CaMKII binding to GluN2B is critical during memory consolidation
2012; Springer Nature; Volume: 31; Issue: 5 Linguagem: Inglês
10.1038/emboj.2011.482
ISSN1460-2075
AutoresAmy R. Halt, Robert F. Dallapiazza, Yu Zhou, Ivar S. Stein, Hai Qian, Scott A. Juntti, Sonja M. Wojcik, Nils Brose, Alcino J. Silva, Johannes Hell,
Tópico(s)Receptor Mechanisms and Signaling
ResumoArticle10 January 2012free access CaMKII binding to GluN2B is critical during memory consolidation Amy R Halt Amy R Halt Department of Pharmacology, Roy J and Lucille A Carver College of Medicine, University of Iowa, Iowa City, IA, USA Search for more papers by this author Robert F Dallapiazza Robert F Dallapiazza Department of Pharmacology, Roy J and Lucille A Carver College of Medicine, University of Iowa, Iowa City, IA, USA Search for more papers by this author Yu Zhou Yu Zhou Department of Neurobiology, Semel Institute, and Department of Psychology, Brain Research Institute, University of California at Los Angeles, Los Angeles, CA, USAPresent address: Department of Physiology, Medical College of Qingdao University, Qingdao 266071, China Search for more papers by this author Ivar S Stein Ivar S Stein Department of Pharmacology, Roy J and Lucille A Carver College of Medicine, University of Iowa, Iowa City, IA, USA Department of Pharmacology, School of Medicine, University of California at Davis, Davis, CA, USA Search for more papers by this author Hai Qian Hai Qian Department of Pharmacology, Roy J and Lucille A Carver College of Medicine, University of Iowa, Iowa City, IA, USA Search for more papers by this author Scott Juntti Scott Juntti Department of Pharmacology, Roy J and Lucille A Carver College of Medicine, University of Iowa, Iowa City, IA, USA Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany Search for more papers by this author Sonja Wojcik Sonja Wojcik Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany Search for more papers by this author Nils Brose Nils Brose Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany Search for more papers by this author Alcino J Silva Alcino J Silva Department of Neurobiology, Semel Institute, and Department of Psychology, Brain Research Institute, University of California at Los Angeles, Los Angeles, CA, USA Search for more papers by this author Johannes W Hell Corresponding Author Johannes W Hell Department of Pharmacology, Roy J and Lucille A Carver College of Medicine, University of Iowa, Iowa City, IA, USA Department of Pharmacology, School of Medicine, University of California at Davis, Davis, CA, USA Search for more papers by this author Amy R Halt Amy R Halt Department of Pharmacology, Roy J and Lucille A Carver College of Medicine, University of Iowa, Iowa City, IA, USA Search for more papers by this author Robert F Dallapiazza Robert F Dallapiazza Department of Pharmacology, Roy J and Lucille A Carver College of Medicine, University of Iowa, Iowa City, IA, USA Search for more papers by this author Yu Zhou Yu Zhou Department of Neurobiology, Semel Institute, and Department of Psychology, Brain Research Institute, University of California at Los Angeles, Los Angeles, CA, USAPresent address: Department of Physiology, Medical College of Qingdao University, Qingdao 266071, China Search for more papers by this author Ivar S Stein Ivar S Stein Department of Pharmacology, Roy J and Lucille A Carver College of Medicine, University of Iowa, Iowa City, IA, USA Department of Pharmacology, School of Medicine, University of California at Davis, Davis, CA, USA Search for more papers by this author Hai Qian Hai Qian Department of Pharmacology, Roy J and Lucille A Carver College of Medicine, University of Iowa, Iowa City, IA, USA Search for more papers by this author Scott Juntti Scott Juntti Department of Pharmacology, Roy J and Lucille A Carver College of Medicine, University of Iowa, Iowa City, IA, USA Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany Search for more papers by this author Sonja Wojcik Sonja