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A Pharmacogenetic Inducible Approach to the Study of NMDA/αCaMKII Signaling in Synaptic Plasticity

2002; Elsevier BV; Volume: 12; Issue: 8 Linguagem: Inglês

10.1016/s0960-9822(02)00767-4

1879-0445

Masuo Ohno, Paul W. Frankland, Alcino J. Silva,

Receptor Mechanisms and Signaling

We recently introduced an inducible pharmacogenetic approach where pharmacological manipulations can be used to reveal recessive mutant phenotypes in a temporally controlled manner [1Ohno M. Frankland P.W. Chen A.P. Costa R.M. Silva A.J. Inducible, pharmacogenetic approaches to the study of learning and memory.Nat. Neurosci. 2001; 4: 1238-1243Crossref PubMed Scopus (92) Google Scholar]. This approach takes advantage of synergisms between pharmacological and genetic manipulations to alter the function of specific signaling pathways. For example, mice heterozygous for a point mutation (T286A) in the α-calcium/calmodulin-dependent kinase II (αCaMKII) gene show normal learning and memory. However, a concentration of an NMDA receptor antagonist (CPP) that does not affect learning in wild-type (WT) littermates, reveals learning deficits in this heterozygote (αCaMKIIT286A+/−) [1Ohno M. Frankland P.W. Chen A.P. Costa R.M. Silva A.J. Inducible, pharmacogenetic approaches to the study of learning and memory.Nat. Neurosci. 2001; 4: 1238-1243Crossref PubMed Scopus (92) Google Scholar]. Here, we show that pretetanic application of a concentration of CPP (0.1 μM) ineffective in WT hippocampal slices induced deficits in αCaMKIIT286A+/− slices in hippocampal long-term potentiation (LTP), a mechanism thought to be involved in learning and memory. Importantly, posttetanic application of CPP (0.1 μM) had no effect on the expression or maintenance of LTP in hippocampal slices from αCaMKIIT286A+/− mice. Thus, this pharmacogenetic approach allowed us to demonstrate that NMDA receptor-dependent autophosphorylation of αCaMKII is required during the induction but not maintenance of LTP. This ability to temporally induce recessive mutant phenotypes could be applicable to a broad range of problems and genetic systems. We recently introduced an inducible pharmacogenetic approach where pharmacological manipulations can be used to reveal recessive mutant phenotypes in a temporally controlled manner [1Ohno M. Frankland P.W. Chen A.P. Costa R.M. Silva A.J. Inducible, pharmacogenetic approaches to the study of learning and memory.Nat. Neurosci. 2001; 4: 1238-1243Crossref PubMed Scopus (92) Google Scholar]. This approach takes advantage of synergisms between pharmacological and genetic manipulations to alter the function of specific signaling pathways. For example, mice heterozygous for a point mutation (T286A) in the α-calcium/calmodulin-dependent kinase II (αCaMKII) gene show normal learning and memory. However, a concentration of an NMDA receptor antagonist (CPP) that does not affect learning in wild-type (WT) littermates, reveals learning deficits in this heterozygote (αCaMKIIT286A+/−) [1Ohno M. Frankland P.W. Chen A.P. Costa R.M. Silva A.J. Inducible, pharmacogenetic approaches to the study of learning and memory.Nat. Neurosci. 2001; 4: 1238-1243Crossref PubMed Scopus (92) Google Scholar]. Here, we show that pretetanic application of a concentration of CPP (0.1 μM) ineffective in WT hippocampal slices induced deficits in αCaMKIIT286A+/− slices in hippocampal long-term potentiation (LTP), a mechanism thought to be involved in learning and memory. Importantly, posttetanic application of CPP (0.1 μM) had no effect on the expression or maintenance of LTP in hippocampal slices from αCaMKIIT286A+/− mice. Thus, this pharmacogenetic approach allowed us to demonstrate that NMDA receptor-dependent autophosphorylation of αCaMKII is required during the induction but not maintenance of LTP. This ability to temporally induce recessive mutant phenotypes could be applicable to a broad range of problems and genetic systems. Recently, we showed that subthreshold doses of drugs that disrupt specific signaling components upstream or downstream of genetically targeted molecules can be used to reveal the effects of recessive mutations in a temporally controlled manner [1Ohno M. Frankland P.W. Chen A.P. Costa R.M. Silva A.J. Inducible, pharmacogenetic approaches to the study of learning and memory.Nat. Neurosci. 