Multiple mechanisms for the potentiation of AMPA receptor-mediated transmission by alpha-Ca2+/calmodulin-dependent protein kinase II

Abstract

Some forms of activity-dependent synaptic potentiation require the activation of postsynaptic Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Activation of CaMKII has been shown to phosphorylate the glutamate receptor 1 subunit of the AMPA receptor (AMPAR), thereby affecting some of the properties of the receptor. Here, a recombinant, constitutively active form of alphaCaMKII tagged with the fluorescent marker green fluorescent protein (GFP) [alphaCaMKII(1-290)-enhanced GFP (EGFP)] was expressed in CA1 pyramidal neurons from hippocampal slices. The changes in glutamatergic transmission onto these cells were analyzed. AMPA but not NMDA receptor-mediated EPSCs were specifically potentiated in infected compared with nearby noninfected neurons. This potentiation was associated with a reduction in the proportion of synapses devoid of AMPARs. In addition, expression of alphaCaMKII(1-290)-EGFP increased the quantal size of AMPAR-mediated responses. This effect reflected, at least in part, an increased unitary conductance of the channels underlying the EPSCs. These results reveal that several key features of long-term potentiation of hippocampal glutamatergic synapses are reproduced by the sole activity of alphaCaMKII.

Fig. 1.

Expression of recombinant αCaMKII in hippocampal slices. A, Top, Hippocampal slice (see Materials and Methods) after overnight infection with Sindbis virus expressing αCaMKII_1–290–EGFP. Several injections of the virus were made in the CA1 region (betweenasterisks) resulting in massive expression of the transgene, as detected by fluorescence microscopy (bottom). B, Western blot of protein extracts from slices at various times after infection. In addition to endogenous αCaMKII (lower band), the recombinant fusion protein of higher molecular weight was detectable with αCaMKII antibody between 5 and 12 hr after infection. C, In agreement with these results, a kinase assay on cell extracts prepared at various times after infection revealed a significant increase in Ca²⁺-independent kinase activity between 5 and 12 hr after infection (3.1 ± 0.7 of controls).

Fig. 2.

Elevated CaMKII activity selectively potentiates AMPAR- but not NMDAR-mediated synaptic transmission.A, Low-density infection of a hippocampal slice.Top, Differential interference contrast micrograph of the infected CA1 region. Middle, Fluorescence micrograph of the same region showing EGFP fluorescence of infected neurons. Note that only a few CA1 pyramidal neurons were infected. Bottom, Superimposition of the two micrographs. B, AMPA and NMDAR EPSCs in neighboring infected and noninfected cells. Top, CA1 pyramidal cells were infected with Sindbis virus expressing EGFP only. AMPAR-mediated (inward) EPSCs were recorded at −60 mV and were not significantly different in two neighboring infected and noninfected cells. Similarly, EPSCs recorded at +40 mV, primarily carried by NMDAR, were equally unaffected by EGFP expression. Bottom, In contrast, when Sindbis virus expressing the fusion protein αCaMKII_1–290–EGFP was used to infect CA1 neurons, AMPAR EPSCs were greatly increased in amplitude, whereas EPSCs recorded at +40 mV remained unchanged. Each trace represents the average of 100 consecutive EPSCs. Filled barsshow the sections of the trace used for amplitude measurements. C, Summary plots representing evoked EPSC amplitude in neighboring control and infected cells. Open circles, Individual experiments; filled circles, averages. The amplitude of both AMPA and NMDAR EPSCs was unchanged in cells expressing EGFP only (p = 0.72,n = 9 and p = 0.84,n = 5, respectively). In contrast, expression of αCaMKII_1–290–EGFP increased the amplitude of AMPAR EPSCs to 3.7 ± 0.9 of controls (p < 0.001; n = 13) with no effect on NMDAR EPSCs (p = 0.54; n = 12).

Fig. 3.

Reduction of the proportion of silent synapses onto pyramidal neurons expressing αCaMKII_1–290–EGFP. Synaptic failures were counted with stimulation delivered in the stratum radiatum, while holding the cell at either −60 or +40 mV. A, One infected cell (bottom) and one noninfected cell (top) were recorded sequentially. Traces were scaled on the amplitude of the EPSC recorded at +40 mV, measured as shown in Figure2B. Each trace represents the average of 200 consecutive synaptic responses. Dotted lines indicate the baseline current. B, Amplitude density estimates of 200 EPSCs from the experiment illustrated inA. Failure rates were estimated from the integral of the peak centered on 0 pA. In the control cell, the failure rate recorded at +40 mV was ∼45% of that recorded at −60 mV (top), whereas no difference was apparent in a neighboring cell expressing αCaMKII_1–290–EGFP (bottom).p.d.f., Probability density function. C, Summary plots showing that the ratio ofF₋₆₀ toF₊₄₀, describing the proportion of silent synapses, was significantly reduced in cells expressing αCaMKII_1–290–EGFP (p < 0.005; n = 10). Open circlesrepresent data from individual experiments; filled circles show means and SEs.

Fig. 4.

Increased quantal size of AMPAR EPSCs in pyramidal cells expressing αCaMKII_1–290–EGFP. EPSCs were evoked in pyramidal cells after substitution of Ca²⁺ by Sr²⁺, resulting in asynchronous quantal events.A, EPSCs recorded in two neighboring CA1 cells (average of 200 consecutive EPSCs). Inset, Five consecutive responses showing asynchronous events after a larger initial event.Open bars show the section of trace that was used to detect quantal EPSCs. Calibration, 10 pA, 100 msec.B, Amplitude distributions of quantal EPSCs recorded in the two cells shown above. Note the shift of the distribution toward larger amplitudes in the cell expressing αCaMKII_1–290–EGFP. Consequently, the mean quantal size of EPSCs recorded in this cell, calculated as the average of quantal EPSC amplitudes, showed a ∼83% increase compared with controls. Individual traces show the average of all detected quantal events (424 in controls, 418 in infected cells). Calibration, 2 pA, 10 msec. p.d.f., Probability density function.C, Summary data for nine independent experiments (open circles). Note the increased variance of quantal size in infected cells. Filled circles indicate the average of all experiments. D, Graph showing the ratio of the quantal size of the first cell of a sequence over that of the second when the first cell was not infected and the second either uninfected as well (filled bar;n = 5) or infected (open bar;n = 9). Note the approximately twofold increase in quantal size when the second cell was infected, as inC.

Fig. 5.

The unitary conductance of channels contributing to AMPAR EPSCs is increased in pyramidal cells expressing αCaMKII_1–290–EGFP. Nonstationary variance analysis was applied to locally evoked EPSCs onto control or infected pyramidal cells maintained at −60 mV, as described in Materials and Methods.A, A total of 30–45 EPSCs of similar amplitude were aligned to their half rising time and scaled in amplitude (arrows). The mean and variance of the currents were then computed for all data points of the decay phase, starting at the peak. B, Variance to mean relationship plotted for neighboring pyramidal neurons, both infected (bottom) and noninfected (top). Data points were binned according to mean current (see Materials and Methods). The unitary conductance of channels contributing to EPSCs in the infected cell was ∼60% larger than the conductance derived from EPSCs in a neighboring control cell.C, Summary plots from nine independent experiments (open circles). Filled circles show the average conductance for control and infected cells (11.0 ± 0.8 and 16.9 ± 1.3 pS, respectively; p < 0.002).