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Excessive NMDA receptor activation and excitotoxicity underlies pathology in many neuropsychiatric and neurological disorders, including hypoxia/ischemia. Thus, the development of effective therapeutics for these disorders demands a complete understanding of NMDA receptor (NMDAR) activation during excitotoxic insults. The extrasynaptic NMDAR hypothesis posits that synaptic NMDARs are neurotrophic/neuroprotective and extrasynaptic NMDARs are neurotoxic. In part, the extrasynaptic hypothesis is built on observed selectivity for extrasynaptic receptors of a neuroprotective use-dependent NMDAR channel blocker, memantine. In rat hippocampal neurons we found that a neuroprotective concentration of memantine shows little selectivity for extrasynaptic NMDARs when all receptors are tonically activated by exogenous glutamate. This led us to test the extrasynaptic NMDAR hypothesis using metabolic challenge, where the source of excitotoxic glutamate buildup may be largely synaptic. Three independent approaches suggest strongly that synaptic receptors participate prominently in hypoxic excitotoxicity. First, block of glutamate transporters with a non-substrate antagonist exacerbated rather than prevented damage, consistent with a primarily synaptic source of glutamate. Second, selective, preblock of synaptic NMDARs with a slowly reversible, use-dependent antagonist protected nearly fully against prolonged hypoxic insult. Third, glutamate pyruvate transaminase (GPT), which degrades ambient but not synaptic glutamate, did not protect against hypoxia but protected against exogenous glutamate damage. Together, these results suggest that synaptic NMDARs can mediate excitotoxicity, particularly when the glutamate source is synaptic and when synaptic receptor contributions are rigorously defined. Moreover, the results suggest that in some situations therapeutically targeting extrasynaptic receptors may be inappropriate.
Excitotoxicity is an important contributor to many neurodegenerative diseases and neuropsychiatric disorders. Excitotoxicity is initiated in part through excessive NMDA receptor (NMDAR) activation (Rothman and Olney, 1987). However, NMDAR antagonists have deleterious side effects due to the critical role NMDARs play in normal synaptic signaling and plasticity (Olney et al., 1989; Ikonomidou et al., 1999). Thus, there is a need for therapeutics to balance preservation of normal function with neuroprotection. One hypothesis, the extrasynaptic NMDAR toxicity hypothesis (Hardingham et al., 2002; Lipton, 2007; Hardingham and Bading, 2010), is attractive in this regard because extrasynaptic receptors could be therapeutically targeted without compromising normal synaptic function. This hypothesis posits that extrasynaptic NMDARs selectively trigger cell death pathways while synaptic receptors trigger trophic pathways. However, the breadth of the extrasynaptic hypothesis in models of excitotoxicity has not been fully tested.
Accumulating evidence suggests that extrasynaptic NMDARs play a unique role in cell death. Extrasynaptic receptors may become activated when excessive levels of glutamate spill out of the synapse during prolonged depolarization. Another source of extrasynaptic glutamate during depolarizing insults is the reverse operation of glutamate transporters (Jabaudon et al., 2000; Rossi et al., 2000). These factors could conspire to initiate signaling cascades uniquely activated by extrasynaptic receptors (Hardingham et al., 2002; Dick and Bading, 2010) and that overwhelm trophic effects of synaptic receptor activation. (Hardingham et al., 2002; Soriano et al., 2006; Papadia et al., 2008). Support for the extrasynaptic receptor hypothesis comes from neuroprotective effects of a drug, memantine, which may selectively target extrasynaptic receptors (Leveille et al., 2008; Okamoto et al., 2009; Xia et al., 2010). However, memantine’s selectivity has not been evaluated under conditions of excessive receptor stimulation that may occur during excitotoxic insults. Other studies supporting the extrasynaptic hypothesis have employed exogenous excitotoxic agonists, which may be prevented from reaching synaptic receptors by glial glutamate uptake (Sattler et al., 2000; Sinor et al., 2000) and therefore provide a somewhat artificial excitotoxic stimulus. Whether the extrasynaptic hypothesis applies to insults involving endogenous glutamate, such as metabolic challenge, remains uncertain.
Here we specifically characterized memantine’s selectivity for receptor populations during excitotoxic paradigms and directly investigated the role synaptic receptors play in hypoxia-induced excitotoxic insults. We demonstrate that memantine blocks synaptic receptors when activated equivalently to extrasynaptic receptors. Furthermore, our findings suggest that memantine’s protection may result from blocking excessively activated synaptic receptors. We demonstrate that synaptic receptors appear to account for virtually all the MK-801-sensitive cell death caused by hypoxia. Our findings argue for a tempered view of the extrasynaptic receptor hypothesis.