Wojcik Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany Search for more papers by this author Nils Brose Nils Brose Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany Search for more papers by this author Alcino J Silva Alcino J Silva Department of Neurobiology, Semel Institute, and Department of Psychology, Brain Research Institute, University of California at Los Angeles, Los Angeles, CA, USA Search for more papers by this author Johannes W Hell Corresponding Author Johannes W Hell Department of Pharmacology, Roy J and Lucille A Carver College of Medicine, University of Iowa, Iowa City, IA, USA Department of Pharmacology, School of Medicine, University of California at Davis, Davis, CA, USA Search for more papers by this author Author Information Amy R Halt1,‡, Robert F Dallapiazza1,‡, Yu Zhou2, Ivar S Stein1,3, Hai Qian1, Scott Juntti1,4, Sonja Wojcik4, Nils Brose4, Alcino J Silva2 and Johannes W Hell 1,3 1Department of Pharmacology, Roy J and Lucille A Carver College of Medicine, University of Iowa, Iowa City, IA, USA 2Department of Neurobiology, Semel Institute, and Department of Psychology, Brain Research Institute, University of California at Los Angeles, Los Angeles, CA, USA 3Department of Pharmacology, School of Medicine, University of California at Davis, Davis, CA, USA 4Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany ‡These authors contributed equally to this work *Corresponding author. Department of Pharmacology, UC Davis School of Medicine, University of California, 451 E Health Sciences Drive, Davis, CA 95616-8636, USA. Tel.: +1 530 752 6540; Fax: +1 530 752 7710; E-mail: [email protected] The EMBO Journal (2012)31:1203-1216https://doi.org/10.1038/emboj.2011.482 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Memory is essential for our normal daily lives and our sense of self. Ca2+ influx through the NMDA-type glutamate receptor (NMDAR) and the ensuing activation of the Ca2+ and calmodulin-dependent protein kinase (CaMKII) are required for memory formation and its physiological correlate, long-term potentiation (LTP). The Ca2+ influx induces CaMKII binding to the NMDAR to strategically recruit CaMKII to synapses that are undergoing potentiation. We generated mice with two point mutations that impair CaMKII binding to the NMDAR GluN2B subunit. Ca2+-triggered postsynaptic accumulation is largely abrogated for CaMKII and destabilized for TARPs, which anchor AMPA-type glutamate receptors (AMPAR). LTP is reduced by 50% and phosphorylation of the AMPAR GluA1 subunit by CaMKII, which enhances AMPAR conductance, impaired. The mutant mice learn the Morris water maze (MWM) as well as WT but show deficiency in recall during the period of early memory consolidation. Accordingly, the activity-driven interaction of CaMKII with the NMDAR is important for recall of MWM memory as early as 24 h, but not 1–2 h, after training potentially due to impaired consolidation. Introduction A large body of evidence indicates that memories are encoded by stable increases in the strength of synaptic transmission (i.e., long-term potentiation (LTP)) as a consequence of temporally heightened synaptic activity (Martin et al, 2000; Lee and Silva, 2009; but see Neves et al, 2008 for exceptions). Although LTP can be induced in different brain regions, it is especially robust in the hippocampus. In general, learning, as well as LTP, requires both, Ca2+ influx through the NMDAR (NMDA-type glutamate receptor) and the ensuing CaMKII (calmodulin-dependent protein kinase II) activation (Martin et al, 2000; Collingridge et al, 2004; Malenka and Bear, 2004; Kerchner and Nicoll, 2008; Lisman and Hell, 2008; Kessels and Malinow, 2009). Ca2+ influx stimulates not only CaMKII activity but also CaMKII binding to the NMDAR (Strack and Colbran, 1998; Leonard et al, 1999; Bayer et al, 2006) and CaMKII accumulation at postsynaptic sites (Shen and Meyer, 1999; Bayer et al, 2006; Strack and Hell, 2008). This mechanism supports selective enrichment of CaMKII at