To examine the contribution of di- and triheteromeric receptors to wild-type EPSCs we used cultured hippocampal neurons from mice that lacked expression of N2A or N2B due to homologous recombination (Sakimura et al., 1995
; Kutsuwada et al., 1996
). Our experimental approach assumes that NMDA receptor-mediated EPSCs in knockout neurons result from a homogenous diheteromeric receptor population. To validate this approach, we examined the kinetic and pharmacological characteristics of EPSCs in excitatory neurons. EPSC deactivations from N2A and N2B knockout neurons were fit with the sum of two exponentials (τfast
) and an average weighted time constant (τw
) was calculated to allow easy comparison of kinetic parameters. As shown in , the EPSC deactivations from N2A and N2B knockout neurons differed by more than an order of magnitude (N2B knockout: 22.7 ± 2.2 ms; N2A knockout: 314.9 ± 73.3; n = 35 for each). The τw
values of EPSC deactivations in the knockout neurons match the deactivation in response to brief application of glutamate (1 mM for 1 ms) at recombinant N1/N2A and N1/N2B receptors, respectively (Vicini et al., 1998
). For brevity, we will refer to N1/N2A and N1/N2B diheteromeric NMDA receptors as A-type and B-type receptors, respectively, and to triheteromeric NMDA receptors containing N2A and N2B as AB-type receptors.
A Model System for Diheteromeric Synaptic NMDA Receptors
Zinc and ifenprodil are canonical ligands that have been used to distinguish between diheteromeric receptor subtypes (Paoletti and Neyton, 2007
). Zinc (100 nM free; see Methods) and ifenprodil (3 μM) reduced the EPSC peak from N2B and N2A knockout neurons () to 44.4 ± 1.4 (n = 9) and 18.2 ± 1.5 % of control (n = 16), respectively, comparable to values reported for A-type or B-type recombinant receptors (Rachline et al., 2005
; Perin-Dureau at el., 2002
). Thus the kinetic and pharmacological properties of synaptic receptors from N2A or N2B knockout neurons are consistent with B-type and A-type NMDA receptors, respectively.
To investigate whether excitatory hippocampal neurons expressed other NMDA receptor subunits, we crossed the N2A and N2B knockout mouse lines to generate double knockout (DKO) mice that lacked N2A and N2B. Neurons from these mice showed no NMDA receptor-mediated EPSCs (0/18), but had normal AMPA receptor-mediated EPSCs (). Additionally, most DKO neurons (38/44) lacked any current in response to exogenously applied NMDA (100 μM, ). In the 6 DKO neurons with NMDA-evoked currents, the mean peak amplitude of this current () was less than 0.5% (0.039 ± 0.013 nA) of the mean wild-type controls (8.2 ± 0.6 nA, n = 18). We were able to determine that 2 of these 6 were GABAergic interneurons; however we did not determine the neurotransmitter phenotype of the other 4 neurons. Our data demonstrate that genetic deletion of both N2A and N2B results in excitatory hippocampal neurons with no NMDA receptor-mediated EPSCs, and no NMDA-evoked currents.
In the absence of N2 subunits, NMDA receptors composed of N1 and N3 can form but these receptors are gated by glycine exclusively rather than glutamate and glycine (Chatterton et al., 2002
). Glycine application (100 μM; 500 ms) in the presence of strychnine (2 – 10 μM, Vhold =
−70 mV) resulted in small inward currents in DKO neurons (−4.6 ± 1.0 pA; n = 19). However, when the holding potential was shifted to −35 mV, glycine application produced small outward currents (32.4 ± 9.5 pA; n = 4), consistent with a glycine receptor-mediated chloride conductance. These results confirm that the predominant NMDA receptor subunits expressed in excitatory hippocampal neurons are N1, N2A and N2B and validate our use of neurons from N2A and N2B knockout mice as models for diheteromeric B-type and A-type NMDA receptors.
Diheteromeric synaptic receptors
To estimate the contribution of A- and B-type receptors to EPSCs in wild-type neurons, we measured the NMDA receptor-mediated EPSC deactivations from N2A and N2B knockout neurons. The EPSC deactivations from wild-type, N2A and N2B knockout neurons were well fit with the sum of two exponentials. As expected for A-type receptors, EPSC deactivations from N2B knockout neurons were much faster than B-type EPSC deactivations from N2A knockout neurons. Wild-type EPSCs had intermediate deactivation kinetics ( and ). The behavior of wild-type EPSCs could result from the combination of two diheteromeric receptor subtypes or it could include a contribution from AB-type receptors. In A-type receptor EPSCs (from N2B knockout neurons) τslow contributes minimally to the deactivation because of the small value of Islow. Therefore the wild-type EPSC amplitude at long latency after the peak (>1000 ms) will lack any contribution from A-type receptors. The amplitude of the wild-type EPSC at this long latency then provides an estimate of the maximal contribution from B-type NMDA receptors to the wild-type EPSC deactivation, if A-and B-type receptors are the only NMDA receptor subtypes present.