Hippocampal cultures were prepared as either mass cultures or microcultures (as indicated in Figure legends) from postnatal day 1–3 rat pups of either sex were anesthetized with isoflurane, under protocols consistent with NIH guidelines and approved by the Washington University Animal Studies Committee. Protocols were adapted from earlier descriptions (Huettner and Baughman, 1986; Bekkers et al., 1990; Tong and Jahr, 1994; Mennerick et al., 1995). Hippocampal slices (500 µm thickness) were digested with 1 mg ml−1 papain in oxygenated Leibovitz L-15 medium (Invitrogen, Gaithersburg, MD, USA). Tissue was mechanically triturated in modified Eagle's medium (Invitrogen) containing 5% horse serum, 5% fetal calf serum, 17 mM D-glucose, 400 µM glutamine, 50 U ml−1 penicillin and 50 µg ml−1 streptomycin. Cells were seeded in modified Eagle's medium at a density of ~650 cells mm−2 as mass cultures (onto 25 mm cover glasses coated with 5 mg ml−1 collagen or 0.1 mg ml−1 poly-D-lysine with 1 mg ml−1 laminin) or 100 cells mm−2 as "microisland" cultures (onto 35 mm plastic culture dishes coated with collagen microdroplets on a layer of 0.15% agarose). Cultures were incubated at 37° C in a humidified chamber with 5%CO2/95% air. Cytosine arabinoside (6.7 µM) was added 3–4 days after plating to inhibit glial proliferation. The following day, half of the culture medium was replaced with Neurobasal medium (Invitrogen) plus B27 supplement (Invitrogen).
DIV 14–15 mass cultures were subjected to hypoxia in a commercially available chamber (Billups-Rothenberg Company) humidified and saturated with 95% nitrogen and 5% CO2 at 37° C for the specified amount of time (2.5 h). The gas exchange followed the specifications of the chamber manufacturer (flow of 20 L per minute for four minutes to achieve 100% gas exchange). After the insult, we removed cells from the chamber and incubated them under standard culture conditions until the cell death assay (24 h later). Procedures for controls included all media changes and incubation times relevant to the insult conditions. For experiments in which exogenous glutamate exposure was used in place of hypoxia, medium was exchanged with conditioned Neurobasal medium from sibling cultures, 50 µM glutamate was added, and cultures were incubated for 2.5 h at 37° C. The concentration of glutamate was chosen to approximate the death observed during the same period (2.5 h) of hypoxia. The cultures were then returned to their original medium for 24 h until the cell death assay was performed. For experiments in which glutamate pyruvate transaminase (GPT) was used, medium was replaced with conditioned medium prepared with GPT (5 U ml−1, Roche) and 2 mM pyruvate (Sigma). The neurons were then exposed to hypoxia or 50 µM glutamate. Following 2.5 h, cultures were returned to their original medium for 24 h until the cell death assay was performed.
All cell death studies were performed on mass cultures at 14–15 DIV. Mass cultures at this age contained an astrocyte monolayer, upon which neurons sit. Thus, neurons have strong diffusional contact with the extracellular medium, but processes also exhibit some glial ensheathment (Mennerick et al., 1996). Absolute ratios of astrocyte numbers to neuronal numbers from 19 fields in 4 platings was 5 ± 1 astrocytes to one neuron, assessed by counts of nuclei stained with Hoechst dye. Although this is a larger astrocyte to neuron ratio than found in vivo, diffusional contact in the cultures is higher, thereby limiting transmitter pooling.
We used trypan blue dye to identify compromised neurons 24 h after the insult. Culture medium was removed and replaced with 1 ml of 0.4% trypan blue dissolved in phosphate buffered saline. Cells were incubated in dye at 37° C for 5 min, washed with phosphate buffered saline, then fixed with 4% paraformaldehyde and 0.2% glutaraldehyde at room temperature. Cells were visualized with a 20× objective using both phase-contrast and brightfield microscopy to confirm healthy neuronal profiles (phase-contrast) and verify trypan blue uptake (brightfield). The total numbers of dead and intact neurons were counted and expressed as a percentage of trypan blue positive dead cells to total cells. The average of ten microscope fields for each condition was treated as a single data point for purposes of statistics. Although trypan blue in principle may miss apoptotic cell death in which membranes remain uncompromised until late in the degeneration process, the total number of viable plus non-viable cells did not differ statistically between control and hypoxia conditions (p = 0.4, n = 6 experiments). Therefore, our assay accounted for all or nearly all cells compromised by the insult. We also cannot exclude that possibility that some trypan-blue positive cells could recover following insult. However, this possibility was minimized by use of the 24 h latent period between insult and assessment. In most cases by this time point trypan-positive cells no longer had discernable plasma membranes under brightfield or phase contrast optics.
