Abstract
Glutamatergic NMDA receptors (NMDAR) are critical for cognitive function, and their reduced expression leads to intellectual disability. Since subpopulations of NMDARs exist in distinct subcellular environments, their functioning may be unevenly vulnerable to genetic disruption. Here, we investigate synaptic and extrasynaptic NMDARs on the major output neurons of the prefrontal cortex in mice deficient for the obligate NMDAR subunit encoded by Grin1 and wild-type littermates. With whole-cell recording in brain slices, we find that single, low-intensity stimuli elicit surprisingly-similar glutamatergic synaptic currents in both genotypes. By contrast, clear genotype differences emerge with manipulations that recruit extrasynaptic NMDARs, including stronger, repetitive, or pharmacological stimulation. These results reveal a disproportionate functional deficit of extrasynaptic NMDARs compared to their synaptic counterparts. To probe the repercussions of this deficit, we examine an NMDAR-dependent phenomenon considered a building block of cognitive integration, basal dendrite plateau potentials. Since we find this phenomenon is readily evoked in wild-type but not in Grin1-deficient mice, we ask whether plateau potentials can be restored by an adult intervention to increase Grin1 expression. This genetic manipulation, previously shown to restore cognitive performance in adulthood, successfully rescues electrically-evoked basal dendrite plateau potentials after a lifetime of NMDAR compromise. Taken together, our work demonstrates NMDAR subpopulations are not uniformly vulnerable to the genetic disruption of their obligate subunit. Furthermore, the window for functional rescue of the more-sensitive integrative NMDARs remains open into adulthood.
Similar content being viewed by others
Introduction
Glutamatergic N-methyl-D-aspartate receptors (NMDARs) are increasingly appreciated for their role in cognitive integration [1,2,3,4]. Mutations that reduce expression or function of NMDARs are a direct cause of intellectual disability [5, 6]. Relatively little is known, however, about whether there is variability across cellular domains in the functional impact of NMDAR genetic compromise. This is a critical area of exploration because NMDARs in different subcellular compartments play distinct neurophysiological roles [2, 7, 8] and experience distinct regulatory environments that may permit differing degrees of homeostatic compensation [9,10,11,12,13,14]. Understanding the relative vulnerability of NMDAR subpopulations to genetic disruption is essential to appreciate mechanisms of cognitive compromise and to identify new treatment approaches.
NMDARs are high affinity ligand-gated channels that are also voltage-dependent and act as coincidence detectors, requiring both glutamate-binding and depolarization to open. It is increasingly appreciated that glutamate travels beyond the synapse and this spillover increases upon strong or repeated stimuli [15,16,17]. Glutamate spillover can activate extrasynaptic NMDARs which are the substrate for integrative phenomena called dendritic plateau potentials. Stimulation of extrasynaptic NMDARs during dendritic plateau potentials allows enhanced cortical output in response to strong, repetitive, or converging inputs [2, 7, 18,19,20] and is critical for cognition and plasticity [2,3,4, 21]. While the fundamental properties of dendritic plateau potentials have been studied extensively, there has been no examination of how they are affected during NMDAR disruption in neurodevelopmental disorders.
Here, we investigated Grin1 knockdown (Grin1KD) mice with a profound deficiency in NMDAR receptor expression and binding [22, 23] and severe cognitive deficits [24]. This mouse is a hypomorph with reduced transcript expression caused by genetic insertion of a Neo cassette in intron 19 of the Grin1 gene [22], resulting in substantially reduced GluN1 protein expression in the brain (<10% of wild-type levels) [24, 25]. Consistent with previous work in this mouse and in other models of developmental cognitive disruption [24, 26], neuronal membrane properties are unaltered in Grin1KD compared to wild type. Furthermore, low-intensity stimuli revealed that neither AMPA receptor (AMPAR) nor NMDAR synaptic currents differed significantly across genotypes. However, a sizable deficit in the Grin1KD NMDAR response was revealed by stronger, repetitive, or pharmacological stimulation. The magnitude of this functional deficit was consistent with deficits observed anatomically in previous receptor binding work. To probe the repercussions of this primarily extrasynaptic deficit in NMDARs, we examined dendritic plateau potentials and found that Grin1KD mice are severely impaired in this integrative domain. In the final experiment, we tested the possibility of restoring cognitively-critical synaptic integration in adulthood, building on recent work showing that adult intervention to increase Grin1 expression achieves meaningful cognitive restoration [24]. We determine that dendritic plateau potentials can indeed be rescued by adult intervention to increase Grin1 expression. Taken together, this work reveals that integrative NMDARs are disproportionately sensitive to genetic disruption but amenable to restoration upon intervention in adulthood.
Materials and methods
Animals
All experiments were approved by the University of Toronto Animal Care and Use Committee and followed Canadian Council on Animal Care guidelines. Mice were group-housed and kept on a 12-h light cycle with food and water access ad libitum Mice for the initial experiments were generated from intercross breeding of C57Bl/6J Grin1 heterozygotes with 129X1Sv/J Grin1 heterozygotes, producing an F1 generation of Grin1KD (Grin1neo/neo) and wild-type (WT) littermate siblings used for experiments [22, 24]. Adult male and female mice were used for experiments (sex-matched and age-matched; age: 102 ± 5 days), with recordings from 56 WT and Grin1KD mice.
For the adult genetic rescue experiments, we used an additional 14 WT, Grin1KD, and Grin1rescue mice of both sexes. The generation of the line permitting adult rescue with tamoxifen is described in greater detail [24]. Starting in adulthood at 84 ± 6 days, all three genotypes of mice for the rescue experiment were treated with tamoxifen chow (TD.140425, 500 mg/kg, Envigo) ad libitum for 14 days. Electrophysiology experiments were conducted upon 38 ± 5 days washout from tamoxifen (sex-matched and age-matched; age:135 ± 3 days). Another cohort of mice similarly treated in adulthood were used after 1 month of washout to measure cortical NMDAR subunit protein levels (details in Supplementary Methods).