2001; 4: 1238-1243Crossref PubMed Scopus (92) Google Scholar]. This ability to temporally control the phenotypes of mutations is critical for experimental design and interpretation. For example, it helps to address concerns that the cognitive effects of certain mutations are not due to the disruption of adult processes but to undetected developmental deficits. With this pharmacogenetic approach, we previously showed that NMDA receptor-dependent activation of αCaMKII during training (but not afterward) is critical for learning [1Ohno M. Frankland P.W. Chen A.P. Costa R.M. Silva A.J. Inducible, pharmacogenetic approaches to the study of learning and memory.Nat. Neurosci. 2001; 4: 1238-1243Crossref PubMed Scopus (92) Google Scholar]. These findings are consistent with accumulating evidence suggesting a close structural and functional link between the NMDA receptor and αCaMKII [2Husi H. Ward M.A. Choudhary J.S. Blackstock W.P. Grant S.G. Proteomic analysis of NMDA receptor-adhesion protein signaling complexes.Nat. Neurosci. 2000; 3: 661-669Crossref PubMed Scopus (994) Google Scholar, 3Kennedy M.B. Signal-processing machines at the postsynaptic density.Science. 2000; 290: 750-754Crossref PubMed Scopus (630) Google Scholar, 4Leonard A.S. Lim I.A. Hemsworth D.E. Horne M.C. Hell J.W. Calcium/calmodulin-dependent protein kinase II is associated with the N-methyl-D-aspartate receptor.Proc. Natl. Acad. Sci. USA. 1999; 96: 3239-3244Crossref PubMed Scopus (314) Google Scholar, 5Silva A.J. Giese K.P. Gene targeting: a novel window into the biology of learning and memory.in: Martinez J. Kesner R. Neurobiology of Learning and Memory. Academic Press, San Diego1998: 89-142Crossref Google Scholar, 6Lisman J. The CaM kinase II hypothesis for the storage of synaptic memory.Trends Neurosci. 1994; 17: 406-412Abstract Full Text PDF PubMed Scopus (435) Google Scholar]. The autophosphorylation of αCaMKII at Thr 286 by Ca2+ influx through NMDA receptors [7Fukunaga K. Soderling T.R. Miyamoto E. Activation of Ca2+/calmodulin-dependent protein kinase II and protein kinase C by glutamate in cultured rat hippocampal neurons.J. Biol. Chem. 1992; 267: 22527-22533Abstract Full Text PDF PubMed Google Scholar, 8Ouyang Y. Kantor D. Harris K.M. Schuman E.M. Kennedy M.B. Visualization of the distribution of autophosphorylated calcium/calmodulin-dependent protein kinase II after tetanic stimulation in the CA1 area of the hippocampus.J. Neurosci. 1997; 17: 5416-5427Crossref PubMed Google Scholar] switches the kinase into a calcium/calmodulin-independent active state [6Lisman J. The CaM kinase II hypothesis for the storage of synaptic memory.Trends Neurosci. 1994; 17: 406-412Abstract Full Text PDF PubMed Scopus (435) Google Scholar, 9Hanson P.I. Schulman H. Neuronal Ca2+/calmodulin-dependent protein kinases.Annu. Rev. Biochem. 1992; 61: 559-601Crossref PubMed Scopus (651) Google Scholar] and is a critical step underlying hippocampus-dependent learning and memory [10Giese K.P. Fedorov N.B. Filipkowski R.K. Silva A.J. Autophosphorylation at Thr286 of the α calcium-calmodulin kinase II in LTP and learning.Science. 1998; 279: 870-873Crossref PubMed Scopus (832) Google Scholar]. Consistent with our previous findings [10Giese K.P. Fedorov N.B. Filipkowski R.K. Silva A.J. Autophosphorylation at Thr286 of the α calcium-calmodulin kinase II in LTP and learning.Science. 