Whole-cell recordings were performed at room temperature from neurons cultured for 10–15 days using a Multiclamp 700B amplifier (Molecular Devices, Sunnyvale, CA, USA). For recordings, cells were transferred to an extracellular solution containing (in mM): 138 NaCl, 4 KCl, 2 CaCl2, 10 glucose, 10 HEPES, 0.01 glycine (a saturating concentration for the co-agonist site on the NMDA receptor), 0.001 2,3-dihydroxy-6-nitro-7-sulfonyl-benzo[f]quinoxaline (NBQX) at pH 7.25. In some experiments, D-2-Amino-5-phosphonovalerate (D-APV, 10–50 µM) was added to block NMDARs as indicated. Experiments in which synaptic NMDARs were preblocked with 10 µM dizocilpine maleate (MK-801) prior to recording or to hypoxic challenge used a bath solution with lowered Ca2+ (1 mM), 50 µM bicuculline, no added NBQX, and no added Mg2+, unless otherwise indicated. The tip resistance of patch pipettes was 3–6 MΩ when filled with an internal solution containing (in mM): 130 potassium gluconate, 2 NaCl, 0.1 EGTA, and 10 HEPES at pH 7.25, adjusted with KOH. In experiments examining current responses to exogenous agonists, cesium methanesulfonate was used in place of potassium gluconate to block potassium channels. Holding voltage was typically −70 mV. Access resistance for synaptic recordings (8–10 MΩ) was compensated 80–100%. For autaptic responses, cells were stimulated with 1.5 ms pulses to 0 mV from −70 mV to evoke transmitter release. In experiments in which NMDAR EPSCs were examined, AMPAR EPSCs and GABAergic IPSCs were blocked with 1 µM NBQX and 25 µM bicuculline respectively. Drugs were applied with a gravity-driven local perfusion system from a common tip. The estimated solution exchange times were <100 ms (10–90% rise), estimated from junction current rises at the tip of an open patch pipette.
Experiments were performed as previously described ((Mennerick et al., 2010). Briefly, multielectrode arrays (MEAs) were coated with poly-D-lysine and laminin per the manufacturer’s instructions, and dispersed cultures were grown as described above. At DIV7 and DIV10, 1/3 of the media was removed and replaced with fresh Neurobasal supplemented with B27 and glutamine. Recordings were made with the MEA-60 recording system (MultiChannel Systems, Reutlingen, Germany) with the headstage in an incubator set at 29°C and equilibrated with 5% CO2 in room air with no additional humidity. The lower temperature was necessary because the electronics in the headstage generate ~7°C of excess heat. The MEA itself rested on a heating plate inside the headstage to maintain the cultures at 37°C. To allow extended recordings in the dry incubator, cultures were covered with a semipermeable membrane that allows diffusion of oxygen and carbon dioxide but not water (Potter and DeMarse, 2001). Data were amplified 1100 times and sampled at 5 kHz. Spikes were detected by threshold crossing of high pass filtered data. The threshold was set individually for each contact at five standard deviations above the average RMS noise level. Baseline data were recorded in culture media. We then recorded activity in culture media containing an additional 50 µM glutamate. Finally, we collected another data set in original growth media. Statistics were performed on 30 minute treatment periods.
Electrophysiology data acquisition and analysis were performed primarily using pCLAMP 10 software (Molecular Devices). All cell counts and electrophysiological measurements were processed with Microsoft Excel and are presented as mean ± S.E.M. Statistical significance was determined using a Student’s two-tailed t test with a Bonferroni correction for multiple comparisons where appropriate. Data plotting, statistical analysis and figure preparation were performed with Sigma Plot (Systat; San Jose, CA) and Adobe Photoshop (San Jose, CA, USA). Decay time constants were measured using standard exponential fitting functions using the variable metric algorithm in pClamp software.
All drugs were obtained from Sigma (St. Louis, MO, USA) except for D-APV, NBQX, and TBOA which were obtained from Tocris (Ellisville, MO, USA) and GPT from Roche (Indianapolis, IN, USA). GPT was supplied as a suspension in 3.2 M ammonium sulfate and was prepared by pelleting out the suspended protein with 32,500 rpm centrifugation, removing the supernatant and dissolving in HEPES-buffered saline (138 mM NaCl, 4 mM KCl, 10 mM HEPES). This procedure resulted in a small volume of residual suspension medium in final working solutions (discussed in Results).