Deactivation Kinetics of NMDA Receptor-mediated EPSCs
To make this estimate, we peak-scaled wild-type EPSCs with a two exponential function made from the mean B-type EPSC deactivation parameters (from N2A knockout neurons) and measured the ratio of the wild-type current amplitude to that of B-type at 1000 ms after the EPSC peak (greater than 8 times τslow for A-type receptors; ). The amplitude of the wild-type EPSC measured in the time window (200 ms) corresponded to a maximal contribution from B-type receptors of 23.9 ± 2.3% (n = 51; ). This estimate assumes there is no long latency contribution from triheteromeric receptors at 1000 ms after the peak. To determine whether linear summation of A-type and B-type receptors was sufficient to approximate the deactivation of wild-type EPSCs, we added the mean waveforms from A-type and B-type EPSCs, with our estimate of the maximal contribution to the EPSC from B-type receptors ± 1 SD (23.9 ± 16.6%) added to a balance from A-type receptors (76.1 ± 16.6%). The summed waveform did not match the wild-type deactivation waveform and was not well fit using a two exponential function constrained by the mean wild-type deactivation time constants (red dashed line; ). Thus, estimates based on combinations of diheteromeric EPSC waveforms fail to recapitulate the wild-type EPSC deactivation.
NVP-AAM077 and NMDA EPSCs
Our kinetic analysis suggests the presence of a third type of synaptic NMDA receptor. We used a pharmacological approach to address this possibility. An ideal subtype-specific competitive antagonist should simply prevent agonist binding and otherwise not affect channel gating. Although zinc or ifenprodil differ in their affinity for different NMDA receptor subtypes by more than 2 orders of magnitude (Neyton and Paoletti, 2006
), these modulatory ligands prolong NMDA receptor-mediated EPSC deactivations (Tovar and Westbrook, 2012
), making them unsuitable for our analysis strategy. We used the competitive antagonist NVP-AAM077 (NVP) because A-type and B-type receptors differ in their IC50s
for NVP by an order of magnitude (Neyton and Paoletti, 2006
). As expected, A-type receptor EPSCs were more sensitive to NVP by a factor of 10 than B-type receptor EPSCs (A-type: IC50
= 10.01 ± 0.82 nM, nH
= 1.05 ± 0.06; B-type: IC50
=119.41 ± 8.34 nM; nH
= 1.14). Wild-type neuron EPSCs had an intermediate sensitivity to NVP (IC50
= 37.4 ± 6.2 nM; nH
= 1.00 ± 0.10). The single-site isotherm fit to the wild-type dose-inhibition curve (in red) was much better than a two component isotherm using the IC50
s for A-type and B-type receptor (in solid black; ).
Using a Competitive Antagonist to Separate NMDA Receptor Subtypes
If wild-type EPSCs result solely from mixtures of A-type and B-type NMDA receptors, then the deactivation kinetics should become progressively slower with increasing NVP concentrations because low NVP concentrations will preferentially block the faster A-type receptors. Thus at high NVP concentrations (≥ 100 nM) the deactivation of the unblocked EPSC should be composed almost exclusively of B-type receptors. The weighted EPSC deactivation time constants of wild-type EPSCs became slower with increasing NVP concentrations (), consistent with block of A-type receptors. However, wild-type EPSCs never became as slow as those from B-type NMDA receptors. Even in an NVP concentration (300 nM) at which 97% of the A-type receptors are expected to be blocked, the wild-type EPSC deactivation (τw = 204.5 ± 16.7 ms; n = 16) was still faster than the deactivation from B-type receptors (τw = 325 ± 22.2 ms; n = 14; p < 0.0001; ). NVP did not modulate receptor gating because increasing the NVP concentration had no effect on EPSC deactivations of diheteromeric receptors in knockout neurons ().
By similar reasoning, if wild-type EPSCs are composed exclusively of A-type and B-type receptors, we should be able to isolate an NVP-sensitive A-type component because low concentrations of NVP (≤10 nM) have little effect on B-type receptors. We therefore subtracted the wild-type EPSC in 10 nM NVP from the control EPSC (). The NVP-sensitive difference current deactivation in wild-type neurons was significantly slower (τw = 68.9 ± 12.6, n = 18) than the deactivation in N2B knockout EPSCs (τw = 22.1 ± 0.5 ms, n = 20; p < 0.0005). Overall the NVP data confirm the kinetic analysis that A-type and B-type receptors cannot be the sole receptor types contributing to wild-type EPSCs. Additionally, the wild-type EPSC dose-inhibition data was well fit with a single-site isotherm, raising the possibility that a third receptor type with intermediate NVP sensitivity is the major component.