Electrophysiological recordings
Prefrontal brain slices were prepared as previously described [27, 28] and as detailed in the Supplementary Methods. Layer 5 pyramidal neurons in the medial prefrontal cortex, including cingulate and prelimbic regions, were visually identified by their pyramidal shape and prominent apical dendrite using infrared differential inference contrast microscopy. Unless otherwise indicated, whole-cell patch clamp electrodes contained potassium-gluconate patch solution (120 K-gluconate, 5 KCl, 2 MgCl2, 4 K2-ATP, 0.4 Na2-GTP, 10 Na2-phosphocreatine, 10 HEPES buffer with the final pH adjusted to 7.3 with KOH, in mM). Normal ACSF (128 NaCl, 10 D-glucose, 26 NaHCO3, 2 CaCl2, 2 MgSO4, 3 KCl, 1.25 NaH2PO4, in mM) was used except for specific experiments where we specify reduced concentration of magnesium ions. All ACSF and pipette solutions used for the following experiments are listed in the Supplementary Methods. Intrinsic membrane properties and excitability were assessed in current-clamp. All recordings were compensated for the liquid junction potential (14 mV).
Evoked excitatory postsynaptic currents
AMPAR-mediated evoked excitatory postsynaptic currents (eEPSCs) were measured in voltage-clamp at a holding potential of −75 mV. A bipolar stimulating electrode (FHC) was located in layer 2/3 for apical dendrite stimulation with pyramidal neurons in layer 5 recorded ~250 µm away from the electrode. For basal dendrite stimulation, the stimulating electrode was placed in the basal dendritic field ~100 µm from the soma of the recorded layer 5 pyramidal neuron. For both apical and basal stimulation paradigms, single pulses of 40 µs duration were delivered at 0.1 Hz, increasing in 10 µA increments. The AMPAR-mediated eEPSCs were analyzed as an average of at least 3 traces with Clampfit (Molecular Devices).
Isolated NMDAR–mediated evoked postsynaptic currents (ePSCs) were measured in voltage-clamp at a holding potential of +60 mV using specialized patch solution (details in Supplementary Methods) to block voltage-gated potassium and sodium channels. These recordings were performed in the presence of modified ACSF (1 mM MgSO4), AMPAR antagonists CNQX (20 µM) or NBQX (20 µM), and GABA receptor antagonists picrotoxin (PTX, 50 µM) and CGP52432 (CGP, 1 µM). Stimulation in the apical or basal dendritic fields were delivered as above. The NMDA receptor–mediated ePSCs were analyzed as an average of 3 traces with Clampfit (Molecular Devices) and D-APV (50 μM) was applied to confirm NMDAR responses.
Enhancing glutamate spillover with high-frequency stimulation
We measured the ability of Grin1KD NMDARs to respond to increasing amounts of glutamate spillover during single pulse and low to high frequency [29, 30] train stimulation at mild and strong stimulus intensities.
Enhancing glutamate spillover pharmacologically
To recruit extrasynaptic NMDA receptors in a different approach, 20 Hz train of mild stimuli was delivered in the apical location. Glutamate spillover was then enhanced with the application of TBOA (30 µM) and LY341495 (1 µM) to block glial glutamate reuptake and mGluR2/3 presynaptic autoreceptors respectively [31, 32].
Pharmacological stimulation with NMDA application
Total synaptic and extrasynaptic NMDAR currents were measured by bath application of NMDA (20 µM, 30 s) in a different subset of brain slices. Voltage-clamp recordings were performed with potassium-gluconate patch solution in a modified ACSF to reduce magnesium blockade (details in Supplementary Methods) as neurons were held at –75 mV. The AMPAR antagonist CNQX (20 µM) was also included. The peak amplitude of the NMDA receptor current was compared to baseline current using Clampfit. In a subset of experiments, D-APV (50 μM) was applied to verify NMDAR mediation of the inward currents.
NMDAR-dependent dendritic plateau potentials
Plateau potentials were generated by stimulation of the basal dendritic field of layer 5 pyramidal neurons, with the stimulating electrode placed within ~100 µm radius of the cell body. Plateau potentials were recorded in current-clamp at an initial membrane potential of −75 mV. They were generated with 10 stimuli at 50 Hz at the minimal stimulus intensity to evoke glutamatergic EPSCs [7, 33]. PTX (20 μM) and CGP52432 (1 μM) were present to block GABA receptors in combination with AMPAR blockers CNQX (20 μM) or NBQX (20 μM) to isolate NMDAR plateau potentials. D-APV (50 μM) was applied to confirm NMDAR dependence of plateau potentials.
Statistics
Statistical tests were performed in Prism 7 (Graphpad). Data are presented as mean ± SEM. Parametric statistical comparisons between responses from different groups of mice were determined using one-way ANOVA, or two-tailed unpaired t tests, and within-cell effects examined with two-tailed paired t tests. Where appropriate, interactions between genotype and other variables were assessed with two-way ANOVA or repeated-measure two-way ANOVA with post hoc Sidak-corrected t tests. Where 3 groups were treated with tamoxifen, the impact of adult intervention to rescue Grin1 expression was assessed with non-parametric Kruskal–Wallis test and Dunn’s post hoc tests due to the distribution of the data. Within cell pharmacological investigations for this dataset were therefore compared with a non-parametric paired test.
Results
Since Grin1KD mice have extremely low NMDAR protein expression (see Supplementary Table S1), we investigated the differential vulnerability of synaptic and extrasynaptic NMDARs to genetic disruption. We performed ex vivo electrophysiology in major output pyramidal neurons of prefrontal cortex from mice deficient in the obligate NMDAR subunit (Grin1KD) and their wild-type (WT) littermate controls (Fig. 1A). We found that neuronal properties, including resting membrane potential, input resistance, capacitance, spike amplitude, and rheobase did not differ significantly between the genotypes (Supplementary Table S2). The input-output relationship showed the expected effect of current (F3,123 = 307.6; p < 0.0001; Fig. 1B, C), but did not differ significantly between the genotypes (F1, 41 = 0.4525; p = 0.50), nor show an interaction F3,123 = 1.123; p = 0.34).