synapses that are undergoing potentiation upon repeated glutamate uncaging, a model for LTP (Zhang et al, 2008; Lee et al, 2009). Work in cultured hippocampal slices indicates that CaMKII binding to GluN2B is important for LTP (Barria and Malinow, 2005). Our previous study on animals expressing an inducible form of the ∼640 residue long GluN2B C-terminus is consistent with this finding (Zhou et al, 2007). However, this mutant mouse must have deficits in addition to disruption CaMKII binding to GluN2B, presumably by affecting binding of other proteins to the GluN2B C-terminus or of CaMKII to other targets, as Morris water maze (MWM) learning in that mouse was severely affected (Zhou et al, 2007) when complete abrogation of the CaMKII–GluN2B interaction in our GluN2B knockin mouse (GluN2B KI) had no effect on this learning at all (see below Figure 8). We created this GluN2B KI mouse by mutating Leu1298 to Ala and Arg1300 to Gln to specifically test the functional role of the CaMKII–GluN2B interaction in vivo. Each mutation individually blocks CaMKII binding to GluN2B as had been elegantly defined by Colbran and coworkers (Strack et al, 2000a). Results Activity-driven association of CaMKII with the NMDAR complex is abrogated in GluN2B KI mice Homozygous GluN2B KI mice showed normal fertility, birth rate, and body weight and normal Mendelian ratio of offspring from heterozygous breeders (Supplementary Figure S5). The amount of GluN1, GluN2B, GluA1, and CaMKIIα present in total brain lysates were as in litter-matched WT mice (Supplementary Figure S1A and B). To biochemically evaluate content of postsynaptic proteins at the postsynaptic density (PSD), we isolated PSDs by differential centrifugation and two sucrose gradient centrifugations, one before and one after extraction of presynaptic and perisynaptic elements with Triton X-100. The content of GluN1, GluN2A, GluN2B, GluA1, PSD-95, and CaMKII in PSD fractions was also comparable between KI and WT mice (Supplementary Figure S1C and D). As expected, co-immunoprecipitation (co-IP) of the NMDAR complex with CaMKII was reduced by 35–40% in KI mice (Figure 1A and B). Co-IP of PSD-95 with GluN2B was unaltered (Figure 1C and D). Accordingly, the KI mutations specifically target CaMKII association with GluN2B without affecting binding of PSD-95 to the very C-terminus of GluN2B. The residual co-IP of the NMDAR with CaMKII was likely due to CaMKII binding to GluN1 (Leonard et al, 1999, 2002; Merrill et al, 2007) as extraction conditions (1% deoxycholate, pH 8.5) were chosen to preserve the overall integrity of the NMDAR complex (Leonard et al, 1999). Figure 1.Impaired activity-induced CaMKII binding to the NMDAR in GluN2B KI mice. (A) The GluN2B LR/AQ mutations reduce the association of the NMDAR complex with CaMKII. Membrane fractions from forebrains of WT and heterozygote (Het) and homozygote GluN2B KI (KI) mice were extracted with 1% deoxycholate before removal of insoluble material by ultracentrifugation, IP with CaMKIIα antibodies or isotype-matched control IgG (‘Mix’; each genotype contributed 33% of the extract for this control IP) and IB for NMDAR subunits and CaMKIIα (Leonard et al, 1999). (B) Immunosignals were quantified and NMDAR signals were divided by CaMKIIα signals and normalized to WT values for the corresponding NMDAR subunit. WT values were normalized to the average WT value over all experiments. Bars represent the average values±s.e.m. for each genotype for the indicated number of experiments (n). Asterisks (*) indicate P 0.05; t-test). (E) Activity-driven CaMKII binding to the NMDAR requires its interaction with GluN2B. Forebrain slices were treated with vehicle or NMDA (200 μM, 5 min; plus 1 μM TTX to prevent overexcitation) immediately before extraction, CaMKIIα IP and IB for GluN2B, GluN1, and CaMKIIα, as detailed earlier (Leonard et al, 1999). Mock IP of a mix of 50% WT and 50% KI lysates (Mix) with control IgG showed specificity of CaMKIIα IP. (F) The NMDA-induced increase