cassette was inserted between exon1 and exon 2 of Eef1a1 genomic locus in mouse ES cells. Recognition site for the Southern blot probe is indicated. b) Southern blot after BamHI restriction enzyme-digested DNA isolated from embryonic stem cells using the probe indicated in a). Modified (6.2 kB kb) and unmodified (10.3 kB) DNA bands are indicated with arrows. In Nes iPKR brains, EGFP-L10 is expressed in the soma of neurons in the anterior cingulate cortex (c), somatosensory cortex (d), CA1 (e), CA3 (f) and dentate gyrus (g) consistent with NeuN expression. Insets show the corresponding brain areas at higher magnification.

Extended Data Fig. 3 Nes.iPKR mice display normal locomotor and anxiety related behavior.

a) Nes.iPKR mice acclimated to the novel environment equivalent to the wildtype and exhibited c) normal locomotor activity in the open field test. d) Nes.iPKR animals displayed normal thigmotaxis as assessed by % distance traveled in the center compared to total distance. n = 5–7 per group; RM One-way ANOVA for (a) and Unpaired t-test for (c) and (d).

Extended Data Fig. 4 General translation inhibition in CamK2α principal neurons.

Blocking protein synthesis in CamK2α principal neurons in LA did not affect acclimation to a novel environment (a), total locomotor activity (b) or thigmotaxis, assessed by % distance traveled in center compared to total distance (c). d) In the elevated plus maze, however, animals with protein synthesis blocked in CamK2α principal neurons exhibited reduced anxiety i.e. increased %open arm duration (*p<0.05) compared to vehicle treated CamK2α iPKR mice and CamK2α wildtype mice even though they make equivalent entries to the open arm (e). n = 4-5 per group. RM Two-way ANOVA for a), One-way ANOVA followed by Bonferroni’s post-hoc test for (b), (c), (d) and (e).

Extended Data Fig. 5 Blocking cap-dependent translation in LA CamK2α principal neurons.

a) Alternate strategy of blocking translation in CamK2α principal neurons in LA using cre-tet regulated synthetic micro-RNA targeted against eIF4E. Col1a1.TRE.GFP.shmir4E mice were bilaterally injected in the lateral amygdala with AAV1.CamK2α.Cre and AAV9.DIO.tTA, and placed off dox diet for 10 days before training. b) eIF4E protein level was significantly decreased in GFP+ neurons that express shmir4E. Two-way ANOVA with Bonferroni’s post-hoc test. Genotype X GFP interaction: F(1,311) = 32.29, ****p<0.0001; GFP: F(1,311) = 45.32, ****p<0.0001. c) CamK2α 4Ekd mice learned the association between CS and US during training. RM Two-way ANOVA with Bonferroni’s post-hoc test. CS: F(2,14) = 17.54, ***p=0.0002. d) Cued LTM was severely impaired across all three CS presentations. n=8 per group; RM Two-way ANOVA with Bonferroni’s post-hoc test. F(10,20) = 15.65, **p=0.0027. d) Mean cTC LTM was significantly impaired in CamK2α 4Ekd mice compared to wildtype (***p<0.001). n = 8 per group; Unpaired t-test. Data are presented as ± SEM.

Extended Data Fig. 6 Characterization of mice expressing phosphomutant eIF2α in LA CamK2α principal neurons.

a) eIF2α phosphorylation at S51 was significantly reduced in GFP+ neurons in CamK2α.eIF2α (A/A) mice compared to GFP- neurons, as well as GFP+ neurons in CamK2α.WT mice (****p<0.001). (n = 60–74 per group, 3 animals); One-way ANOVA with Bonferroni’s post-hoc test. F (2, 154) = 1055, ****p<0.0001. b) Representative motion traces from the open field test for CamK2α.WT, CamK2α.eIF2α(A/+) and CamK2α eIF2α (A/A) mice. c) In the open field test, CamK2α.eIF2α (A/A) mice acclimated to the novel environment and had comparable spontaneous locomotion compared to the CamK2α.WT mice and CamK2α. eIF2α (A/+) mice. RM One-way ANOVA. d) Bar graphs representing thigmotaxis, i.e. %time spent in center compared to total distance traversed in the open field arena for the three groups. One-way ANOVA. f) In the elevated plus maze, CamK2α.eIF2α (A/A) mice spent a significantly higher duration in the open arm compared to CamK2α WT mice (g) (*p<0.05) indicating anxiety like behavior, even though they made equivalent entries to the open arm. One-way ANOVA with Bonferroni’s post-hoc test. F(2,17) = 3.775, *p=0.0440. Data are presented as +/- SEM.