Preserved synaptic glutamatergic responses in Grin1KD mice
To test AMPAR synaptic responses from stimulation in the apical dendritic field, we recorded from layer 5 pyramidal neurons at a holding potential of −75 mV and applied electrically-evoked stimulation in layer 2/3 (Fig. 1D). There was no significant difference between genotypes in the electrical stimulus required to elicit the minimal response (t27 = 0.3; p = 0.8), and response amplitudes were similar in both genotypes (Fig. 1E, F). We observed the expected effect of stimulus strength on response amplitude (F3,115 = 11.04; p < 0.0001), but not an effect of genotype (F1,115 = 2.354; p = 0.13), nor an interaction between genotype and stimulus strength (F3, 115 = 0.20; p = 0.9). These results show that AMPAR-mediated synaptic transmission in response to low-intensity stimulation is similar in WT and Grin1KD prefrontal cortex. Furthermore, miniature EPSCs measured in the presence of TTX did not differ in amplitude or frequency between WT and Grin1KD (Supplementary Fig. S1), confirming that AMPAR synaptic transmission is not altered in Grin1KD.
To isolate NMDAR synaptic responses, we next recorded evoked currents at a holding potential of +60 mV in the presence of AMPAR and GABAA receptor antagonists, using recording pipette solution designed to block voltage-gated potassium and sodium channels. Again, there was no genotype difference in the minimal current required to elicit a response (t24 = 0.4; p = 0.71), nor in response amplitudes across an increasing range of stimuli (Fig. 1G, H). We observed the expected effect stimulus strength on response amplitude (F3,87 = 12.53; p < 0.0001), but no effect of genotype (F1,87 = 0.1926; p = 0.66), nor an interaction between genotype and stimulus strength (F3,87 = 0.1485; p = 0.93). There was no effect of sex nor an interaction between sex and genotype on the amplitude of NMDAR ePSCs (Interaction: F1,54 = 0.081, p = 0.78; Sex: F1,54 = 1.838, p = 0.181). Consistent with the intended NMDAR-mediation of these ePSCs, the evoked currents were strongly suppressed by the selective NMDAR antagonist, D-APV (50 µM): t(10) = 6.1, p = 0.0001). The GluN2B selective antagonist ifenprodil also caused significant suppression of NMDAR ePSCs (F1,25 = 5.879, p = 0.023) in both WT and Grin1KD without any genotype effect (F1,25 = 0.003, p = 0.957). These results demonstrate that the amplitudes of isolated NMDAR currents are similar between genotypes in response to low-intensity stimulation. This unexpected finding was surprising because of the prominent differences in the expression of the obligate subunit and NMDAR binding between the genotypes in previous reports [22, 23].
Deficient extrasynaptic NMDAR responses in Grin1KD mice
We hypothesized that deficits are more prominent in the extrasynaptic NMDAR subpopulation, which can be recruited by stronger electrical stimulation to increase glutamate spillover [34, 35]. Therefore, we delivered stronger single stimuli (80 µA) (Fig. 2A, B). In contrast to the relatively-homogenous effects of low-intensity stimulation, stronger stimuli elicited a significant and substantial difference in NMDAR ePSC amplitude between genotypes (WT: 599 ± 105 pA, n = 9; Grin1KD: 339 ± 62 pA, n = 16; t23 = 2.29; p = 0.032). This result prompted a detailed characterization of extrasynaptic NMDAR in Grin1KD mice using multiple approaches.
To physiologically evoke glutamate spillover and recruit extrasynaptic NMDARs, we used repetitive stimulus trains delivered in layer 2/3. High-frequency stimulation (100 Hz) is particularly known to stimulate glutamate spillover and activate extrasynaptic NMDARs [29, 30]. We measured the ability of Grin1KD NMDARs to proportionately respond to increasing amounts of glutamate spillover during 100 Hz train stimulation at mild vs. stronger stimulus intensities by quantifying the area of the response. Grin1KD neurons showed deficient integration at 100 Hz stimulation compared to WT and were unable to proportionately increase their responses at stronger stimulus strengths (Fig. 2C, D, effect of genotype: F1,36 = 5.478, p = 0.025). The train NMDAR response evoked by strong 100 Hz stimulation is significantly reduced in Grin1KD (Sidak’s post hoc test: t36 = 2.804, p = 0.024). Single pulse or 20 Hz stimulation that release far lesser glutamate compared to 100 Hz, did not reveal a major deficit in Grin1KD (1 pulse: t36 = 0.434, p = 0.962; 20 Hz: t36 = 0.813, p = 0.807).
To further probe extrasynaptic NMDAR involvement, we examined whether pharmacologically boosting glutamate spillover with TBOA and suppression of autoinhibition with LY341495 [31, 32] even at lower frequency stimulation (20 Hz) would reveal a deficit in Grin1KD (Fig. 2E, F). In wild-type mice, repetitive stimulation led to summation of postsynaptic responses, yielding a higher peak response compared to the first input, with further potentiation of peak response caused by glutamate spillover in the presence of TBOA. In Grin1KD, by contrast, pharmacologically boosting spillover did not increase the peak response, leading to a significant interaction between the genotype and spillover conditions (F2,16 = 11.37; p = 0.0008). Repetitive stimulation in the presence of TBOA significantly potentiated the peak response compared to the first stimulus in WT (Sidak’s post hoc test, p = 0.0001) but not in Grin1KD (p = 0.2). These results suggest a lack of extrasynaptic NMDARs in Grin1KD available to be recruited by glutamate spillover.
To reach an even broader group of extrasynaptic receptors, we activated NMDARs using direct pharmacological manipulation with the agonist NMDA. For these experiments, we bath-applied NMDA to the prefrontal slice in the presence of AMPAR antagonist CNQX and low-Mg2+ to permit NMDAR activation at a holding potential of −75 mV. As anticipated [24], pharmacological NMDAR currents were substantially and significantly reduced in Grin1KD mice compared to their littermates (WT: 87 ± 5 pA, n = 23; Grin1KD1: 24 ± 2 pA, n = 21; t42 = 10.6, p = 0.0001; Fig. 2G, H). These pharmacologically-elicited inward currents were suppressed by D-APV (50 µM; WT: n = 5, t4 = 6.2, p = 0.003; Grin1KD mice: n = 7, t6 = 3.5, p = 0.01). Of note, the 3-fold genotype difference in the response to bath NMDA mirrors the difference in NMDAR binding observed in prefrontal cortex in Grin1KD compared to wild-type controls [24].