1998; 279: 870-873Crossref PubMed Scopus (832) Google Scholar], mice homozygous for a point mutation that blocks the autophosphorylation of αCaMKII at Thr286 (αCaMKIIT286A−/−) showed severe deficits in hippocampal LTP at Schaffer collateral-CA1 synapses, while the heterozygous mutation (αCaMKIIT286A+/−) only attenuated the magnitude of LTP (F2,29 = 11.6, p < 0.05) (Figure 1A). Although these results indicate that the autophosphorylation of αCaMKII is required for the induction of LTP, it is unclear whether the NMDA receptor-dependent autophosphorylation/activation of αCaMKII is required for the maintenance and/or expression of LTP. As it has been previously shown in rats, pretetanic application of the NMDA receptor antagonist CPP blocked the induction of LTP in hippocampal slices from WT mice (F2,31 = 10.4, p < 0.05) (Figure 1B), confirming the importance of NMDA receptor-dependent signaling in the induction of hippocampal CA1 LTP. To test the role of NMDA receptor-dependent activation of αCaMKII in LTP, we used the pharmacogenetic approach outlined above. Although pretetanic application of 0.1 μM CPP did not affect hippocampal LTP in WT slices, the same concentration of CPP induced severe LTP deficits in slices from heterozygous αCaMKIIT286A mutants (F3,46 = 16.9, p < 0.05) (Figures 1B and 1C). It is important to note that the αCaMKIIT286A mutation does not affect NMDA currents [10Giese K.P. Fedorov N.B. Filipkowski R.K. Silva A.J. Autophosphorylation at Thr286 of the α calcium-calmodulin kinase II in LTP and learning.Science. 1998; 279: 870-873Crossref PubMed Scopus (832) Google Scholar], suggesting that the effects revealed by CPP in the heterozygous mutants are due to lower levels of kinase activation. Interestingly, the residual LTP observed in αCaMKIIT286A heterozygous slices under 0.1 μM CPP (110.2% ± 2.8%, mean ± SEM, n = 13) was similar to the magnitude of LTP in αCaMKIIT286A homozygous slices (110.6% ± 4.5%, n = 6) and indistinguishable from the magnitude of LTP present in WT mice after full NMDA receptor blockade (1 μM CPP; 110.5% ± 4.6%, n = 6). These results suggest that the LTP left in the heterozygous mutants under 0.1 μM CPP is NMDA receptor independent. Taken together, these findings suggest that the autophosphorylation of αCaMKII is required for the induction of NMDA receptor-dependent LTP in area CA1 of the hippocampus and that decreasing NMDA signaling reveals LTP deficits in αCaMKIIT286A heterozygous mutants. It is also important to note that 0.1 μM CPP had a negligible effect on posttetanic potentiation measured 30 s after a tetanus in αCaMKIIT286A heterozygous slices (194.1% ± 11.5%, n = 13) (Figure 1C), as compared with the effects of either 1 μM CPP in WT slices (156.6% ± 6.6%, n = 6) (Figure 1B) or the αCaMKIIT286A homozygous mutation (158.9% ± 5.7%, n = 6) (Figure 1A). In contrast, all three manipulations block LTP to the same extent. These findings indicate that posttetanic potentiation is far less dependent on NMDA receptor-activated αCaMKII than LTP. Posttetanic (20 min) application of 0.1 μM CPP, however, did not affect the expression of established hippocampal LTP in αCaMKIIT286A heterozygous slices (Figure 2). Consistent with our findings, it was previously shown that application of CaMKII peptide inhibitor into the postsynaptic cell blocked the induction of LTP but did not affect LTP maintenance in the hippocampal CA1 region [11Otmakhov N. Griffith L.C. Lisman J.E. Postsynaptic inhibitors of calcium/calmodulin-dependent protein kinase type II block induction but not maintenance of pairing-induced long-term potentiation.J. Neurosci. 1997; 17: 5357-5365Crossref PubMed Google Scholar]. These results indicate that NMDA receptor/αCaMKII signaling is required for the induction of hippocampal LTP rather than the expression or maintenance of LTP. These hippocampal LTP results parallel our previous findings with the hippocampus-dependent learning task [1Ohno M. Frankland P.W. Chen A.P. Costa R.M. Silva A.J. Inducible, pharmacogenetic approaches to the study of learning and memory.Nat. Neurosci. 