To begin to assess the potential role of synaptic and extrasynaptic receptors in excitotoxic cell death mediated by endogenous glutamate, we first examined effects of the neuroprotective open-channel blocker memantine. Cultures were subjected to 2.5 h of hypoxia and allowed to recover for 24 h to capture delayed cell loss using a trypan blue stain. Under these conditions, we consistently achieved robust cell death of approximately 50% (Figure 1). We found that memantine at concentrations below 10 µM conferred only variable and trend-level protection from hypoxic excitotoxicity, but 10 µM memantine clearly protected neurons (Figure 1). This is consistent with previous reports suggesting that concentrations ≥ 5 µM memantine are neuroprotective (Chen et al., 1998; Volbracht et al., 2006). We therefore focused additional studies on 10 µM memantine.
Memantine is selective for extrasynaptic receptors over synaptic receptors when the presentation of glutamate to synaptic receptors is transient, which is the case under physiological conditions (Lester et al., 1990; Xia et al., 2010). However, the basis for memantine’s apparent selectivity remains unclear. Memantine could possess a higher affinity for extrasynaptic versus synaptic receptor populations. Alternatively, as an open-channel blocker, memantine’s selectivity could arise as a result of phasic versus sustained patterns of synaptic versus extrasynaptic receptor activation (Johnson and Kotermanski, 2006; Xia et al., 2010). To distinguish these alternatives, we developed protocols to equivalently activate synaptic and extrasynaptic receptor populations, using sustained agonist presentation.
During hypoxia, glutamate may accumulate tonically to activate both synaptic and extrasynaptic receptors. To partly simulate tonic glutamate build up, we used sustained glutamate (1 µM) application onto NMDAR populations and evaluated antagonism by 10 µM memantine. The concentration of glutamate was chosen to be less than the EC50 concentration of 2.3 µM (Patneau and Mayer, 1990). We used a low concentration of NMDA to probe the availability of receptors before and after conditioning with glutamate plus memantine (Figure 2A). When NMDA was re-applied following conditioning, currents exhibited an initially reduced response (red trace inset in Figure 2A). This initial suppression (red trace in Figure 2A inset) is attributable to memantine bound to (trapped in) closed NMDAR channels (Johnson and Kotermanski, 2006; Kotermanski et al., 2009). When all NMDAR populations were activated with glutamate, memantine inhibited currents by approximately 80% compared to the baseline NMDA currents (Figure 2A, D). During the sustained NMDA re-application, current re-emerged gradually as channels were re-opened and allowed trapped memantine to dissociate.
We next enriched responses for extrasynaptic NMDARs by eliciting action potential-dependent EPSCs in the presence of the slowly dissociating open-channel blocker MK-801 (Figure 2B, left inset). Memantine antagonism at the enriched extrasynaptic NMDAR pool was again assessed by NMDA re-application and showed similar initial memantine inhibition compared to the total NMDAR pool (Figure 2A, B, D). To determine whether memantine blocked synaptic receptor channels during sustained exogenous glutamate application, we examined evoked NMDAR EPSCs on neurons before and after the memantine antagonism protocol (Figure 2C). Memantine inhibition of EPSCs was strong, although slightly weaker than antagonism exhibited at isolated extrasynaptic receptors (Figure 2D). These results are consistent with the idea that extrasynaptic receptors are slightly more sensitive to memantine than synaptic receptors (Okamoto et al., 2009; Xia et al., 2010). However, the strong inhibition of synaptic receptors at neuroprotective memantine concentrations suggested that synaptic receptors deserve attention as possible targets of memantine and as mediators of toxicity.
Figure 2 examines effects of memantine on tonically activated NMDAR populations. It is also possible that synaptic receptors are phasically but repeatedly activated during endogenous insults. Although low memantine concentrations (1 µM) have been comprehensively evaluated for effects on repetitive activation of synaptic receptors, neuroprotective concentrations of memantine (10 µM) have not been fully investigated. We found that 10 µM memantine applied continuously while evoking NMDA EPSCs initially produced only about 30% inhibition. However, over the course of multiple stimuli, inhibition reached ~80% (n = 5; Figure 3) and recovered very little with memantine washout (Figure 3B), suggesting inhibition by memantine bound to closed channels. Steady-state inhibition was likely not achieved in these experiments; with additional stimulation it is possible that memantine might inhibit EPSCs more completely. Nevertheless, the observations demonstrate that neuroprotective concentrations of memantine strongly antagonize synaptic receptors when receptors are repetitively activated.