Stronger, repetitive, and pharmacological stimulations that recruit extrasynaptic NMDARs all unmask genotype differences between the wild-type littermates and Grin1KD mice, consistent with the interpretation that Grin1KD mice have a specific and disproportionate deficit in extrasynaptic NMDARs.
Impaired dendritic plateau potentials in Grin1KD mice: further evidence for extrasynaptic NMDAR disruption
Dendritic plateau potentials can be evoked by spillover of glutamate onto extrasynaptic NMDARs under conditions of high-frequency repetitive stimulation of inputs to basal dendrites [7, 33]. This integrative phenomenon depends on the recruitment of extrasynaptic NMDARs (Fig. 3A) and would be vulnerable if this population were compromised (Fig. 3B). Dendritic plateau potentials are considered an important cognitive substrate to link multiple streams of incoming information and generate burst firing [16, 19, 20, 36], an output signal predicted to exert stronger downstream consequences [37, 38]. Deficient extrasynaptic NMDARs are predicted to have profound consequences for such signaling [7, 33].
To examine basal dendrite plateau potentials in both genotypes, we recorded from layer 5 pyramidal neurons while electrically stimulating inputs in the basal field. AMPAR eEPSCs evoked by basal dendritic stimulation were similar between wild-type and Grin1KD mice, had the expected effect of stimulus strength (F2,66 = 12.7; p = 0.0001), but no effect of genotype (F1, 66 = 0.148, p = 0.7) nor interaction between genotype and stimulus strength (F2,66 = 0.127, p = 0.88, data not shown). Next, we recorded NMDAR plateau potentials in current-clamp in response to trains of stimuli (50 Hz, 10 pulses) in the presence of AMPA and GABA receptor blockade and observed a marked genotype difference (Fig. 3C, D). While wild-type neurons showed clear NMDAR plateau potentials (peak amplitude: 2.15 ± 0.27 mV, n = 8), the train of stimuli did not elicit dendritic plateau potentials in Grin1KD neurons (0.48 ± 0.10 mV, n = 8; t14 = 5.8, p < 0.0001; Fig. 3C, D). This genotype effect was seen in both sexes with no interaction between genotype and sex (Interaction: F1,12 = 0.143, p = 0.711; Sex: F1,12 = 0.061, p = 0.81). Plateau potentials in wild-type neurons could be eliminated by the NMDAR antagonist APV (significant genotype × D-APV interaction: F1,7 = 7.53, p = 0.029; peak amplitude at baseline vs. APV in WT: t7 = 4.12, p = 0.009, Sidak’s post hoc test, data not shown). Grin1KD prefrontal pyramidal neurons have a significant deficit in dendritic plateau potentials compared to those recorded in brain slices from wild-type littermate mice. This measure confirms a profound physiological impact of insensitivity to glutamate spillover in Grin1KD.
Electrophysiological examination of consequences of adult Grin1 rescue
To identify whether a genetic intervention in adulthood could restore crucial aspects of NMDAR function in Grin1KD mice, we tested a tamoxifen-induced Cre-based approach to rescue Grin1 expression in adult mice (as described in Supplementary Methods). This has previously been shown to increase prefrontal NMDAR radioligand binding and reverse key behavioral deficits [24]. These mice are referred to as Grin1rescue mice. In order to ensure equivalent comparison, all 3 genotypes (WT, Grin1KD, Grin1rescue) were treated with tamoxifen at the same age and for the same time course. Western blot of synaptoneurosomal protein extracts confirmed that NMDAR subunit protein levels are significantly reduced in the Grin1KD and increased in Grin1rescue upon tamoxifen treatment (Supplementary Fig S2). Intrinsic electrophysiological properties of prefrontal layer 5 pyramidal neurons including the resting membrane potential, input resistance, capacitance, and action potential amplitude were not significantly different across the tamoxifen-treated, littermate wild-type, Grin1KD and Grin1rescue mice (Supplementary Table S3). Consistent with Fig. 1, synaptic NMDAR ePSCs evoked by minimal stimuli were not significantly different in amplitude across the three genotypes (Supplementary Fig. S3). Furthermore NMDAR ePSC kinetics were also similar (tau decay slow: F2,23 = 1.74, p = 0.197) suggesting that tamoxifen treatment to rescue NMDARs did not result in lasting changes in NMDAR molecular composition [39, 40].
Adult intervention rescues dendritic plateau potentials in prefrontal cortex
To identify whether an adult intervention to boost Grin1 expression can restore dendritic plateau potentials in mice after a lifelong deficit, we examined NMDAR plateau potentials in the three groups of tamoxifen-treated mice. Under these conditions, Grin1KD mice again showed significantly smaller NMDAR plateau potentials compared to wild-type mice, but there was a striking increase in the amplitude of the NMDAR plateau potentials in the Grin1rescue mice compared to the Grin1KD (Fig. 4B). The distribution of the data prompted non-parametric analysis (Kruskal–Wallis test = 11.30, p = 0.003; Dunn’s post hoc tests: WT vs. Grin1KD, Z = 3.18, p = 0.004; Grin1KD vs. Grin1rescue, Z = 2.55, p = 0.032; but no significant difference WT vs. Grin1rescue, Z = 0.76, p = 0.99). Correspondingly, dendritic plateau potentials were significantly suppressed by D-APV in both WT and Grin1rescue mice (Wilcoxon matched-pairs signed rank test: p = 0.016, n = 7, data not shown).
Here we show that increasing expression of the obligate NMDAR subunit in adulthood is sufficient to restore dendritic plateau potentials, consistent with the significant behavioral improvement observed previously [24]. These findings suggest that the boost in Grin1 expression results in an increase in functional extrasynaptic NMDARs, as illustrated in the working model in Fig. 5. This work demonstrates the potential for adult treatments to restore NMDAR function critical for signal integration.
Discussion
Our data reveal that developmental deficiency in the obligate Grin1 subunit leads to a profound bias in NMDAR function in the prefrontal cortex. The subpopulation of synaptic NMDARs recruited by mild stimulation shows markedly greater functional preservation than the extrasynaptic receptors recruited by stronger, repetitive, or pharmacological stimuli. To probe the physiological implications of this uneven pattern of NMDAR disruption, we examined dendritic plateau potentials and identified striking deficits in this integrative phenomenon in Grin1KD mice. Lastly, we discovered that genetic rescue of Grin1 expression restores this form of integrative neurophysiology in the mature brain. Our work suggests that, in mice with NMDAR insufficiency, the window for functional improvement remains open into adulthood.