Receptor open probability and the NMDA receptor subtype
The opening kinetics of A-type and B-type synaptic receptors are distinct and result in differences in channel open probability, as measured in knockout neurons (Tovar and Westbrook, 2012
). We asked whether differences in the opening kinetics between receptor subtypes could be used to reveal a third NMDA receptor population. To examine the relative contribution of different synaptic NMDA receptor subtypes we measured the latency to first opening (following neurotransmitter binding) and the conditional channel open probability of knockout and wild-type neurons. We used the high affinity NMDA receptor open channel blocker MK-801 for this purpose (Huettner and Bean, 1988
; ) because only channels that open in response to synaptically-released glutamate will be blocked and because blocked channels do not become unblocked during the experiment. Block by MK-801 is limited by the channel opening rate following glutamate binding (Jahr, 1992; Tovar and Westbrook, 2012
). Thus the EPSC in high concentrations of MK-801 (> 20 μM) is a distribution of how long it takes for channels to open following neurotransmitter binding (first latency), as shown in (top red trace). We used two measures to compare receptor types. We integrated the EPSC in time to measure the charge transfer and used the time at which 60% of charge transfer (Q60
) in MK-801 has occurred (, lower trace). We also measured the probability of a channel having opened by the time of the control EPSC peak (Po
*), which is the ratio of charge at control EPSC peak to the total charge.
The Opening Kinetics of Synaptic NMDA Receptor Subtypes
The values of Q60 and Po* for wild-type EPSC differed significantly from either A-type or B-type receptors (, respectively). To expand this analysis, when we eliminated fast A-type receptors in wild-type neurons with increasing concentrations of NVP, the Q60 increased, as expected from an increasing contribution from B-type receptors (). However, even when we blocked 92% of A-type receptors with 100 nM NVP, the Q60 of the remaining wild-type EPSC (47.8 ± 2.2 ms; n = 7) was still much faster than for B-type receptors (109.7 ± 6.7 ms; n = 14; p < 10−6). Moreover, increasing NVP from 30 to 100 nM halved the EPSC peak, but the Q60 did not change (30 nM: 50.2 ± 8.2 ms; n = 7, p = 0.69). Similarly, the Po* of wild-type EPSCs changed only slightly in NVP (; control: 0.35 ± 0.01; 100 nM NVP: 0.30 ± 0.01; p < 0.05). Thus the opening kinetics of wild-type EPSCs in the absence of A-type receptors must reflect a significant population of receptors other than exclusively B-type receptors.
The relative contribution of A-, B- and AB-type receptors to wild-type EPSCs
We used the NVP dose-inhibition data and the deactivation kinetics of A-type and B-type receptors to estimate the relative contribution of NMDA receptor subtypes to the wild-type EPSCs. We tested five NVP concentrations on each neuron because wild-type EPSC deactivations were considerably more variable than EPSCs from knockout neurons (), as expected if two or more receptor types contribute to wild-type EPSCs. Dose-inhibition data from several wild-type neurons is shown superimposed on fits to the dose-inhibition data from N2A and N2B knockout neurons (). The single-site isotherm fits for each individual neuron were distinctly steeper than the theoretical mixes of A- and B-type receptors. The mean Hill coefficient for the individual neurons was 1.04 ± 0.03 (n = 10), consistent with a predominant contribution from a novel receptor-antagonist interaction.
A Quantitative Estimate of the Contribution of NMDA Receptor Subtypes to Wild-type EPSCs
The pharmacological and kinetic properties of A- and B-type receptor EPSCs were used to estimate the contribution of each NMDA receptor subtype to wild-type EPSCs. This estimate was based on the known reduction of A- and B-type receptors by NVP and the fact that the deactivation of wild-type EPSCs became slower with increasing NVP concentrations, whereas the deactivations of pure A-type or B-type in knockout neurons did not. We used this data to constrain a series of two-exponential fits (A-type, B-type and unconstrained) of the wild-type EPSC deactivation and dose-inhibition data at each of the 5 NVP concentrations tested. The unconstrained variables provided the kinetic and dose-inhibition characteristics of AB-type receptors (see Methods). The contribution of diheteromeric receptors to the wild-type EPSC was surprisingly small (A-type: 20.1 ± 2.8%; B-type: 16.8 ± 3.0%) whereas the contribution of triheteromeric AB-type NMDA receptors predominated (, AB-type: 63.1 ± 4.1%). The model predicts that AB-type receptors have an NVP IC50 of 28.7 ± 1.2 nM with a Hill coefficient of 1.17 ± 0.08 (n = 9), comparable to the mean IC50 and Hill coefficient from fits of each of the 5-point dose-inhibition curves (30.0 ± 1.4 nM). Thus our data indicate that at least two thirds of the wild-type EPSC results from activation of triheteromeric AB-type receptors.
