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Oct 7, 2002 - Daniel R. Storm. Department of Pharmacology, Box 357280, Health Sciences Building,. University of Washington, Seattle, Washington ...
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Hippocampal CREmediated gene expression is required for contextual memory formation Jaime Athos, Soren Impey, Victor V. Pineda, Xi Chen and Daniel R. Storm Department of Pharmacology, Box 357280, Health Sciences Building, University of Washington, Seattle, Washington 98195-7280, USA Correspondence should be addressed to D.R.S. ([email protected])

Published online 7 October 2002; doi:10.1038/nn951

One of the molecular events associated with contextual longterm memory (LTM) formation is the induction of cyclic AMP response element (CRE)-mediated transcription1. Here we report that activation of NMDA receptors and of extracellular signal–regulated kinase (ERK) were necessary for stimulation of CRE-mediated transcription during contextual fear conditioning. In addition, we found that inhibition of CRE-regulated transcription during learning blocked LTM, which indicates that this transcriptional activity is critical for memory formation. The formation of LTM, including contextual memory, requires de novo synthesis of mRNA and proteins that modify the efficacy of synaptic transmission 2,3. Using a transgenic CRE–lacZ reporter mouse strain, we have previously shown that training for contextual learning stimulates CRE-mediated transcription and phosphorylation of CRE binding protein (CREB) in area CA1 of the dorsal hippocampus1. Moreover, genetic perturbations of CREB function in Drosophila4 and mice5,6 implicate CREB-mediated transcription in LTM. Likewise, microinjection of CRE oligodeoxynucleotides (ODNs) into the nuclei of Aplysia sensory neurons blocks long-term facilitation, a model for LTM7, and infusion of antisense CREB ODNs into the dorsal hippocampus of rats interferes with spatial memory formation8. Therefore, it is surprising that expression of dominant-

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negative CREB in area CA1 of the dorsal hippocampus of mice does not interfere with contextual memory9. However, interpretation of the behavioral phenotypes of CREB transgenic or knockout mice is complicated by the compensation between CRE-binding transcription factors10. In this study, our objectives were (i) to identify the signal transduction pathway that promotes training-induced increases in CRE-mediated transcription during contextual memory formation and (ii) to determine if CRE-mediated transcription is required for contextual memory. We examined the importance of NMDA receptor and ERK activities for training induction of CRE-mediated transcription because these signaling elements have been implicated in formation of hippocampus-dependent LTM11–13. Bilaterally cannulated CRE–lacZ mice were infused with 5 mM APV, an NMDA receptor antagonist, 15 minutes before training for contextual fear conditioning (Supplementary Methods online; animal protocols were approved by the University of Washington IACUC). In this protocol, animals learn to associate the context of the training chamber with an aversive foot shock, and memory for this association is inferred from an observed increase in freezing behavior or an overall decrease in movement during the testing session. The pre-training, intrahippocampal infusion of APV blocked LTM formation; the behavior of APV-treated mice was indistinguishable from control (naïve) mice during the training and testing sessions (Fig. 1a). Immediately after behavioral testing, animals were killed, and β-galactosidase immunostaining showed that the learning-induced increases in CRE-mediated gene expression were completely blocked by APV infusion (Fig. 1b; see Supplementary Fig. 1a online for confocal images). These data indicate that NMDA receptor activity is required for contextual LTM formation as well as for learning-induced increases in CREregulated transcription. In a similar set of experiments, bilaterally cannulated CRE–lacZ mice were infused with PD98059, an inhibitor of the ERK kinase MEK, 1 hour before contextual training. Traininginduced increases in ERK activity were significantly reduced by intrahippocampal infusion of PD98059 (data not shown). As with APV, PD98059 blocked both LTM formation (Fig. 2a) and learning-induced increases in CRE-mediated gene expression (Fig. 2b and Supplementary Fig. 1b), demonstrating the necessity for ERK activity in both of these processes. To test whether increases in CRE-mediated gene expression in the CA1 region of the dorsal hippocampus are required for contextual memory formation, bilaterally cannulated mice were infused 6 hours before behavioral training with either CRE decoy ODNs or control ODNs of the same composition with a scrambled sequence. The diffusion and cellular uptake of ODNs was restricted to the CA1 region of the dorsal hippocampus (Supplementary Fig. 2). The behavior of mice that were infused with the CRE decoy 6 hours before training was unaffected during the training session; these mice responded to the foot shock similar-