in NMDAR association with CaMKII was significantly different between WT and GluN2B KI (t-test: *P 0.05; t-test). Download figure Download PowerPoint We found earlier that Ca2+ influx through the NMDAR increased the NMDAR–CaMKII interaction by about two-fold in acute hippocampal slices from WT rats (Leonard et al, 1999). We treated acute forebrain slices with NMDA in the presence of TTX, the latter preventing overexcitation of the slices, as described earlier (Hell et al, 1996; Leonard et al, 1999). This treatment increased co-IP of the NMDAR with CaMKII in our WT but not KI mice (Figure 1E and F). Hence, CaMKII binding to GluN2B is essential for increased receptor–kinase association following NMDAR activity. To control for potential alterations in access of Ca2+ to CaMKII upon NMDAR stimulation, we determined CaMKIIα T286 autophosphorylation, a measure of CaMKII activation, which did not differ between genotypes under basal conditions and following NMDA stimulation (Figure 1G and H). Activity-driven postsynaptic accumulation of CaMKII is abolished in GluN2B KI mice Ca2+ influx through the NMDAR induces clustering of ectopically expressed GFP–CaMKIIα in primary hippocampal rat cultures (Shen and Meyer, 1999; Shen et al, 2000) and organotypic hippocampal slice cultures (Lee et al, 2009; Otmakhov et al, 2004; Zhang et al, 2008). We demonstrated more recently that untagged, endogenous CaMKIIα and CaMKIIβ also cluster upon NMDAR-mediated Ca2+ influx in primary hippocampal cultures (Merrill et al, 2005; Strack and Hell, 2008). Hippocampal cultures were prepared in parallel from littermate WT and KI pubs, treated at 20 DIV with vehicle or glutamate and fixed immediately. Immunofluorescence analysis illustrates that CaMKII was relatively smoothly distributed under control conditions in WT and KI neurons (Figure 2A, D, M1, and N1). Although glutamate induced CaMKII clustering in WT and KI neurons (Figure 2G, J, O1, and P1), double labelling for the synaptic marker synapsin showed that extensive activity-induced CaMKII clustering took place at postsynaptic sites only in WT cultures (Figure 2H, I, O2, and O3). In KI neurons, the lack of increase in Pearson's coefficient as well as in the fraction of CaMKII immunofluorescent pixels that colocalizes with the synaptic marker synapsin (Mander's coefficient) indicates that, contrasting the CaMKII clusters that were present under basal conditions, the numerous newly formed CaMKII clusters were mostly not colocalized with or juxtaposed to synapsin puncta and were, therefore, formed mainly outside synapses (Figure 2K, L, P2, and P3). In fact, such activity-induced CaMKII clustering can occur in dendritic shafts under certain pathological conditions (Hudmon et al, 2005). Colocalization of CaMKII and synapsin indicative of synaptic localization of CaMKII was unaffected in KI neurons under non-stimulated conditions as CaMKII binds to two other major postsynaptic components (densin-180 and α-actinin) and several other postsynaptic proteins without requiring activation by Ca2+ (Colbran, 2004; Robison et al, 2005; Strack and Hell, 2008; Nikandrova et al, 2010). Thus, GluN2B KI mice did not show aberrant targeting of CaMKII under basal conditions that could have otherwise contributed to the reduction in CaMKII binding to postsynaptic NMDARs upon stimulation. Furthermore, the cytoarchitecture was normal throughout the brains of KI mice as illustrated by Nissl staining for Cortex and Hippocampus (Supplementary Figure S2A–H). The GluN2B distribution was also normal indicating that the point mutations in GluN2B did not affect its postsynaptic targeting (Supplementary Figure S2I–R). We conclude that CaMKII binding to GluN2B is the main determinant for activity-driven association of CaMKII with the NMDAR and for enhanced postsynaptic CaMKII accumulation following NMDAR-mediated Ca2+ influx in vivo. Figure 2.Loss of activity-driven postsynaptic CaMKII accumulation in GluN2B KI neurons. (A–P) In all, 20 DIV hippocampal