Novel Role for the NMDA Receptor Redox Modulatory Site in the Pathophysiology of Seizures

Inhibition of evoked epileptiform field potentials in Mg²⁺-free ACSF by PQQ and DTNB. The datatraces show the emergence of multiple peaks in the evoked response during exposure to Mg²⁺-free media and the inhibition of these later peaks by subsequent addition of 1 mm DTNB to the bath. As with the spontaneous ictal bursts, the evoked late peaks were not observed during DTNB washout but were restored by DTT application.

Redox modulation of bicuculline-induced epileptiform activity. A, The toptrace shows multiple CA1 population spikes evoked by a single stimulus applied to the Schaffer collateral afferents in the presence of 7 μm BMI. Application of 500 μmDTT resulted in an increased number of population spikes in the evoked response (secondtrace fromtop), and this effect persisted after DTT washout (thirdtrace from top). Subsequent application of 100 μm PQQ reduced the number of spikes to the pre-DTT baseline level (fourthtrace from top), and this effect also persisted after PQQ washout (bottomtrace). B, Similar recordings obtained in the presence of 100 μm AP-5 (APV) showed no modulation by redox agents when NMDARs were blocked.C, The bar graphs summarize the effects of DTT and PQQ on evoked CA1 field potentials in low BMI. DTT (0.5 mm) significantly increased the number of population spikes in the evoked response, and this increase persisted after DTT washout. The effect of DTT was completely reversed by PQQ (100 μm), but the number of spikes in the evoked response did not decrease beyond pre-DTT levels. D, The bar graphs illustrate the lack of redox modulation of epileptiform responses in the presence of 100 μm AP-5 (n = 4). E,Identical experiments in the presence of SOD (100 units/ml) and CAT (210 units/ml) showed no differences from those obtained in the absence of SOD and CAT (n = 4), indicating that the effects of PQQ were not mediated by the generation of free radicals.

NMDA-evoked whole-cell currents are modulated by redox reagents at concentrations that modulated epileptiform activity. A,I–V curves for NMDA-evoked currents were obtained by subtracting the responses to command voltage ramps from −90 to +30 mV before and during brief NMDA superfusion. Subtracted current responses revealed characteristic voltage-dependent currents that reversed between 0 and +10 mV, and the superimposed subtracted ramps for three consecutive NMDA applications show the reproducibility of responses obtained in this manner.B, Superimposed I–V curves show that baseline NMDA-evoked currents (trace 1) were significantly potentiated by exposure to 500 μm TCEP (trace 2) and subsequently diminished by 200 μm PQQ (trace 3). Eachtrace is an average of three subtracted ramp responses.C, Ramp currents from B scaled to the largest current at −20 mV showed that TCEP and PQQ exerted modulation with no effect on the voltage dependence or reversal potential of the NMDA-evoked currents. D, Persistence of the effects of redox reagents on currents evoked by focal NMDA application are shown. The graph shows the peak amplitude of individual current responses to focal NMDA application (holding potential = +30 mV) as a function of time for a CA1 pyramidal neuron that was recorded for 3 hr. (Horizontalbars show the time of superfusion of each redox agent.) The initial application of 400 μm TCEP caused a gradual increase in the peak NMDA-evoked current that remained unchanged during TCEP washout but very rapidly returned to baseline after subsequent exposure to 200 μm PQQ. Responses were potentiated again by a second TCEP application, and this effect was reversed by exposure to 500 μm DTNB, mimicking the effect of PQQ. Individualtraces are shown at the top for thepoints labeled in the graph. Calibration: vertical, 100 pA; horizontal, 10 sec.

NMDAR-mediated synaptic currents were more sensitive to inhibition by DTNB after ictal activity.A, Shown superimposed are averaged (4–6 events) NMDAR-mediated EPSCs recorded at a holding potential of −15 mV in a CA1 neuron from a control slice before and after exposure to 500 μm DTNB. B, The same is shown for a neuron recorded from a slice that had been exposed to Mg²⁺-free media and exhibited spontaneous field activity for 30 min before the initiation of whole-cell recording (see Materials and Methods). C, The bar graphs show summary data. The increased sensitivity of NMDAR-mediated EPSCs to DTNB-induced inhibition indicated that the epileptogenic condition had caused NMDAR redox sites to be reduced by endogenous factors.

Anticonvulsant concentrations of PQQ do not interfere with NMDAR-dependent synaptic plasticity. LTP of the CA1 field EPSP evoked by Schaffer collateral stimulation was induced by tetanic stimulation in control slices (n = 4) and in slices maintained in 200 μm PQQ (n= 4) for 30 min before tetanus and for the duration of recording.A, The superimposed traces show averaged field EPSPs (5 events) before and after tetanus in a representative slice recorded in the presence of PQQ. B, The summary graph shows the mean increase in EPSP slope as a function of time from the tetanus for the two conditions. The magnitude of LTP was not statistically different in the presence versus the absence of PQQ (two-way repeated measures ANOVA with post hoccomparison).