The properties of triheteromeric NMDA receptor-mediated EPSCs
Although triheteromeric receptors have been implicated in synaptic transmission (Gray et al., 2011; Rauner and Köhr, 2011
) it has not been possible to directly examine the properties of these receptors. To investigate triheteromeric receptors in relative isolation, we first evoked wild-type EPSCs in the presence of NVP (100 – 200 nM) and MK-801 (25 μM). At this concentration, NVP prevents opening of 92 – 97% of A-type receptors, thus masking their contribution to the EPSC. The remaining EPSC (resulting from B- and AB-type receptors) was repeatedly evoked in the presence of MK-801 (50 to 75 episodes; ), resulting in a progressive and permanent block of the receptors not protected by NVP (Rosenmund, et al., 1993; see Methods for details). Because B-type receptors are less NVP-sensitive, they should become permanently blocked during stimulation in NVP/MK-801, thus enriching the contribution of A-type receptors to the remaining EPSC. Following removal of NVP and MK-801, the A- and AB-type receptors that had been masked by NVP constituted 36.4 ± 2.8% of the control EPSC (n = 17). Consistent with enrichment of A-type receptors, the τw
of the remaining EPSC after removal of NVP/MK-801 was faster (44.2 ± 1.9 ms) than before NVP/MK-801 application (88.5 ± 5.8 ms; p < 10−6
; ). Finally, to isolate AB-type receptors, EPSCs were recorded in 50 nM NVP which blocks 86% of A-type receptors. With this enrichment method, coupled with the reduction of the EPSC by 50 nM NVP, we estimate the maximal contamination of A-type receptors in the triheteromeric-enriched EPSC to be less than 6% (see Methods).
The weighted deactivation time constant of the triheteromeric AB-type receptor-enriched EPSC was 78.7 ± 3.0 ms (n = 18). The deactivation of AB-type receptor EPSCs was similar to the EPSC kinetics preceding NVP/MK-801 application (; p = 0.31; paired comparison), but substantially faster than B-type receptors. Because removal of the B-type receptors did not affect the deactivation, this result provides further evidence that AB-type NMDA receptors constitute the major receptor type at wild-type hippocampal neuron synapses. Because τslow contributes significantly to the deactivation of enriched AB-type EPSCs (), the contribution of B-type receptors to the wild type EPSC may actually be much less than our previous maximal estimate based on EPSC deactivation (). Consistent with this idea, the estimate of the maximal contribution of B-type receptors in the wild-type EPSCs from these neurons (), done as in , was not affected by isolation of AB-type receptors (18.8 ± 1.9% before and 21.5 ± 1.7% after, n = 17, p = 0.23).
Functional isolation of AB-type receptors permitted us to directly examine their basic properties (; ). The Po
* from these receptors, as measured using the MK-801 method, was 0.28 ± 0.02 (n = 9), closer to the value for B-type than A-type receptors (). The NMDA receptor ligands zinc and ifenprodil bind to homologous regions of the N2A and N2B amino-terminal domain, respectively (Paoletti, 2011
) These ligands have been used extensively to examine A- and B-type receptors, but how these ligands directly affect AB-type receptors at synapses is unknown. As shown in , both ligands decreased the peak of the AB-type receptor-enriched EPSC but only zinc prolonged the deactivation. This result indicates that in AB-type receptors, occupancy of a single high affinity zinc binding site produces synaptic charge redistribution.
Kinetic and Pharmacological Characteristics of AB-type Receptors
To compare the actions of these amino-terminal domain ligands, we plotted the ligand-induced change in total charge transfer as a function of the change in the EPSC peak amplitude (). Competitive antagonists like NVP are not expected to alter the EPSC deactivation kinetics. Thus for any dose of NVP, the reduction in EPSC peak and charge should fall on the unity line, as shown for NVP on A-type EPSCs. Ifenprodil (3 μM) reduced the EPSC peak and charge of AB-type receptors to similar degrees, consistent with its lack of effect on the deactivation, producing a result comparable to that of a competitive antagonist. In contrast, zinc (100 nM, free) greatly prolonged the EPSC deactivation of AB-receptors, resulting in a significant redistribution of charge attributed to the τslow component of deactivation (60.3 ± 1.3% in control; 72.1 ± 1.9% in 100 nM zinc; n = 9; p < 0.001). AB-type receptors thus showed a departure from unity in that was comparable to A-type receptors.