cultures from WT and litter-mate KI mice were treated with vehicle (water; A–F, M1–N3) or glutamate (100 μM, 5 min; G–L, O1–P3), immediately fixed (4% paraformaldehyde, 30 min), and stained for CaMKIIα (green in overlay) and synapsin (red in overlay). Areas outlined in C, F, I, and L are shown at larger magnification in (M–P), respectively. Scale bar for (A–L) in (A) is 25 μm and for (M–P) in M1 is 5 μm. (Q, R) Glutamate treatment significantly increased CaMKII colocalization with synapsin in WT but not KI cultures as indicated by Pearson's coefficient (Q), as well as quantification of the fraction of CaMKII clusters colocalized with synapsin clusters determined after thresholding (R; two-way ANOVA, P<0.05; n=3 with 10 neurons analysed per experiment and condition; *P<0.05; error bars: s.e.m.). Download figure Download PowerPoint Basal synaptic transmission is normal but CA1 LTP reduced in adult GluN2B KI mice GluN2B KI mice had normal paired-pulse facilitation over the full range of interpulse intervals with maximal facilitation at 50 ms in both genotypes (Figure 3A). Synaptic responses to repetitive stimulation at 10 and 100 Hz were unaffected showing that the totally releasable pool and the readily releasable pool of synaptic vesicles are normal in KI mice (Figure 3B and C). The mEPSC frequency and amplitude were comparable for the two genotypes (Figure 3D). Input–output relationships as determined by plotting initial slope of fEPSPs against fibre volley were virtually identical for the two genotypes (Figure 3E). To evaluate whether postsynaptic NMDAR activity is affected in KI mice, we measured NMDAR-mediated fEPSPs. For this purpose, Mg2+ was removed while CNQX added to inhibit AMPAR (AMPA-type glutamate receptor) and at the same time prevent epileptiform activity due to lack of Mg2+ (Huang et al, 2006; Lu et al, 2007). The resulting input–output relationships were indistinguishable for WT and KI mice (Figure 3F). Also, NMDAR resulting from ectopically expressed GluN1 plus GluN2B with R1330Q/S1303D double mutation, which, like our L1298A/R1300Q double mutation, abrogates CaMKII binding, have normal decay τ and current/voltage relationship (Barria and Malinow, 2005). Accordingly, CaMKII binding to GluN2B does not overtly affect NMDAR properties. These results, together with normal CaMKII activation upon NMDAR stimulation (Figure 1G and H) and normal LTD (Figure 4F), indicate that postsynaptic NMDAR functions are unchanged under basal conditions in KI mice. Figure 3.Basal synaptic transmission is normal in adult GluN2B KI mice. (A) Paired-pulse facilitations of fEPSPs for 20–500 ms interstimulus intervals in the CA1 area in acute hippocampal slices were virtually identical for WT and KI mice, suggesting unaltered excitation–exocytosis coupling. (B) Decreases in fEPSPs in response to repetitive stimulations at frequencies of 10 Hz were comparable for WT and KI mice, indicating that the totally releasable pool of synaptic vesicles is unchanged. (C) Decreases in fEPSPs in response to repetitive stimulations at frequencies of 100 Hz were comparable for WT and KI mice, indicating that the readily releasable pool of synaptic vesicles is unchanged. (D) AMPAR-mediated mEPSCs were monitored by whole-cell patch recording from pyramidal cells in CA1 in the presence of 200 nM TTX to block Na+ channels and thereby prevent spontaneous action potentials, 10 μM bicuculline to block GABAA receptors, and 50 μM AP-5 to block NMDARs. Amplitude (middle) and frequency (right) of mEPSCs are statistically not different in neurons from WT and KI mice. Representative traces from WT and KI mice are shown to the left of the bar diagrams. (E, F) Averages of initial fEPSP slopes (±s.e.m.) under standard conditions reflecting AMPAR response strength (E) or with ACSF containing 8 μM CNQX (to block AMPAR) and no Mg2+ indicative of NMDAR response strength (F) are plotted versus presynaptic fibre volley amplitudes (as a measure of presynaptic activation level) obtained