Non-ionotropic cross-talk between AMPA and NMDA receptors in rodent hippocampal neurones

Abstract

Many fast excitatory synapses in the hippocampus are enriched with both AMPARs (alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate receptors) and NMDARs (N-methyl-D-aspartate receptors). Their proximity allows them to be activated simultaneously by the same neurotransmitter, L-glutamate. Activation of AMPARs leads to influx of sodium and calcium ions, which can increase or decrease NMDAR activity through sodium concentration-dependent cascades or a calcium-calmodulin-dependent inactivation process, respectively. Here we provide evidence that the activation of AMPARs inhibits NMDARs through a non-ionotropic mechanism. NMDA-induced current in isolated rat CA1 hippocampal cells and nucleated patches of cultured mouse hippocampal neurones decreased when AMPARs were activated. Conversely, when AMPARs were blocked, the NMDA component of glutamate-induced current increased. The inhibitory action of AMPAR activation on NMDAR-mediated current depends upon the open state of AMPA channels and rapidly diminishes after deactivation of AMPARs. The inhibitory action was independent of membrane voltage, univalent cation fluxes and calcium influx. The AMPA-NMDA cross-inhibition also occurred in evoked synaptic current in CA1 neurones from intact mouse hippocampal slices. This cross-talk may play a role in preventing overexcitation during bursting activities in the hippocampus.

Figure 1. Kainate and NMDA-induced currents are sub-additive in hippocampal neurones

A, current traces recorded from the same acutely isolated CA1 hippocampal neurone (V_H = −60 mV) in response to rapid application of kainate (KA, 200 μM for 250 ms), NMDA (100 μM for 250 ms) and both agonists (NMDA + KA at the same concentrations for 250 ms). The grey trace is a calculated sum of the two individual current traces in response to kainate alone and NMDA alone. Note the difference between the current of the calculated sum (grey trace) and the actual current response to co-application of kainate and NMDA. To obtain full-sized NMDAR-mediated currents, the bath contained 20 μM glycine and zero magnesium. B, bar graph showing the average current amplitude induced by kainate alone (KA), NMDA alone (NMDA), the calculated sum (grey bar) and co-application of the two agonists (NMDA + KA, black bar). The current amplitude for co-application of NMDA and KA was significantly less than the calculated sum of the two currents induced by these two agonists individually (P < 0.01, n = 7).

Figure 2. Kainate dose-dependently reduces NMDA-induced current in hippocampal neurones

A, superimposed current records from a single isolated CA1 hippocampal neurone (V_H = −60 mV) in response to a series of kainate pulses of different concentrations (KA, filled bar, 0, 12, 40, 120, 400 and 1200 μM). A 400 ms pulse of 100 μM NMDA (open bar) was delivered during the middle of each 1200 ms kainate pulse. B, dose-response curve for kainate-induced current (I_KA). C, NMDAR current (I_NMDA) induced by 100 μM NMDA was significantly reduced by co-application of kainate at concentrations of 40 μM or higher (*P < 0.05; **P < 0.01, n = 7). I_NMDA is the difference between the peak of the current during NMDA application and the current before application of NMDA. D, all current recordings are from the same isolated hippocampal neurone in response to eight different concentrations of NMDA during control conditions (top traces) and in the presence of 200 μM kainate (bottom traces). E, dose-response relationships of NMDA-induced current are illustrated during control conditions (•) and in the presence of kainate (200 μM, ○). A logistic equation was used to estimate the maximum response (I_NMDA,max) and EC₅₀ for each individual cell. In the presence of kainate I_NMDA,max was reduced from 1.66 ± 0.37 nA to 0.89 ± 0.15 nA (n = 5, P = 0.038). However, there was no difference in the estimated EC₅₀ during control (61 ± 5 μM) and in the presence of kainate (47 ± 7 μM, n = 5, P = 0.18).

Figure 3. Kainate reduces NMDA-induced current in nucleated patches of hippocampal neurones

A, current traces obtained from the same nucleated patch of a cultured hippocampal neurone with KMeSO₄-based pipette solution to show NMDA-induced (2 mm) current under control conditions is larger than the same concentration of NMDA-induced current in the presence of kainate (2 mm). B, currents induced by different concentrations (indicated under the bars) of NMDA are shown in control conditions (open bars) and during kainite-induced (concentrations indicated) response (filled bars). Note that the current scale is different for the left and right panels of the histogram. NMDA-induced current was significantly lower in the presence of kainate than in control conditions for all concentrations tested.

