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) PSDs were highly purified from WT and KI forebrains by differential and sucrose gradient centrifugation, extraction of PSDs with 0.5% Triton X-100 and a final sucrose gradient centrifugation. Crude lysates (Lys), P2 fraction (P2), synaptosome-enriched fraction (Syn), and highly PSD-enriched fraction (PSD) underwent sequential IB for CaMKIIα pT286 and CaMKIIα. (E) Phosphorylation of CaMKII T286 relative to total CaMKII is significantly reduced in highly purified PSDs from KI mice compared with WT (mean values±s.e.m.; t-test: ^*P<0.05; n=3). (F) Acute cortical slices were treated and crude PSD fractions isolated as in (A) before IB for PSD-95 and TARPs (stg/γ2, γ3, γ4, γ8). (G) Accumulation of TARP γ-8 (55 kDa) was increased in crude PSD fractions from both WT and KI slices following cLTP treatment but, contrasting WT, did not persist in KI fractions (mean values±s.e.m.; two-way ANOVA: ^*P<0.05; n=3).