Although the absolute magnitude of memantine’s effect were likely exaggerated in these experiments by the absence of Mg2+, which is known to interact with memantine antagonism (Kotermanski and Johnson, 2009), the results confirm that memantine’s selectivity arises largely as a result of different dynamics of agonist presentation at synaptic and extrasynaptic receptors. If during hypoxic insult the mode of agonist presentation is synaptic but becomes more sustained or repetitive than under physiological conditions, then it is possible that memantine (and other neuroprotective NMDAR antagonists) act by blocking synaptic receptors. This would contradict recent evidence that extrasynaptic receptors play a privileged role in excitotoxicity.
In contemplating a role for synaptic receptors in toxicity during energy deprivation, we started by considering the assumption that the source of hypoxic glutamate is synaptic. There is some experimental support for the idea that synaptic release is a major source of glutamate during energetic insults (Rothman, 1983; Kaplan et al., 1989; Monyer et al., 1992; Andrade and Rossi, 2010). The major alternative mechanism of glutamate release is glial reverse glutamate uptake, which may occur under particularly severe conditions of oxygen-glucose deprivation combined with chemical ischemia (Jabaudon et al., 2000; Rossi et al., 2000). Reverse transport could produce excessive tonic glutamate presumably most concentrated at extrasynaptic regions where transporters are primarily localized (Danbolt, 2001; Gouix et al., 2009) although synaptic NMDARs could also be recruited since they comprise the majority of cellular receptors (Rosenmund et al., 1995; Harris and Pettit, 2007). We reasoned that if reverse uptake participates strongly in hypoxic glutamate release, blocking the (reverse) activity of transporters during hypoxia would be protective. However, if transporters remove toxic levels of glutamate released synaptically, then blocking transporters would exacerbate the damage. Thus, to test the role of glutamate transporters during hypoxia, we co-applied DL-threo-β-Benzyloxyaspartic acid (TBOA), a broad-spectrum, non-substrate glutamate transporter inhibitor (Shimamoto et al., 2004), during hypoxia. We found that application of TBOA significantly exacerbated cell death, suggesting that glutamate transporters normally play a protective role in hypoxia (p < 0.05, n = 5, Figure 4). Although these results do not directly determine the NMDAR populations responsible for cell death, the data are consistent with a synaptic rather than an extrasynaptic source of glutamate.
To initially test whether milder synaptic receptor activity can be toxic to neurons, we queried whether bicuculline, commonly used to enhance synaptic activity, could kill neurons. Indeed, 5 h treatment of cells with 50 µM bicuculline, 10 µM glycine, 2 mM Ca2+ and no added Mg2+ produced mild but reliable D-APV sensitive neurotoxicity measured 24 h later (82 +/− 3% vs. 90 +/− 2% cell survival, p<0.05, n=6). Although this is a mild effect, it demonstrates that prolonged and excessive synaptic activity may kill neurons, in contrast to the prevailing idea that any synaptic receptor activation is neuroprotective (Hardingham and Bading, 2010).
The evidence that neuroprotective memantine exhibits strong synaptic receptor (EPSC) block during sustained or repetitive receptor activation, coupled with data suggesting that the glutamate source in hypoxia is synaptic, prompted us to investigate whether synaptic NMDARs may mediate cell death during hypoxia. Hypoxia is an insult that we and others have employed as a model of excitotoxic damage (Rothman, 1983; Hogins et al., 2011). Unlike bath application of NMDA or glutamate, other common experimental excitotoxic challenges, hypoxia triggers endogenous glutamate release. In this sense, then, energetic insults like hypoxia are more similar to in vivo insults such as ischemia and stroke (Choi and Rothman, 1990; Lipton, 1999). To test the hypothesis that excessive activation of synaptic NMDARs during hypoxia mediates cell death, we selectively blocked synaptic receptors prior to hypoxia using the very slowly dissociating, use-dependent antagonist, MK-801. This protocol has been previously employed extensively (Hardingham and Bading, 2002; Ivanov et al., 2006; Liu et al., 2007; Stanika et al., 2009; Bordji et al., 2010; Stark and Bazan, 2011; Zhang et al., 2011). First, we exchanged the culture medium for isoosmotic defined recording solution (see Materials and Methods) containing 10 µM MK-801 for 15 minutes. The preblock bath also contained low (1 mM) Ca2+ concentration, 50 µM bicuculline (to decrease network inhibition), no Mg2+ and 10µM glycine. This solution exchange prevented background glutamate in the culture medium from activating receptors, thus minimizing the chance of non-specific MK-801 block. Low Ca2+ concentration ensured a low quantal content for each action potential, minimizing spillover onto extrasynaptic receptors (Mennerick and Zorumski, 1995). Furthermore, we omitted 4-aminopyridine, a potassium-channel blocker commonly used in the preblock protocol (Hardingham et al., 2002; Bordji et al., 2010; Zhang et al., 2011), but which may enhance quantal content and spillover onto extrasynaptic receptors.