Broader relevance of this model of NMDAR insufficiency
The Grin1KD mouse has been used as a model to study aspects of schizophrenia, autism spectrum disorder, and most recently as a general model for variants in Grin1 that cause GRIN disorder [41, 42]. Grin1KD mice most closely model Grin1 haploinsufficiency, since they have a genetic modification causing a dramatic reduction in the amount of GluN1 protein and NMDAR without a change in amino acid sequence or in the biophysical properties of the receptor. The Grin1KD mouse expresses low levels of the obligate NMDAR subunit and greatly reduced cortical NMDARs, as measured by radioligand binding [22,23,24]. Understanding the cellular electrophysiological consequences of this substantial deficit is relevant beyond GRIN disorder, since perturbed NMDAR levels are also a key contributing factor to the symptoms of other neurodevelopmental disorders, including those arising from variants in DLG3, SHANK3, and FMRP [5, 43,44,45,46,47]. Our investigation of Grin1KD mice suggest that patients with reduced NMDARs are likely to have a functional deficit in extrasynaptic NMDAR, with a relative preservation of synaptic receptors. Given the historical focus on synaptic NMDAR for neural communication and extrasynaptic receptors for excitotoxicity, it is remarkable that the profound cognitive impairments of Grin1KD mice could be attributed to extrasynaptic deficits. It is also striking that rescue experiments in adulthood, which improve executive function and sensory integration [24], appear to boost functioning of this extrasynaptic population to restore dendritic plateau potentials, a measure of integrative neurophysiology. Future work needs to examine whether adult rescue of Grin1 is sufficient to restore extrasynaptic NMDAR function across brain circuits or whether earlier age at rescue might be required for proper rescue in specific brain circuits. This combination of findings urges greater attention to extrasynaptic NMDARs in developmental disorders and their treatment.
New perspectives on extrasynaptic NMDARs and their integrative role
Extrasynaptic NMDARs, located perisynaptically [10], or non-synaptically on dendritic shafts [10], used to be predominantly described in terms of pathology and their role in activating excitotoxic cell death pathways [48]. However, this view is shifting as growing preclinical research demonstrates the physiological conditions under which extrasynaptic NMDARs are recruited [15,16,17]. This recent body of work points to their role in normal brain function via generation of dendritic plateau potentials [3, 4, 49]. Extrasynaptic receptors bind the small amount of glutamate that escapes the synapse, to become “primed” and ready for rapid activation by subsequent depolarizing input(s). NMDARs on small dendritic branches are thus positioned to detect the activation of multiple synapses close together in space and time. Such temporal and spatial integration is required to generate dendritic plateau potentials [2, 7, 18, 19]. These NMDAR-mediated integration events trigger burst firing [7, 19], a robust neuronal response [37, 38], thought to be essential for behavior-evoked network activity [4, 20, 50]. Our results indicate that developmental disorders with reduced NMDARs are likely to have compromised neurophysiological integration resulting from disrupted extrasynaptic NMDAR population. Intriguingly, an adult intervention yielding an increase in Grin1 expression and NMDAR radioligand binding [24] (to ~60% of wild-type), restores the neurophysiological phenomenon of dendritic plateau potentials. This integrative recovery is consistent with the marked improvement of cognitive performance observed after treatment in adulthood [24].
Subcompartment-specific NMDAR alterations: potential mechanisms and caveats
Microscopic examination of synaptic substructure has shown that NMDARs localized within dendritic spines are organized in highly heterogenous and dynamic nanodomains [51,52,53] that are functionally segregated [54, 55]. By examining function, we show that despite substantial reduction in GluN1 protein level, there is remarkable preservation of synaptic but not extrasynaptic NMDARs in Grin1KD. Protein levels or localization alone cannot explain this disparity. Reduction and preservation of NMDAR function in extrasynaptic vs. synaptic regions respectively may involve a reorganization of nanodomain architecture or stability [56, 57]. Disparate functional consequences across NMDAR populations have been observed in response to different perturbations [58,59,60,61,62]. Research in cell systems demonstrates that NMDARs move between synaptic and extrasynaptic compartments upon pharmacological manipulation [58,59,60,61], or exposure to antibodies from people with anti-NMDAR encephalitis [63]. Receptor trafficking, however, is not the only path to achieve divergent functional outcomes for synaptic and extrasynaptic NMDAR populations. Multiple mechanisms for functional NMDAR enhancement display compartmental specificity, including post-translational modification pathways [64, 65], co-agonism [66,67,68], and mechanisms of receptor desensitization [69,70,71]. The functional preservation of synaptic NMDAR responses in Grin1KD mice may therefore be caused by multiple complex mechanisms, and not necessarily reflect wild-type levels of receptor density in this compartment [24]. Future work also needs to examine such mechanisms that operate to restore extrasynaptic NMDAR function after adult genetic rescue.
While NMDARs are the focus of a large body of work in models of neurodevelopmental disorders, many characterizations use relatively strong stimuli under conditions where “synaptic” measures may inadvertently include a broader population. Here, we pursued carefully calibrated electrical stimulation under several conditions to isolate synaptic NMDARs from their extrasynaptic counterparts. Our strategy was adopted due to the inherent challenges in separating these contributions with pharmacological tools [72, 73]. This problem is particularly difficult to overcome in the prefrontal cortex, where synaptic and extrasynaptic NMDARs show a high degree of overlap in molecular composition and pharmacological affinities [74, 75], complicating specific manipulations. Differentiating synaptic and extrasynaptic NMDAR populations remains challenging, but it will be an increasingly-important focus for future work into the mechanisms of cognitive compromise arising from NMDAR insufficiency.
