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The synaptic response waveform, which determines signal integration properties in the brain, depends on the spatiotemporal profile of neurotransmitter in the synaptic cleft. Here, we show that electrophoretic interactions between AMPA-receptor-mediated excitatory currents and negatively charged glutamate molecules accelerate the clearance of glutamate from the synaptic cleft, speeding-up synaptic responses. This phenomenon is reversed upon depolarization and diminished when intra-cleft electric fields are weakened through a decrease in the AMPA receptor density. In contrast, the kinetics of receptor-mediated currents evoked by direct application of glutamate are voltage-independent, as are synaptic currents mediated by the electrically neutral neurotransmitter GABA. Voltage-dependent temporal tuning of excitatory synaptic responses may thus contribute to signal integration in neural circuits.
Although ion currents through postsynaptic receptors are small (~10−11 A), they can exert a lateral voltage gradient (electric field) of ~104 V/m inside the synaptic cleft (1, 2) raising the possibility that they can affect the dwell time of electrically charged neurotransmitters (3). Does electrodiffusion therefore play any role in synaptic transmission?
The excitatory neurotransmitter glutamate is negatively charged at physiological pH (pK = 4.4), implying that postsynaptic depolarization should in principle retard its escape from the synaptic cleft (Fig. 1, A). AMPA-receptor-mediated excitatory postsynaptic currents (AMPAR EPSCs) decay more slowly at positive than at negative holding voltages in hippocampal basket cells (4) and in cerebellar granule cells (5). However, this has not been reported for AMPAR EPSCs generated at perisomatic synapses on CA1 or CA3 pyramidal cells (6-8). We evoked dendritic AMPAR EPSCs in CA1 pyramidal cells by stimulating Schaffer collaterals: the EPSC decay time (defined here as the area/peak ratio) increased monotonically with depolarization (Fig. 1, B). The ratio between recorded at +40 mV and at −70 mV (+40/ −70) was consistently above one (average ± SEM: 2.17 ± 0.09, n = 49, p < 0.001; fig. S1, A). This asymmetry was independent of the EPSC amplitude, glutamate transport or recording temperature, and could not be accounted for by unknown voltage-dependent properties of receptor antagonists (fig. S1, A and B).
A trivial possible explanation for this phenomenon is that AMPARs themselves have voltage-dependent kinetics. This has indeed been reported for AMPARs activated by brief pulses of glutamate applied to outside-out patches excised from brainstem neurons (9, 10), but not from hippocampal or dentate granule neurons (11). We confirmed that the decay of AMPAR currents evoked by 1 ms / 1 mM glutamate pulses in outside-out patches excised from somata (n = 9) or dendrites (n = 6) of CA1 pyramidal cells was indistinguishable at positive and negative voltages. Symmetrical decay kinetics were also observed when the AMPAR density was decreased in the patch with 0.1 μM NBQX (Fig. 1, C).
How can the slowed EPSCs at positive voltages observed here be reconciled with the reported voltage independence of the EPSC decay in CA1 pyramidal cells (6)? A possible explanation is that EPSCs in previous studies were mainly elicited at proximal synaptic inputs, to ensure optimal voltage clamp (6, 12, 13). Because in CA1 pyramidal cells the density of dendritic synaptic AMPARs increases with the distance from the soma (12-14), the influence of intra-cleft electric fields on glutamate may be far smaller at perisomatic synapses. To determine whether this explanation is plausible, we simulated the motion of glutamate molecules in the characteristic environment of small hippocampal synapses. Our Monte Carlo approach (15) was broadly consistent with previous models (16, 17), with the difference that, at each elemental time step dt, each molecule underwent a small displacement due to the local electric field generated by the currents flowing through postsynaptic AMPARs (18). Classically, this displacement is given by , where E is the electric field (15), D is the diffusion coefficient (19), q = −1 for glutamate, F is Faraday's constant, R is the gas constant, and T is temperature. The simulations confirmed that reversal of the AMPAR-mediated synaptic current (by switching to a positive membrane potential) retards the rate of escape of glutamate from the cleft, and consequently slows the EPSC decay (Fig. 1, D; fig. S2, A and B). This effect is consistent with the experimentally observed voltage asymmetry of and depends strongly on the number of available synaptic AMPARs (Fig. 1, E; fig. S2, C). When N is relatively high (>20 open AMPARs at the peak), the effect of electrodiffusion is comparable with that of a two-fold change in the glutamate diffusion coefficient (fig. S3). Conversely, the predicted voltage asymmetry of is much smaller when N is lower, as expected for proximal synapses.