This synaptic NMDAR preblock protocol abolished NMDA mediated EPSCs in mass cultures in which toxicity experiments were performed with relatively little reduction in normal synaptic activity, measured by AMPA receptor-mediated EPSCs (Figure 5A). AMPA EPSCs sometimes changed amplitude or frequency following MK-801 preblock (Figure 5A2), but there was no consistent direction of these changes, and the overall charge transfer mediated by AMPAR over 30 s was not statistically different than pre-treatment levels (Figure 5C). Preblock with MK-801 preserved approximately 30% of bath-applied NMDA current (Figure 5B–C), consistent with previous studies suggesting that ~70% of cellular NMDARs are synaptic (Rosenmund et al., 1995; Harris and Pettit, 2007). Following this synaptic preblock protocol, we replaced the original culture medium and subjected the neurons to hypoxia. Preblocking synaptic receptors successfully reduced cell death, evaluated 24 h following insult, from 45 ± 4% to 17 ± 2% (p < 0.05, n = 10) during a 2.5 h hypoxic insult (Figure 5D).
Previous studies investigating the role of synaptic and extrasynaptic receptors in excitotoxic insults typically used exogenous insults in which glutamate or NMDA are bath applied to neurons (Sattler et al., 2000; Hardingham et al., 2001; Hardingham et al., 2002; Papadia et al., 2008). This insult may activate more extrasynaptic receptors than hypoxic insult. Therefore, we hypothesized that selectively blocking synaptic receptors should not protect against an exogenous insult (Hardingham et al., 2002). We selectively blocked synaptic receptors prior to insult with 50 µM glutamate, which we found killed approximately as many neurons as hypoxia over 2.5 h. Surprisingly, we found that selective synaptic receptor block significantly protected against exogenous glutamate (Figure 5E). We hypothesized that this may partly result from increased network activity promoted by exogenous glutamate. This would lead to excessive synaptic receptor activation that could contribute to glutamate-mediated death. To test this we recorded from a network of dissociated neurons using a multi-electrode array (MEA) before and during treatment with 50 µM glutamate. Exogenous glutamate produced a mild but reliable and reversible increase in network activity in our cultures (p<0.05 in 4 out of 4 experiments analyzed with ANOVA and pairwise t-test, Figure 5F). Over the four experiments, the mean increase in array-wide spike rate was 45 ± 22 % over baseline. It seems unlikely that this degree of enhanced synaptic activity accounts fully for the neuroprotective effects of synaptic NMDAR preblock (see Discussion). In aggregate, these data suggest that synaptic NMDARs play a critical excitotoxic role across a variety of insult paradigms.
MK-801 synaptic preblock protects strongly against hypoxic damage, but protection is incomplete (Figure 5D2). Residual cell death could be caused by NMDAR-independent toxicity, rendering even complete MK-801 antagonism ineffective. To test this we applied 10 µM MK-801 before and during the hypoxic insult, and we continued the MK-801 incubation during the post-insult recovery period (24 h). Despite the constant presence of MK-801, we still found incomplete protection (Figure 6A). We also directly examined whether total (synaptic and extrasynaptic) NMDAR preblock could confer full protection. This was achieved by co-incubation with 50 µM NMDA plus 10 µM MK-801 prior to hypoxic insult. We found that the total receptor preblock protocol was also incompletely protective against hypoxic damage and exerted protection quantitatively similar to synaptic preblock (Figure 6B). To verify that MK-801 unblocking is unlikely to underlie the incomplete block, we recorded NMDAR currents immediately after total preblock and immediately after total preblock then 2.5 h hypoxia. Currents immediately following the insult showed only modest recovery (Figure 6C). Average current amplitudes were −57.5 ± 6.8 pA (recorded immediately following preblock, n = 10), − 194.0 ± 29.0 pA (preblocked, recorded post-hypoxia, n = 8), and −2146.9 ± 306.7 pA (control, unblocked, n = 5). These observations are consistent with the idea that little relief of block occurs as a result of insult-induced depolarization and channel activation. Finally, the small residual death associated with MK-801 antagonism in Figures 5D, 5E, 6A, and 6B is also similar to the cell death we previously observed with complete block of all NMDA and AMPA-type glutamate receptors during hypoxia (Hogins et al., 2011). Taken together, this previous and present evidence is consistent with the idea that non-glutamatergic mechanisms contribute to hypoxic damage in this model (Newell et al., 1995; Besancon et al., 2008), and selective synaptic preblock confers nearly the full measure of possible NMDAR-related protection in this model. Extrasynaptic NMDARs do not appear to be significantly involved in the residual toxicity.