Clinical relevance and future implications
Current treatments for cognitive disability arising from genetic disruption of NMDARs focus on supportive therapies because it is assumed that lifting cognitive restrictions hard-wired by abnormal brain development is impossible. However, this assumption has recently been challenged. Promising preclinical data [24, 76, 77] suggest the potential for cognitive improvement, even when intervention is delayed until adulthood. If adult treatments are to be seriously pursued, it is essential to appreciate what neural components are functionally compromised and what may be preserved. Here, we address a critical knowledge gap about the specific cellular and circuit mechanisms by which genetic NMDAR disruption impairs cognitive function. We demonstrate that two important NMDAR subpopulations do not suffer equal consequences from genetic disruption of the obligate subunit Grin1. Extrasynaptic NMDARs are disproportionately compromised with resulting disruption of the integrative capacity required for the generation of dendritic plateau potentials. This deficit, strikingly, proves amenable to rescue by intervention in adulthood. Developing effective treatments for the cognitive impairments caused by NMDAR disruption requires the identification of the most efficient targets. Our discovery underscores the need for research into additional approaches to safely enhance extrasynaptic NMDAR functioning. Overall, our findings suggest that deficient integrative mechanisms are amenable to improvement, even with adult intervention.
References
Xu NL, Harnett MT, Williams SR, Huber D, O’Connor DH, Svoboda K, et al. Nonlinear dendritic integration of sensory and motor input during an active sensing task. Nature. 2012;492:247–51.
Palmer LM, Shai AS, Reeve JE, Anderson HL, Paulsen O, Larkum ME. NMDA spikes enhance action potential generation during sensory input. Nat Neurosci. 2014;17:383.
Gambino F, Pages S, Kehayas V, Baptista D, Tatti R, Carleton A, et al. Sensory-evoked LTP driven by dendritic plateau potentials in vivo. Nature. 2014;515:116–9.
Pages S, Chenouard N, Chereau R, Kouskoff V, Gambino F, Holtmaat A. An increase in dendritic plateau potentials is associated with experience-dependent cortical map reorganization. Proc Natl Acad Sci USA. 2021;118:e2024920118.
Lemke JR, Geider K, Helbig KL, Heyne HO, Schutz H, Hentschel J, et al. Delineating the GRIN1 phenotypic spectrum: a distinct genetic NMDA receptor encephalopathy. Neurology. 2016;86:2171–8.
Chen W, Shieh C, Swanger SA, Tankovic A, Au M, McGuire M, et al. GRIN1 mutation associated with intellectual disability alters NMDA receptor trafficking and function. J Hum Genet. 2017;62:589–97.
Polsky A, Mel B, Schiller J. Encoding and decoding bursts by NMDA spikes in basal dendrites of layer 5 pyramidal neurons. J Neurosci. 2009;29:11891–903.
Lafourcade M, van der Goes MH, Vardalaki D, Brown NJ, Voigts J, Yun DH, et al. Differential dendritic integration of long-range inputs in association cortex via subcellular changes in synaptic AMPA-to-NMDA receptor ratio. Neuron. 2022;110:1532–46.e4.
Lau CG, Zukin RS. NMDA receptor trafficking in synaptic plasticity and neuropsychiatric disorders. Nat Rev Neurosci. 2007;8:413–26.
Petralia RS, Wang YX, Hua F, Yi Z, Zhou A, Ge L, et al. Organization of NMDA receptors at extrasynaptic locations. Neuroscience. 2010;167:68–87.
Rao A, Craig AM. Activity regulates the synaptic localization of the NMDA receptor in hippocampal neurons. Neuron. 1997;19:801–12.
Crump FT, Dillman KS, Craig AM. cAMP-dependent protein kinase mediates activity-regulated synaptic targeting of NMDA receptors. J Neurosci. 2001;21:5079–88.
Tse YC, Lopez J, Moquin A, Wong SA, Maysinger D, Wong TP. The susceptibility to chronic social defeat stress is related to low hippocampal extrasynaptic NMDA receptor function. Neuropsychopharmacology. 2019;44:1310–8.
Rajani V, Sengar AS, Salter MW. Src and Fyn regulation of NMDA receptors in health and disease. Neuropharmacology. 2021;193:108615.
Hires SA, Zhu Y, Tsien RY. Optical measurement of synaptic glutamate spillover and reuptake by linker optimized glutamate-sensitive fluorescent reporters. Proc Natl Acad Sci USA. 2008;105:4411–6.
Chalifoux JR, Carter AG. Glutamate spillover promotes the generation of NMDA spikes. J Neurosci. 2011;31:16435–46.
Armbruster M, Hanson E, Dulla CG. Glutamate clearance is locally modulated by presynaptic neuronal activity in the cerebral cortex. J Neurosci. 2016;36:10404–15.
Schiller J, Major G, Koester HJ, Schiller Y. NMDA spikes in basal dendrites of cortical pyramidal neurons. Nature. 2000;404:285–9.
Rhodes P. The properties and implications of NMDA spikes in neocortical pyramidal cells. J Neurosci. 2006;26:6704–15.
Gao PP, Graham JW, Zhou WL, Jang J, Angulo S, Dura-Bernal S, et al. Local glutamate-mediated dendritic plateau potentials change the state of the cortical pyramidal neuron. J Neurophysiol. 2021;125:23–42.
Larkum ME, Wu J, Duverdin SA, Gidon A. The guide to dendritic spikes of the mammalian cortex in vitro and in vivo. Neuroscience. 2022;489:15–33.
Mohn AR, Gainetdinov RR, Caron MG, Koller BH. Mice with reduced NMDA receptor expression display behaviors related to schizophrenia. Cell. 1999;98:427–36.
Duncan G, Miyamoto S, Gu H, Lieberman J, Koller B, Snouwaert J. Alterations in regional brain metabolism in genetic and pharmacological models of reduced NMDA receptor function. Brain Res. 2002;951:166–76.
Mielnik CA, Binko MA, Chen Y, Funk AJ, Johansson EM, Intson K, et al. Consequences of NMDA receptor deficiency can be rescued in the adult brain. Mol Psychiatry. 2021;26:2929–42.
Ramsey AJ, Laakso A, Cyr M, Sotnikova TD, Salahpour A, Medvedev IO, et al. Genetic NMDA receptor deficiency disrupts acute and chronic effects of cocaine but not amphetamine. Neuropsychopharmacology. 2008;33:2701–14.
Antoine MW, Langberg T, Schnepel P, Feldman DE. Increased excitation-inhibition ratio stabilizes synapse and circuit excitability in four autism mouse models. Neuron. 2019;101:648–61.e4.