To test experimentally if reducing the density of activated AMPARs indeed attenuates the voltage asymmetry of , we evoked EPSCs at different dendritic locations while visualizing the stimulating pipette position with two-photon excitation microscopy (Fig. 2, A). We confirmed that stimulation stochastically evoked synaptic events in dendritic spines (fig. S4). Having documented the +40/ −70 ratio in baseline conditions, we blocked a proportion of AMPARs by 0.1 μM NBQX which decreased the EPSC amplitude by 40 ± 4% (n = 7; Fig. 2, B). Because this decrease could also alter voltage clamp conditions of our recordings, we increased the stimulus strength to restore the EPSC amplitude to its baseline value (we thus recruited more local synapses operating at lower AMPAR densities; Fig. 2, B). At all dendritic sites >100 μm from the soma, partial AMPAR blockade substantially reduced the +40/ −70 ratio (by 37 ± 5%, n = 7, p < 0.005; Fig. 2, C). In contrast, at more proximal sites, this ratio was initially much smaller than that at distal sites (1.30 ± 0.06 and 2.30 ± 0.18, n = 4 and n = 7, respectively; p < 0.003) and it was not altered by NBQX application even though the EPSC amplitude was reduced to the same degree as at distal sites (by 39 ± 3%, n = 4; Fig. 2, D). Because a reduction in AMPAR density has no effect on receptor kinetics in outside-out patches (Fig. 1, C), our observations are consistent with intra-cleft electric fields decelerating glutamate escape from the cleft upon depolarization at distal, but not proximal excitatory synapses in CA1 pyramidal cells.
We designed an alternative approach to test whether the average dwell time of intra-cleft glutamate following exocytosis is greater at positive than at negative holding voltages (Fig. 1, D). If this is indeed case, the rapidly dissociating competitive antagonist γ-D-glutamylglycine (γ-DGG) should block a higher fraction AMPARs at negative than at positive voltages (16, 20, 21), as predicted by modeling (Fig. 3, A). First, we confirmed that the kinetics of AMPAR-mediated currents evoked by a brief glutamate pulses in outside-out patches in 0.5 mM γ-DGG were voltage-independent (Fig. 3, B). In keeping with our prediction, the reduction of AMPAR EPSC amplitudes by 0.5 mM γ-DGG was 20 ± 7 % greater at negative than at positive holding voltages (n = 8, p < 0.02; Fig. 3, C and D). Blocking glutamate uptake with 50 μM TBOA, to rule out any possible contribution of voltage-dependent transporters, increased this difference to 45 ± 16 % (n = 18, p < 0.001; Fig. 3, D) while reducing the overall effects of 0.5 mM γ-DGG, probably due to an increase in the ambient level of glutamate. In contrast, 0.1 μM NBQX reduced the EPSC amplitude equally at both positive and negative holding voltages, in accordance with modeling predictions (fig. S5). These results further confirm that the sign of the synaptic current influences the rate of escape of glutamate from the synaptic cleft.