Although we took care to minimize spillover during our blocking protocol by reducing bath calcium concentration, reducing calcium also enhances excitability (Frankenhaeuser and Hodgkin, 1957). This could counteract the effect of lowering quantal content and result in perisynaptic receptor activation and MK-801 block. To minimize any enhancement in excitability caused by low divalent cation concentration, we omitted bicuculline in our preblock solution and instead added 200 nM of the positive allosteric GABAA receptor modulator allopregnanolone (3α5αP) to the low-calcium bath. This protocol significantly reduced the frequency of spontaneous EPSCs but did not abolish activity (p<0.05, n=8, Figure 7A). When MK-801 was added to this more conservative preblock protocol, we still observed significant protection against 2.5 hypoxia (46.2 +/− 6.5% vs. 69.7 +/− 5.6 % cell survival, p<0.05, n=5, Figure 7B). This offers further evidence that synaptic receptors play a vital role in hypoxic excitotoxic death.
As an independent approach to evaluating the role of synaptic receptors in hypoxic damage, we next queried whether selectively reducing contributions of extrasynaptic NMDARs would protect against hypoxic damage. If damage arises mainly through synaptic receptors, then reducing extrasynaptic receptor contributions should offer little protection. If, however, extrasynaptic receptors contribute strongly to damage (Hardingham et al., 2002), perhaps in concert with synaptic receptors, reducing their activation would be protective. To selectively eliminate sustained extrasynaptic NMDAR contributions, we used glutamate pyruvate transaminase (GPT), which catalyzes pyruvate-dependent glutamate degradation. Catalysis is too slow for GPT to strongly affect the transient, high glutamate concentration in the synaptic cleft, but GPT significantly depresses the activation of extrasynaptic receptors by synaptic overflow (Rossi and Slater, 1993; Rusakov and Kullmann, 1998; Turecek and Trussell, 2000). Consistent with this previous literature, we found that 5 U ml−1 GPT plus 2 mM pyruvate strongly reduced currents in response to exogenous application of glutamate (1 µM, representing a sub-EC50 concentration), but GPT had a minimal effect on NMDA EPSCs (Figure 8).
As previously demonstrated in other preparations (Rossi et al., 2002), we found that GPT significantly protected neurons against excitotoxicity induced by 2.5 h of 50 µM glutamate (Figure 9A). This suggests that the GPT strategy is effective against toxicity mediated by tonic glutamate buildup. However, when tested against hypoxic excitotoxicity, GPT was not protective and instead exacerbated death (Figure 9B). This exacerbation resulted from residual contaminants in the supplier’s enzyme formulation (see Materials and Methods), since equivalent vehicle suspension alone resulted in indistinguishable exacerbation (21 ± 1% survival in hypoxia + GPT, 24 ± 4% survival with vehicle suspension control in hypoxia (p > 0.05), while hypoxia alone yielded 46 ± 2% survival in 6 independent, paired experiments on sibling cultures). As with GPT, the vehicle control did not affect survival in the absence of hypoxia, and the exacerbation was D-APV insensitive (data not shown). Although the basis of the vehicle effects on hypoxic damage remains unclear, the quantitative similarity of the effect in the presence and absence of GPT suggests that exacerbation did not mask a protective effect of GPT during hypoxia. These results are again consistent with the idea that synaptic receptors can mediate excitotoxicity when the source of glutamate is synaptic.
The extrasynaptic NMDAR excitotoxicity hypothesis proposes an attractive dichotomy between trophic synaptic receptor activation and toxic extrasynaptic receptor activation (Hardingham et al., 2002; Papadia et al., 2008). In part the extrasynaptic hypothesis is built on neuroprotection by the use-dependent NMDAR channel blocker memantine, which has been suggested to be selective for extrasynaptic receptors (Lipton, 2007; Leveille et al., 2008; Xia et al., 2010). Here we demonstrated that a neuroprotective concentration of memantine shows only weak selectivity for extrasynaptic NMDARs when the pattern of synaptic and extrasynaptic receptor activation (channel opening) is identical. The extrasynaptic receptor hypothesis also posits that synaptic receptor blockade should not exhibit neuroprotection during excitotoxic insults and may in fact exacerbate damage. In contrast, we demonstrated that synaptic NMDAR blockade is neuroprotective, especially when the excitotoxic insult is mediated by synaptic glutamate release. Our results contradict the strong form of the extrasynaptic NMDAR excitotoxicity hypothesis and suggest that at least some excitotoxic insults act mainly through over-activation of synaptic NMDARs.