Venkatesan S, Lambe EK. Chrna5 is essential for a rapid and protected response to optogenetic release of endogenous acetylcholine in prefrontal cortex. J Neurosci. 2020;40:7255–68.
Power SK, Venkatesan S, Lambe EK. Xanomeline restores endogenous nicotinic acetylcholine receptor signaling in mouse prefrontal cortex. Neuropsychopharmacology. 2023;48:671–82.
Carter AG, Regehr WG. Prolonged synaptic currents and glutamate spillover at the parallel fiber to stellate cell synapse. J Neurosci. 2000;20:4423–34.
Shen HW, Scofield MD, Boger H, Hensley M, Kalivas PW. Synaptic glutamate spillover due to impaired glutamate uptake mediates heroin relapse. J Neurosci. 2014;34:5649–57.
Chen S, Diamond JS. Synaptically released glutamate activates extrasynaptic NMDA receptors on cells in the ganglion cell layer of rat retina. J Neurosci. 2002;22:2165–73.
Wild AR, Bollands M, Morris PG, Jones S. Mechanisms regulating spill-over of synaptic glutamate to extrasynaptic NMDA receptors in mouse substantia nigra dopaminergic neurons. Eur J Neurosci. 2015;42:2633–43.
Major G, Polsky A, Denk W, Schiller J, Tank DW. Spatiotemporally graded NMDA spike/plateau potentials in basal dendrites of neocortical pyramidal neurons. J Neurophysiol. 2008;99:2584–601.
Nie H, Weng HR. Glutamate transporters prevent excessive activation of NMDA receptors and extrasynaptic glutamate spillover in the spinal dorsal horn. J Neurophysiol. 2009;101:2041–51.
Anderson CT, Radford RJ, Zastrow ML, Zhang DY, Apfel UP, Lippard SJ, et al. Modulation of extrasynaptic NMDA receptors by synaptic and tonic zinc. Proc Natl Acad Sci USA. 2015;112:E2705–14.
Little JP, Carter AG. Subcellular synaptic connectivity of layer 2 pyramidal neurons in the medial prefrontal cortex. J Neurosci. 2012;32:12808–19.
Snider RK, Kabara JF, Roig BR, Bonds AB. Burst firing and modulation of functional connectivity in cat striate cortex. J Neurophysiol. 1998;80:730–44.
Chan HK, Yang DP, Zhou C, Nowotny T. Burst firing enhances neural output correlation. Front Comput Neurosci. 2016;10:42.
Gray JA, Shi Y, Usui H, During MJ, Sakimura K, Nicoll RA. Distinct modes of AMPA receptor suppression at developing synapses by GluN2A and GluN2B: single-cell NMDA receptor subunit deletion in vivo. Neuron. 2011;71:1085–101.
Vicini S, Wang JF, Li JH, Zhu WJ, Wang YH, Luo JH, et al. Functional and pharmacological differences between recombinant N-methyl-D-aspartate receptors. J Neurophysiol. 1998;79:555–66.
Benke TA, Park K, Krey I, Camp CR, Song R, Ramsey AJ, et al. Clinical and therapeutic significance of genetic variation in the GRIN gene family encoding NMDARs. Neuropharmacology. 2021;199:108805.
Intson K, van Eede MC, Islam R, Milenkovic M, Yan Y, Salahpour A, et al. Progressive neuroanatomical changes caused by Grin1 loss-of-function mutation. Neurobiol Dis. 2019;132:104527.
Tarpey P, Parnau J, Blow M, Woffendin H, Bignell G, Cox C, et al. Mutations in the DLG3 gene cause nonsyndromic X-linked mental retardation. Am J Hum Genet. 2004;75:318–24.
Duffney LJ, Wei J, Cheng J, Liu W, Smith KR, Kittler JT, et al. Shank3 deficiency induces NMDA receptor hypofunction via an actin-dependent mechanism. J Neurosci. 2013;33:15767–78.
Gonatopoulos-Pournatzis T, Niibori R, Salter EW, Weatheritt RJ, Tsang B, Farhangmehr S, et al. Autism-misregulated eIF4G microexons control synaptic translation and higher order cognitive functions. Mol Cell. 2020;77:1176–92.e16.
Uzunova G, Hollander E, Shepherd J. The role of ionotropic glutamate receptors in childhood neurodevelopmental disorders: autism spectrum disorders and fragile x syndrome. Curr Neuropharmacol. 2014;12:71–98.
Ohba C, Shiina M, Tohyama J, Haginoya K, Lerman-Sagie T, Okamoto N, et al. GRIN1 mutations cause encephalopathy with infantile-onset epilepsy, and hyperkinetic and stereotyped movement disorders. Epilepsia. 2015;56:841–8.
Parsons MP, Raymond LA. Extrasynaptic NMDA receptor involvement in central nervous system disorders. Neuron. 2014;82:279–93.
Kerlin A, Mohar B, Flickinger D, MacLennan BJ, Dean MB, Davis C, et al. Functional clustering of dendritic activity during decision-making. Elife. 2019;8:e45966.
Oikonomou KD, Singh MB, Sterjanaj EV, Antic SD. Spiny neurons of amygdala, striatum, and cortex use dendritic plateau potentials to detect network UP states. Front Cell Neurosci. 2014;8:292.
Nair D, Hosy E, Petersen JD, Constals A, Giannone G, Choquet D, et al. Super-resolution imaging reveals that AMPA receptors inside synapses are dynamically organized in nanodomains regulated by PSD95. J Neurosci. 2013;33:13204–24.
MacGillavry HD, Song Y, Raghavachari S, Blanpied TA. Nanoscale scaffolding domains within the postsynaptic density concentrate synaptic AMPA receptors. Neuron. 2013;78:615–22.
Kellermayer B, Ferreira JS, Dupuis J, Levet F, Grillo-Bosch D, Bard L, et al. Differential nanoscale topography and functional role of GluN2-NMDA receptor subtypes at glutamatergic synapses. Neuron. 2018;100:106–19.e7.
Hruska M, Cain RE, Dalva MB. Nanoscale rules governing the organization of glutamate receptors in spine synapses are subunit specific. Nat Commun. 2022;13:920.