Finally, if synaptic currents do influence glutamate diffusion, holding the postsynaptic cell at the receptor reversal potential (zero current) and immediately after presynaptic glutamate release should abolish the effect of voltage on EPSC decay. We therefore switched the holding voltage from the AMPAR reversal potential (0 mV) to either −70 mV or +40 mV during the EPSC decay phase (Fig. 3, E) and compared the outcome with the EPSCs recorded without the voltage jump. The voltage-jump responses showed a 20 ± 5% slower decay at −70 mV (p < 0.005, n = 11), and a 34 ± 10% faster decay at +40 mV (p < 0.01, n = 12; Fig. 3, F) (18). The EPSC waveform was thus influenced by the recent history of current flow (22), consistent with electrodiffusion.
Does electrodiffusion of glutamate influence the activation of other receptors? High-affinity NMDA receptors (NMDARs) both within and outside synapses may be activated by synaptic releases of glutamate (23, 24). This, in addition to voltage-dependent blockade by Mg2+, is likely to mask the effects of electrodiffusion on NMDAR responses. Nevertheless, we observed a voltage-dependent asymmetry of NMDAR EPSCs in the absence of extracellular Mg2+ in cultured hippocampal neurons, which are not surrounded by dense neuropil (Fig. 4, A; figs. S6 and S7). This was again consistent with modeling (fig. S8). In contrast, a relatively low concentration of glutamate applied diffusely to the dendrites - to activate NMDARs at a lower density - evoked responses that showed voltage-independent kinetics but otherwise were similar to those evoked synaptically (Fig. 4, A; fig. S9). Furthermore, reduction of NMDAR EPSCs by the fast-dissociating competitive antagonist Daminoadipate (D-AA) was greater at negative than at positive voltages (fig. S10). Finally, both slowly- and fast-dissociating NMDAR antagonists, 0.4 μM D-CPP and 50 μM D-AA, reduced the voltage-dependent asymmetry of (fig. S11).
These phenomena should play no role in the activation of GABAA receptors because GABA is a zwitterion. We tested this prediction in neuronal cultures, again, to avoid the confounding effects of extrasynaptic and/or tonically active GABAA receptors in slices. Although GABAA receptor-mediated IPSCs did decelerate at positive voltages (with respect to the Cl− reversal potential), the responses were symmetrical when GABA transporters were blocked with 25 μM SKF-89976A (Fig. 4, B). Thus, we observed no evidence that electric fields affect the synaptic dwell time of GABA.
Electrodiffusion of glutamate thus may explain, at least in part, why AMPAR EPSCs at some central synapses are retarded by depolarization (4, 5) and why EPSCs recorded locally at distal dendrites of CA1 pyramidal cells have faster decays than those at proximal dendrites (12). The extent of this phenomenon is likely to vary among synapses, depending for instance on the density or numbers of synaptic receptors. Although electrodiffusion is thus a fundamental feature of AMPAR-mediated synaptic transmission, does it play a physiologically significant role in synaptic signal integration? Distal dendrites of pyramidal neurons can undergo extensive depolarization (including spiking) without exciting the soma (25) (Fig. 4, C), and even modest changes in due to electrodiffusion should in principle affect the time interval over which the input coincidence triggers an action potential. Indeed, simulations with a NEURON (26) model of a CA1 pyramidal cell (12, 27) suggest that, for an arbitrary sample of Schaffer collateral input locations (Fig. 4, C), a ~20% shortening of the synaptic conductance decay could reduce the coincidence detection window by 52 ± 6 % (n = 16, p < 0.001; Fig. 4, D). Another potential consequence of electrodiffusion is that local postsynaptic depolarization, by extending the dwell time of intra-cleft glutamate (Fig. 1, D), may enhance activation of NMDARs. This is likely to interact synergistically with the depolarization-dependent attenuation of postsynaptic glutamate transport (16) and relief of Mg2+ block, thus potentially facilitating induction of NMDAR-dependent synaptic plasticity.
We thank Bengt Gustafsson and Eric Hanse for their comments and support. This work was supported by the Wellcome Trust, the MRC (UK), EU (Promemoria 512012) and HFSP (RGP50/2006), and also by the Swedish Research Council, the Swedish Society of Medicine and the Göteborg Medical Society.