N-methyl-D-aspartate receptors (NMDARs) mediate ischemic brain damage but also mediate essential neuronal excitation. To treat stroke without blocking NMDARs, we transduced neurons with peptides that disrupted the interaction of NMDARs with the postsynaptic density protein PSD-95. This procedure dissociated NMDARs from downstream neurotoxic signaling without blocking synaptic activity or calcium influx. The peptides, when applied either before or 1 hour after an insult, protected cultured neurons from excitotoxicity, reduced focal ischemic brain damage in rats, and improved their neurological function. This approach circumvents the negative consequences associated with blocking NMDARs and may constitute a practical stroke therapy.

Ischemia results in increased phosphorylation of NMDA receptors. To investigate the possible role of lipid rafts in this increase, lipid rafts and post-synaptic densities (PSDs) were isolated by the extraction of rat brain synaptosomes with Triton X-100 followed by sucrose density gradient centrifugation. Lipid rafts accounted for the majority of PSD-95, whereas SAP102 was predominantly located in PSDs. Between 50 and 60% of NMDA receptors were associated with lipid rafts. Greater than 85-90% of Src and Fyn were present in lipid rafts, whereas Pyk2 was mainly associated with PSDs. Lipid rafts and PSDs were isolated from animals subjected to 15 min of global ischemia followed by 6 h of recovery. Ischemia did not affect the yield, density, flotillin-1 or cholesterol content of lipid rafts. Following ischemia, the phosphorylation of NR1 by protein kinase C and tyrosine phosphorylation of NR2A and NR2B was increased in both lipid rafts and PSDs, with a greater increase in tyrosine phosphorylation occurring in the raft fraction. Following ischemia, NR1, NR2A and NR2B levels were elevated in PSDs and reduced in lipid rafts. The findings are consistent with a model involving close interaction between lipid rafts and PSDs and a role for lipid rafts in ischemiainduced signaling pathways.

Both serine/threonine and tyrosine phosphorylation of receptor proteins have been implicated in the process of long-term potentiation (LTP), but there has been no direct demonstration of a change in receptor phosphorylation after LTP induction. We show that, after induction of LTP in the dentate gyrus of anesthetized adult rats, there is an increase in the tyrosine phosphorylation of the 2B subunit of the N-methyl-D-aspartate (NMDA) receptor (NR2B), as well as several other unidentified proteins. Tyrosine phosphorylation of NR2B was measured in two ways: binding of antiphosphotyrosine antibodies (PY20) to glycoprotein(s) of 180 kDa (GP180) purified on Con A-Sepharose and binding of anti-NR2B antibodies to tyrosine-phosphorylated proteins purified on PY20-agarose. Three hours after LTP induction, anti-NR2B binding to tyrosine phosphorylated proteins, expressed as a ratio of tetanized to control dentate (Tet/Con), was 2.21 ± 0.50 and PY20 binding to GP180 was 1.68 ± 0.16. This increase in the number of tyrosine phosphorylated NR2B subunits occurred without a change in the total number of NR2B subunits. When the induction of LTP was blocked by pretreatment of the animal with the NMDA receptor antagonist MK801, the increase in PY20 binding to GP180 was also blocked (Tet/Con = 1.09 ± 0.26). The increased PY20 binding to GP180 was also apparent 15 min after LTP induction (Tet/Con = 1.41 ± 0.16) but not detectable 5 min after LTP induction (Tet/Con = 1.01 ± 0.19). These results suggest that tyrosine phosphorylation of the NMDA receptor contributes to the maintenance of LTP.Long-term potentiation (LTP) is a form of use-dependent synaptic plasticity that has been proposed as a cellular model for learning (1). The most extensive characterization of the molecular mechanisms involved in the induction and maintenance of LTP has been undertaken in two groups of glutamatergic synapses in mammalian hippocampus: the synapses formed between the axons of the Schaffer collaterals and the dendrites of the pyramidal neurons in the CAl region, which have mainly been investigated in slices in vitro, and the synapses between the axons of the perforant path and the dendrites of the granule cells of the dentate gyrus, which have mainly been investigated in vivo. Induction of LTP at both of these synapses requires the activation of the N-methyl-D-aspartate (NMDA) subtype of the glutamate receptor and a rise in the intracellular concentration of Ca2+ in the postsynaptic neuron (1-3). This rise in intracellular Ca2+ is believed to activate a series of biochemical events involving changes in protein phosphorylation that result in the induction and maintenance of LTP by mechanisms that may be post-and/or presynaptic (1).The (12) show greatly impaired or altered LTP, respectively. The evidence for the involvement of tyrosine kinases in LTP induction and/or maintenance is based on the ability of broad-spectrum tyrosine kinase inhibitors to inhibit the induction of a longlasting potentiation (13) and the specific deficits in LTP inductio...