with increasing stimulus strengths. Inserts on the right of each panel show sample traces with increasing stimulus strengths for WT (top) and KI (bottom). Each individual panel shows data (±s.e.m.) from n slices obtained from 3 to 5 mice for each genotype. Download figure Download PowerPoint Figure 4.LTP is reduced in adult GluN2B KI mice. (A) LTP (2 × 100 Hz/1 s) in CA1 in acute hippocampal slices stabilized at 156±6% (WT) and 121±4% (KI) of baseline (mean±s.e.m.) showing a significant difference between slices from WT and KI mice (t-test: P<0.05). Inserts show fEPSPs sample traces before (black) and 60 min after LTP induction (red) from WT (top) and KI (bottom) mice. (B) LTP is blocked by 100 μM AP5 in both genotypes. Recordings from WT and KI slices plateaued at 107±6% and 104±4%, respectively, 60 min after LTP induction. (C) LTP is blocked by 10 μM KN93 in both genotypes. Recordings from WT and KI slices leveled off at 108±8% and 108±5%, respectively. (D) LTP induced by θ burst stimulation (10 trains of 4 stimuli at 100 Hz; trains were 200 ms apart) in CA1 showed a significant difference (t-test: P<0.05) between slices from WT (156±7%) and KI (131+7%) mice. (E) LTP induced by 10 Hz/15 s stimulation showed a 38±2% increase in fEPSP in slices from WT mice and a 17±2 % increase in slices from KI mice (t-test: P 0.05). Inserts show fEPSPs sample traces before (black) and 60 min after LTD induction (red) from WT (top) and KI (bottom) mice. Each individual panel shows data from n slices obtained from 3 to 5 mice for each genotype. Download figure Download PowerPoint Although basal synaptic transmission is normal in GluN2B KI mice, LTP induced by two tetani of 100 Hz/1 s was reduced by about 50% in the KI mice (Figure 4A). The remaining LTP in KI slices was completely abolished by AP5 and KN93 (Figure 4B and C), indicating that the residual LTP is both NMDAR- and CaMK-dependent and not due to compensatory mechanisms that would circumvent CaMKII. We recently showed in young adult mice that LTP induced by a single tetanus is sensitive to blockage of PKA whereas a two-tetanus LTP as measured here is not (Lu et al, 2007). As in WT (Lu et al, 2007), two-tetanus LTP was not sensitive to H-89, which inhibits PKA (Supplementary Figure S3). The residual LTP thus appears to rely on the same main regulatory mechanisms as the LTP in WT mice, that is, Ca2+ influx through NMDAR and the ensuing activation of CaMKII rather than requiring additional support from PKA, which is otherwise important for single-tetanus LTP. These results provide further evidence for the notion that postsynaptic signalling by Ca2+ is normal although the binding deficiency of GluN2B for activated CaMKII results in reduced LTP. Other forms of LTP as triggered by θ burst stimulation and by a 10 Hz/15 s stimulus train were also reduced by about half (Figure 4D and E). Finally, LTD was normal in 2-week-old KI mice (Figure 4F) further supporting that most synaptic properties were unaltered and the reduction in LTP was a highly specific deficit. We conclude that NMDAR and CaMKII play important roles in LTP independent of their interaction but binding of CaMKII to GluN2B is necessary for LTP to fully develop. Activity-induced phosphorylation of GluA1 on S831 by CaMKII is abolished in GluN2B KI mice LTP is, to a large extent, mediated by upregulation of postsynaptic AMPAR activity (Collingridge et al, 2004; Malenka and Bear, 2004; Kerchner and Nicoll, 2008; Lisman and Hell, 2008; Kessels and Malinow, 2009). However, the molecular basis of LTP in general, and specifically how CaMKII upregulates postsynaptic AMPAR activity, remains largely unknown. Because phosphorylation of GluA1 on S831 by CaMKII is thought to contribute to LTP, at least under certain conditions (Lisman and Hell, 2008), we evaluated the impact of reduced CaMKII binding to the NMDAR on S831 phosphorylation. Ca2+ influx through the NMDAR increased S831 phosphorylation in WT but not GluN2B KI slices (Figure 5A and B). Accordingly, the