Figure 4. The amplitude of NMDAR current in isolated CA1 pyramidal cells is larger when AMPARs are blocked

A, currents (V_H = −60 mV) from an isolated CA1 hippocampal cell induced by a brief pulse (400 ms) of a mixture of kainate (200 μM) and NMDA (50 μM, open bars): 1, under control conditions; 2, in the presence of 25 μM CPP; 3, after wash-out of CPP; 4, in the presence of 5 μM NBQX; 5, in the presence of NBQX + CPP. B, CPP-sensitive current in the control condition (I_NMDA = I₁ – I₂) and in the presence of NBQX (I_NMDA* = I₄ – I₅). C, fraction of total current carried by NMDAR channels in the absence (fI_NMDA = (I₁ – I₂) / I₁) and presence of NBQX (fI_NMDA* = (I₄ – I₅) / I₃). fI_NMDA* is significantly larger than fI_NMDA. D, currents induced by a brief pulse of l-glutamate (0.5 mm for 100 ms, filled bars) from a single isolated hippocampal neurone during: 1, control conditions; 2, in the presence of 50 μM CPP; 3, wash; 4, in the presence of 10 μM NBQX; 5, in the presence of NBQX + CPP. E, CPP-sensitive current in the control condition (I_NMDA = I₁ – I₂) and in the presence of NBQX (I_NMDA* = I₄ – I₅). F, fraction of total current carried by NMDAR channels in the absence (fI_NMDA) and presence of NBQX (fI_NMDA*). fI_NMDA* was reliably larger than fI_NMDA regardless of which antagonist pair was used. The smaller values for fI_NMDA and fI_NMDA* observed with the NMDAR antagonist D-AP5 (20 μM) are due to incomplete block of NMDAR current at this D-AP5 concentration. The concentrations of the antagonists used were: CPP (25-50 μM), NBQX (10 μM), CNQX (10 μM), D-AP5 (20 μM). A near-saturating concentration of l-glutamate (3 mm) and higher concentrations of CPP (Hi CPP, 100 μM) and NBQX (Hi NBQX, 20 μM) were used for the last pair of bars.

Figure 5. The recovery time course of NMDA current after activation of AMPARs

A, two sets of superimposed current recordings obtained from the same isolated neurones in response to a brief pulse of NMDA (100 μM, open bars) in control conditions (left panel) and at variable times after a prepulse of kainate (200 μM, filled bar, right panel). B, summarized data show the time course of the recovery process of NMDA-induced current shortly after kainate-induced current (•, n = 10). ○, inhibition of NMDA-induced current during the kainate-induced current.

Figure 6. The inhibition of NMDAR current by AMPARs is independent of voltage, the direction of monovalent cation flow and the availability of extracellular monovalent cations

A, NMDAR current in the absence (I_NMDA, •) and presence of CNQX (10 μM, I_NMDA*, ○) plotted against voltage clamp holding potential. B, fI_NMDA* is larger than fI_NMDA at all holding potentials (-60≈+60 mV, P < 0.01, two-way ANOVA, n = 4). D-AP5 (20 μM) was used to isolate NMDAR current. C and D, current responses to glutamate pulses at different holding potentials (70, 40, 10, −20, −50, −80, −110 mV) in Na⁺- and K⁺-free, 10 mm Ca²⁺ extracellular medium. C, the AMPAR component of the glutamate-induced current was measured after blocking the NMDAR component with 25 μM CPP. D, the NMDAR component of the glutamate-induced response was measured after blocking the AMPAR component with 10 μM CNQX. E, I-V curves for the AMPAR and NMDAR current components under conditions described for C, and D. Note the very different reversal potentials for the two channel subtypes. F, after voltage clamping at the AMPAR channel reversal potential (V_H −77 mV), when the average AMPAR channel current is zero, the NMDAR current increased significantly in the presence of 10 μM CNQX (n = 7). A later application of CPP abolished the glutamate-induced current in the presence of CNQX (data not shown).

Figure 7. The role of intracellular calcium, G-proteins and protein kinases in AMPAR channel-induced inhibition of NMDAR current

In each case, the fraction of current through the NMDAR channels was calculated as described in Fig. 3. In all cases, AMPARs were blocked with 10 μM CNQX and NMDARs with 50 μM CPP. The fI_NMDA was significantly lower than fI_NMDA* in the presence of divalent ion chelators (11 mm EGTA and 10 mm BAPTA), in the presence of either an agonist (400 μM GTP-γ-S) or an antagonist (2 mm GDP-β-S) of G-protein activity, in the presence of the protein tyrosine kinase inhibitor Lavendustin A (1 μM), protein kinase C inhibitor chelerythrine (10 μM) and also a general protein kinase inhibitor H7 (100 μM). The number of cells in each group of data is indicated at the top of the bars (*P < 0.05 and **P < 0.01; Student's paired t test).

Figure 8. The inhibitory effect of AMPAR activation on NMDAR channel current occurs at intact synapses in hippocampal slices

A, composite EPSCs from CA1 pyramidal cells during whole cell voltage clamp at +60 mV holding potential. The responses are evoked by 10 stimuli of the Schaffer-collateral pathway at 100 Hz. Responses before (con) and after the addition of 20 μM D-AP5 are shown (left panel). Responses before the addition of any drug (con), after the addition of 10 μM CNQX and after the further addition of 20 μM D-AP5 (right panel). B, fraction of the peak EPSC blocked by D-AP5. This fraction was significantly greater in the presence (n = 5) than in the absence of CNQX (n = 4, P = 0.04; Student's t test).