Extended Data Fig. 7 Artificial chemogenetic activation of LA CamK2α principal neurons.

a) All groups of mice - CamK2α. iPKR hM3Dq, CamK2α.hM3Dq and CamK2α WT, exhibited low freezing during ITI in LTM1. XY plots showing %freezing during individual ITIs and Post-CS (left; RM Two-way ANOVA) and bar graphs showing mean %freezing during ITI (right; One-way ANOVA). n = 6–9 per group. b) During LTM2, administration of DREADD agonist C21 caused an increase in freezing during ITI for both CamK2α.hM3Dq and CamK2α. iPKR hM3Dq groups compared to CamK2α WT mice. XY plots showing %freezing during individual ITIs and Post-CS (left). n = 5–7 per group. RM Two-way ANOVA genotype: F(2,15)=12.63, ***p=0.0006). Bar graphs showing mean %freezing during ITI (right). One-way ANOVA with Bonferroni’s post-hoc test. *p<0.05 and **p<0.01. c) CamK2α. eIF2α (A/A) mice displayed comparable learning in the diferential threat conditioning training for both CS+ (right) and CS- (left). d) However, in the LTM test, they displayed significant increase in CS- response compared to CamK2α WT mice (**p<0.01). Two-way ANOVA with Bonferroni’s post-hoc test. CS: F(1,28) = 49.18, ****p<0.0001; Genotype: F(1,28) = 15.26, ***p=0.0005. e) The cTD discrimination index was significantly lower for CamK2α. eIF2α (A/A) mice (**p<0.01) relative to controls. n=7–10 per group; Unpaired t-test. f) Besides stimulus generalization, CamK2α. eIF2α (A/A) mice also displayed cognitive inflexibility and could not stop freezing after the tone offset, and thus had significantly higher freezing rate during the ITIs. RM Two-way ANOVA with Bonferroni’s post-hoc test. Genotype: F(1,10) = 16.70, **p=0.0022. g) Mean freezing response during ITI is significantly increased in CamK2α. eIF2α (A/A) mice (**p=0.0097) . n=7-10 per group; Unpaired t-test. Data are presented as mean +/- SEM.

Extended Data Fig. 8 Model for protein synthesis regulation during long-term memory consolidation.

a) In wild-type mice, threat conditioning leads to a spatiotemporally regulated increase in somatic and dendritic protein synthesis that stabilizes the memory trace. b) Application of ciPSI system prevents the coordinated increase in cell-wide translation leading to impaired LTM. c) Dephosphorylation of eIF2α enhances general translation, but it is dysregulated and unable to coordinate the cell-wide translation program to store a complex memory trace, resulting in memory generalization. d) Artificial reactivation of the amygdala principal neurons after protein synthesis inhibition-mediated amnesia leads to an increase in translation but does not restore synapse specificity and thus leads to memory generalization.

Supplementary information

Supplementary Data 1

Sequence information for iPKR targeting construct.

Reporting Summary

Source data

Source Data Fig. 2

Unprocessed western blots.

Source Data Extended Data Fig. 1

Unprocessed western blots and/or gels.

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Shrestha, P., Ayata, P., Herrero-Vidal, P. et al. Cell-type-specific drug-inducible protein synthesis inhibition demonstrates that memory consolidation requires rapid neuronal translation. Nat Neurosci 23, 281–292 (2020). https://doi.org/10.1038/s41593-019-0568-z

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