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To elucidate mechanisms that control and execute activity-dependent synaptic plasticity, ot-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPA-Rs) with an electrophysiological tag were expressed in rat hippocampal neurons. Long-term potentiation (LTP) or increased activity of the calcium/ calmodulin-dependent protein kinase II (CaMKII) induced delivery of tagged AMPA-Rs into synapses. This effect was not diminished by mutating the CaMKII phosphorylation site on the GluR1 AMPA-R subunit, but was blocked by mutating a predicted PDZ domain interaction site. These results show that LTP and CaMKII activity drive AMPA-Rs to synapses by a mechanism that requires the association between GluR1 and a PDZ domain protein.
Long-term potentiation (LTP) of synaptic transmission is a well-characterized form of activity-- dependent plasticity likely to play important roles in learning and memory (1). A key mediator of this plasticity is CaMKII, an enzyme that is strongly expressed at excitatory synapses (2). Although the cell biological processes underlying this form of plasticity are poorly understood, the trafficking of synaptic receptors appears to play a crucial role (3).
Genes of interest were delivered to neurons in organotypically cultured hippocampal slices, using the Sindbis virus expression system (3-5). Neurons carrying foreign genes were identified by green fluorescent protein (GFP) expression and whole-cell recordings were obtained (Fig. IA) (6). To examine the effect of elevated CaMKII activity on neuronal function, we generated a construct encoding the catalytic domain of this enzyme fused with GFP (tCaMKII-GFP). The expression of this construct increased constitutive CaMKII activity in baby hamster kidney (BHK) cells (Fig. 113) (7). In hippocampal slice neurons expressing this construct, fluorescence was detected in dendritic arbors and spines (8). To determine the effect on synaptic transmission, we measured synaptic responses in two nearby neurons, one infected with tCaMKIIGFP (indicated by GFP fluorescence) and the other uninfected (Fig. 1, A and D). Such pairwise comparisons of synaptic responses to stimuli delivered at the same site showed that tCaMKII-GFP enhanced transmission (Fig. ID) (9-11). Cells infected with GFP alone did not show any change in synaptic response (Fig. IC).
To examine if this increase in AMPA-R-- mediated transmission was due to a delivery of receptors to synapses, we developed an electrophysiological assay. The current-voltage (I-V) relationship of AMPA-Rs is determined by the GluR2 subunit: AMPA-Rs with GluR2 show linear I-V relations;...