Reese AL, Kavalali ET. Single synapse evaluation of the postsynaptic NMDA receptors targeted by evoked and spontaneous neurotransmission. Elife. 2016;5:e21170.
Tang AH, Chen H, Li TP, Metzbower SR, MacGillavry HD, Blanpied TA. A trans-synaptic nanocolumn aligns neurotransmitter release to receptors. Nature. 2016;536:210–4.
Groc L, Choquet D. Linking glutamate receptor movements and synapse function. Science. 2020;368:eaay4631.
Fong DK, Rao A, Crump FT, Craig AM. Rapid synaptic remodeling by protein kinase C: reciprocal translocation of NMDA receptors and calcium/calmodulin-dependent kinase II. J Neurosci. 2002;22:2153–64.
Tovar KR, Westbrook GL. Mobile NMDA receptors at hippocampal synapses. Neuron. 2002;34:255–64.
Groc L, Heine M, Cousins SL, Stephenson FA, Lounis B, Cognet L, et al. NMDA receptor surface mobility depends on NR2A-2B subunits. Proc Natl Acad Sci USA. 2006;103:18769–74.
Ferreira JS, Papouin T, Ladépêche L, Yao A, Langlais VC, Bouchet D, et al. Co-agonists differentially tune GluN2B-NMDA receptor trafficking at hippocampal synapses. eLife. 2017;6:e25492.
Jézéquel J, Johansson EM, Dupuis JP, Rogemond V, Gréa H, Kellermayer B, et al. Dynamic disorganization of synaptic NMDA receptors triggered by autoantibodies from psychotic patients. Nat Commun. 2017;8:1791.
Ladépêche L, Planagumà J, Thakur S, Suárez I, Hara M, Borbely JS, et al. NMDA receptor autoantibodies in autoimmune encephalitis cause a subunit-specific nanoscale redistribution of NMDA receptors. Cell Rep. 2018;23:3759–68.
Yu XM, Askalan R, Keil GJ 2nd, Salter MW. NMDA channel regulation by channel-associated protein tyrosine kinase Src. Science. 1997;275:674–8.
Yu XM, Salter MW. Gain control of NMDA-receptor currents by intracellular sodium. Nature. 1998;396:469–74.
Fossat P, Turpin FR, Sacchi S, Dulong J, Shi T, Rivet JM, et al. Glial D-serine gates NMDA receptors at excitatory synapses in prefrontal cortex. Cereb Cortex. 2012;22:595–606.
Papouin T, Ladepeche L, Ruel J, Sacchi S, Labasque M, Hanini M, et al. Synaptic and extrasynaptic NMDA receptors are gated by different endogenous coagonists. Cell. 2012;150:633–46.
Martineau M, Parpura V, Mothet JP. Cell-type specific mechanisms of D-serine uptake and release in the brain. Front Synaptic Neurosci. 2014;6:12.
Tong G, Shepherd D, Jahr CE. Synaptic desensitization of NMDA receptors by calcineurin. Science. 1995;267:1510–2.
Ehlers MD, Zhang S, Bernhadt JP, Huganir RL. Inactivation of NMDA receptors by direct interaction of calmodulin with the NR1 subunit. Cell. 1996;84:745–55.
Lau LF, Mammen A, Ehlers MD, Kindler S, Chung WJ, Garner CC, et al. Interaction of the N-methyl-D-aspartate receptor complex with a novel synapse-associated protein, SAP102. J Biol Chem. 1996;271:21622–8.
Cull-Candy SG, Leszkiewicz DN. Role of distinct NMDA receptor subtypes at central synapses. Sci STKE. 2004;2004:re16.
Neyton J, Paoletti P. Relating NMDA receptor function to receptor subunit composition: limitations of the pharmacological approach. J Neurosci. 2006;26:1331–3.
Wang H, Stradtman GG 3rd, Wang XJ, Gao WJ. A specialized NMDA receptor function in layer 5 recurrent microcircuitry of the adult rat prefrontal cortex. Proc Natl Acad Sci USA. 2008;105:16791–6.
Wang M, Yang Y, Wang CJ, Gamo NJ, Jin LE, Mazer JA, et al. NMDA receptors subserve persistent neuronal firing during working memory in dorsolateral prefrontal cortex. Neuron. 2013;77:736–49.
Guy J, Gan J, Selfridge J, Cobb S, Bird A. Reversal of neurological defects in a mouse model of Rett syndrome. Science. 2007;315:1143–7.
Mei Y, Monteiro P, Zhou Y, Kim JA, Gao X, Fu Z, et al. Adult restoration of Shank3 expression rescues selective autistic-like phenotypes. Nature. 2016;530:481–4.
Salter MW, Kalia LV. Src kinases: a hub for NMDA receptor regulation. Nat Rev Neurosci. 2004;5:317–28.
Salter MW, Pitcher GM. Dysregulated Src upregulation of NMDA receptor activity: a common link in chronic pain and schizophrenia. FEBS J. 2012;279:2–11.
Acknowledgements
We gratefully thank Wendy Horsfall, Duong Chu, Janice McNabb, and Marija Milenkovic for expert technical assistance.
Funding
This work was generously supported by the Canadian Institutes of Health Research (EKL, PJT-178372, EKL MOP-89825; AJR MOP-119298), Simons Foundation for Autism Research (AJR), CureGRIN Research Foundation (AJR), the Canada Research Chairs program (EKL, Canada Research Chair in Developmental Cortical Physiology), a Pathway Award from the University of Toronto Faculty of Medicine (EKL), as well as multiple fellowships from the Ontario Graduate Scholarship programs (SV, MAB, CAM).
Author information
Authors and Affiliations
Contributions
MAB, SV, and EKL designed study; SV, MAB, and CAM conducted experiments and analyzed data; AJR contributed tools; MAB and SV wrote first draft of paper; MAB, SV, CAM, AJR, and EKL wrote and revised paper.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Venkatesan, S., Binko, M.A., Mielnik, C.A. et al. Deficits in integrative NMDA receptors caused by Grin1 disruption can be rescued in adulthood. Neuropsychopharmacol. 48, 1742–1751 (2023). https://doi.org/10.1038/s41386-023-01619-y
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41386-023-01619-y