CaMKII–GluN2B interaction is critical for S831 phosphorylation by CaMKII upon Ca2+ influx, perhaps secondarily to correct placement of the kinase. Abrogation of this mechanism likely contributes to some, but likely limited, degree to the reduction of LTP in GluN2B KI mice. Figure 5.Deficits in stimulation of GluA1 S831 phosphorylation by CaMKII. (A) Forebrain slices were treated in the presence of GF109203X (10 μM) to inhibit PKC with vehicle or NMDA (100 μM, 5 min) immediately prior to extraction of AMPAR with 1% Triton X-100, ultracentrifugation to remove non-solubilized material, GluA1 IP, and sequential IB for GluA1 pS831 and total GluA1 (Leonard et al, 1999). Mock IP of a mix of WT and KI lysates (Mix) with control IgG showed specificity of GluA1 IP. (B) NMDA significantly increased S831 phosphorylation in WT but not KI slices (shown are mean values±s.e.m.; two-way ANOVA, *P<0.01; n=3). Download figure Download PowerPoint Chemical LTP-induced autophosphorylation of postsynaptic CaMKII on T286 lasts longer in WT than GluN2B KI mice LTP induction triggers persistent T286 autophosphorylation and thereby activation of CaMKII, which likely contributes to LTP (Fukunaga et al, 1995; Barria et al, 1997; Lee et al, 2000). We thus monitored CaMKII T286 autophosphorylation in acute hippocampal slices following chemically induced LTP (cLTP) using forskolin-induced neuronal stimulation (Makhinson et al, 1999; Lu et al, 2007) (see also Kopec et al, 2006). GluN2B KI and WT mice showed the same relative degree of CaMKII activation (i.e., phospho-T286) in PSD fractions from forebrain slices following cLTP (Figure 6A). However, T286 phosphorylation lasted for at least 30 min only in WT but not in KI slices (Figure 6B). CaMKII content within the PSD relative to PSD-95 increased transiently after cLTP induction in WT slices (Figure 6C). The absence of a similar transient increase in KI slices further supports the above findings that activity-dependent postsynaptic recruitment of CaMKII is impaired by the mutations (Figure 1). Notably, our data do not exclude the possibility of a prolonged increase in total CaMKII within the PSD following LTP in WT mice in parallel to an overall increase in PSD size. In fact, CaMKII content persistently increases in parallel with the size of dendritic spines following LTP (Otmakhov et al, 2004; Zhang et al, 2008; Lee et al, 2009). However, because PSD yields vary substantially, we can only quantify levels of CaMKII relative to a PSD marker protein such as PSD-95 and not changes in total PSD protein content (Ehlers, 2003). Accordingly, we would not be able to detect a more permanent increase in CaMKII content of PSD fractions if PSD-95 content would increase in parallel, as would be expected for stable potentiation. Figure 6.Deficits in postsynaptic maintenance of CaMKIIα T286 phosphorylation and of TARP accumulation following chemical LTP in GluN2B KI mice. (A) Forebrain slices underwent control (Ctl), or cLTP treatment (cLTP; sequential incubation with forskolin, followed by increased K+ in the absence of Mg2+ (Lu et al, 2007)), with subsequent 30 min recovery in ACSF if indicated (30′). P2 fractions were isolated by differential centrifugation, presynaptic and perisynaptic elements removed with 0.5% Triton X-100 and crude PSD fractions collected by ultracentrifugation. PSD-95, CaMKIIα pT286, CaMKIIα, and CaMKIIβ were detected in PSD fractions by IB. (B) The ratio CaMKIIα pT286 to total CaMKIIα, normalized to WT control and expressed as mean values±s.e.m., increased in the PSD fractions from WT and KI slices upon cLTP treatment but this increase persisted only in WT but not KI fractions (two-way ANOVA, **P<0.01; n=3). (C) Relative CaMKIIα abundance in the PSD fractions as obtained by normalizing total CaMKIIα signals to PSD-95 signals (expressed as mean values±s.e.m.) is increased after cLTP treatment in WT but not KI slices (two-way ANOVA, *P<0.05; n=3). (D
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