2001; 4: 1238-1243Crossref PubMed Scopus (92) Google Scholar]. First, the αCaMKIIT286A heterozygous mutants show nearly normal hippocampal LTP and hippocampus-dependent learning. Second, decreasing NMDA receptor signaling revealed LTP and learning deficits in these heterozygotes. Third, our pharmacogenetic findings suggest that NMDA receptor-dependent autophosphorylation of αCaMKII is specifically required for the induction of behavioral and synaptic plasticity in the hippocampus but not for the stability of these processes. Recent pharmacological experiments indicate that CPP administered after LTP induction blocks the subsequent decay of LTP at perforant path-dentate gyrus synapses, a result suggesting that LTP maintenance is a persistent process, and its eventual decay is an active process mediated by NMDA receptor activation [12Villarreal D.M. Do V. Haddad E. Derrick B.E. NMDA receptor antagonists sustain LTP and spatial memory: active processes mediate LTP decay.Nat. Neurosci. 2002; 5: 48-52Crossref PubMed Scopus (148) Google Scholar]. Interestingly, they also demonstrate that posttraining administration of CPP at doses that enhance LTP longevity also enhance retention of spatial memory [12Villarreal D.M. Do V. Haddad E. Derrick B.E. NMDA receptor antagonists sustain LTP and spatial memory: active processes mediate LTP decay.Nat. Neurosci. 2002; 5: 48-52Crossref PubMed Scopus (148) Google Scholar]. The inducible pharmacogenetic approach introduced here could be applied to the study of NMDA receptor-dependent activation of CaMKII in LTP decay and memory extinction. In conclusion, our results demonstrate that NMDA receptor-dependent autophosphorylation of αCaMKII is required for the induction of LTP. The pharmacogenetic approach used here combines the temporal flexibility that pharmacological manipulations offer, with the molecular specificity of genetic disruptions. It is also important to note that since the pharmacogenetic approach uses drugs at lower concentrations that are ineffective in WT controls, the nonspecific effects of these drugs should be reduced. This approach should be applicable to a broad range of biological problems and genetic systems. Starting with the αCaMKIIT286A+/− chimeras (contributing to 129 background) [10Giese K.P. Fedorov N.B. Filipkowski R.K. Silva A.J. Autophosphorylation at Thr286 of the α calcium-calmodulin kinase II in LTP and learning.Science. 1998; 279: 870-873Crossref PubMed Scopus (832) Google Scholar], this mutation was backcrossed five to six consecutive times into the C57Bl/6 genetic background. The αCaMKIIT286A+/− and αCaMKIIT286A−/− mice used in the experiments were F2 progeny derived from a cross between these heterozygotes. At 4–5 weeks postnatally, the mice were weaned, and their genotypes were determined with PCR analysis of tail DNA samples. All experiments were done with mice 3–7 months old, and a similar number of males and females was used. Transverse hippocampal slices (400 μm thick) were maintained in a submerged recording chamber perfused with ACSF equilibrated with 95% O2 and 5% CO2 at 30°C. The ACSF contained (in mM) 120 NaCl, 3.5 KCl, 2.5 CaCl2, 1.3 MgSO4, 1.25 NaH2PO4, 26 NaHCO3, and 10 D-glucose. Extracellular field EPSPs were recorded with a Pt/Ir electrode (FHC, Bowdoinham, ME) from the stratum radiatum layer of the area CA1, and the Schaffer collaterals were stimulated with a bipolar electrode (FHC). The intensity of stimulation (100 μs duration) was adjusted to give field EPSP ∼33% of maximum. LTP was induced by a tetanic stimulation (100 Hz, 1 s) delivered at the test intensity. After the responses were monitored at least for 20 min to ensure a stable baseline, [±]-3-[2-carboxypiperazin-4-yl]propanephosphonic acid (CPP; Sigma, St. Louis, MO) was applied for 25 min starting 20 min before the tetanus or for 40 min posttetanically (20 min after tetanization). Experiments were conducted with the experimenter blind to the drug treatments as well as the genotype of mice. To determine whether the magnitude of LTP differed significantly between the groups, responses from the last 10 min block of recordings (50–60 min) were compared by a one-way analysis of variance followed by post-hoc Newman-Keuls test when F ratios reached significance (p < 0.05). We thank N.B. Fedorov and K.P. Giese for helpful discussions; and R. Chen and M. Lacuesta for help with genotyping. M.O. was partially supported by a research fellowship from the Uehara Memorial Foundation for Life Sciences. This work was funded by a grant from the National Institutes of Health (AG13622) to A.J.S.