Fig. 1. NMDA receptor activity is required for learning-induced CREmediated gene expression. (a) The competitive NMDA receptor antagonist APV was infused bilaterally into the dorsal hippocampi of mice 15 min before training in contextual fear conditioning. Vehicleinfused, conditioned mice showed an increase in the proportion of freezing time in the testing session (black) compared with the training session (gray), indicating memory for context. This memory formation was attenuated in APV-infused, conditioned mice. (b) Quantification of normalized β-galactosidase immunostaining in the CA1 region of the hippocampus (naïve: vehicle-infused n = 6, APV-infused n = 6; conditioned: vehicle-infused n = 6, APV-infused n = 7; ****P < 0.001). 1119

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Fig. 2. Inhibition of ERK activation blocks LTM formation and learninginduced CRE-mediated gene expression. (a) Bilateral, intrahippocampal infusion of PD98059 (5 mM), an MEK inhibitor, 1 h before training completely blocked LTM formation. (b) Quantification of normalized βgalactosidase immunostaining in the CA1 region of the hippocampus (naïve: vehicle-infused n = 5, PD98059-infused n = 6; conditioned: vehicle-infused n = 7, PD98059-infused n = 7; ***P < 0.005).

ly to the uninfused and scrambled oligonucleotide–infused control mice (Supplementary Fig. 3). When tested 24 hours later, however, CRE decoy–treated mice showed no memory for context and behaved like naive animals (Fig. 3a). Dorsal hippocampal extracts were taken immediately after the testing session and assayed for the CRE-regulated gene product C/EBPβ14, a learning-induced gene product15. Pre-training infusion of the CRE decoy completely blocked training-induced increases in C/EBPβ expression (Fig. 3b and c). These data show that the learninginduced expression of CRE-regulated genes such as C/EBPβ is blocked by CRE-decoy oligonucleotides and that this expression is necessary for LTM formation. In summary, our data indicate that a critical function of learning-induced NMDA receptor and ERK activities within area CA1 of the dorsal hippocampus is to promote the expression of CRE-regulated genes; the expression of these genes is required for the establishment of contextual LTM. This hypothesis, however, does not exclude contributions to LTM from other signaling events; for example, modulation of ion channel activity may also depend on activation of NMDA receptors or the ERK pathway. Note: Supplementary information is available on the Nature Neuroscience website.

Acknowledgments Microscopy and image analysis was performed in the W.M. Keck Center for Neural Signaling, University of Washington. This research was supported by National Institutes of Health grants NS 20498 and DC 04158. J.A. was supported in part by PHS NRSA T32 GM07270 from NIGMS and PHS NRSA 1F31MH064311-01 from NIMH. V.P. was supported in part by PHS NRSA 1F31NS042475-01 from NINDS.

Competing interests statement The authors declare that they have no competing financial interests.

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Fig. 3. CRE-mediated gene expression is required for contextual longterm memory formation and learning-induced increases in C/EBPβ expression. (a) Animals infused with CRE-decoy ODNs 6 h before training behaved like naive animals during the testing session 24 h later and did not show a decrease in mean velocity that would indicate LTM formation. (b) Intrahippocampal infusion of CRE-decoy ODNs 6 h before training blocked learning-induced increases in C/EBPβ expression 24 h after training. (c) Quantification of normalized C/EBPβ expression indicates that CRE-decoy ODNs completely blocked learning-induced expression, whereas scrambled control ODNs had no effect on expression (naïve n = 5, conditioned n = 6; +CRE decoy n = 7, +scrambled ODN n = 6; ****P < 0.001).

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nature neuroscience • volume 5 no 11 • november 2002