Our data suggest that memantine possesses similar actions at extrasynaptic and synaptic receptors. Although extrasynaptic receptors may be enriched in NR2B subunits (Groc et al., 2006), offering a potential substrate for selectivity, memantine exhibits no clear subunit selectivity (Blanpied et al., 1997). A previous study of memantine found evidence for selectivity at extrasynaptic receptors, but this study did not directly test the contribution of transient versus sustained receptor activation in memantine’s apparent selectivity (Xia et al., 2010). Our results suggest that the pattern of receptor activation is the most important determinant of memantine’s apparent selectivity, consistent with memantine’s open-channel block mechanism (Chen and Lipton, 1997). Transient receptor activation during brief transmitter presence limits the amount of memantine channel block during synaptic activation, especially at low memantine concentrations. By contrast, sustained activation of receptors facilitates memantine inhibition. Previous studies assessed memantine’s selectivity using a low concentration (1 µM) (Okamoto et al., 2009; Xia et al., 2010), whereas we focused on a 10-fold higher concentration that we and others have documented to be neuroprotective (Figure 1) (Chen et al., 1998; Volbracht et al., 2006). Our results demonstrate that synaptic receptors (EPSCs) can be strongly blocked by this concentration if they are activated by either sustained or repetitive agonist presentation. Either pattern or both patterns could apply to excitotoxic insults such as hypoxia. It should be noted that our measurements (Figures 2 and and3)3) were performed at −70 mV in the absence of extracellular Mg2+. It is likely that under pathophysiological conditions such as hypoxia, the absolute level of memantine block for both extrasynaptic and synaptic receptors will be reduced by physiological Mg2+ and by hypoxic depolarization (Kotermanski and Johnson, 2009; Xia et al., 2010).
We also provide evidence that synaptic receptors can play a critical role in mediating excitotoxic death during a moderate hypoxic challenge. Blocking synaptic NMDARs protected against hypoxia. Key to interpreting this result is the selectivity of our strategy for preblocking synaptic receptors. For our MK-801 preblocking protocol, we followed protocols established in the literature (Hardingham et al., 2002; Papadia et al., 2008). We also defined the synaptic receptor population conservatively by preblocking in bath solutions designed to limit glutamate spillover during synaptic activity, and we ensured minimal background glutamate levels by preblocking in fresh, defined bath solution. Most importantly, we directly verified the selectivity of our preblock strategy (Figure 5). Finally, we complemented MK-801 preblock experiments with tests of the effect of enzymatic degradation of glutamate. GPT failed to protect against hypoxic damage but significantly reduced damage to exogenous glutamate. This result suggests strongly that tonic buildup at extrasynaptic receptors is not a major source of toxic glutamate under conditions of our hypoxia experiments. A recent study has also shown that tonic NMDAR activity may not play a critical role in excitotoxic insults, particularly in certain neuronal populations vulnerable to excitotoxic insult (Povysheva and Johnson, 2012).
We cannot exclude the possibility that under some conditions extrasynaptic NMDARs may play a more pronounced role in excitotoxicity than we observed here. Insults of altered severity may promote a larger toxic role for extrasynaptic receptors. For instance, reverse glutamate transport has been shown to play a prominent role in glutamate release under very extreme conditions of metabolic poisoning plus oxygen/glucose deprivation (Rossi et al., 2000). Because neuronal and glial transporters are mainly excluded from synapses (Danbolt, 2001), release by reverse glutamate transport may promote a more prominent role for extrasynaptic receptors in toxicity than in our studies. Additional conditions fostering an extrasynaptic receptor role could include cell types with a large percentage of extrasynaptic receptors or networks with weak synaptic connectivity. Finally, we note that previous studies have rarely rigorously verified the degree of synaptic receptor block in the oft-used MK-801 preblock protocol. This leaves open the possibility that significant numbers of synaptic receptors remained unblocked in these studies and may have contributed to toxicity attributed to extrasynaptic receptors.
A related issue concerns the definition of synaptic receptors. Our studies, like previous work, used an operational definition of synaptic NMDARs: receptors that are blocked by MK-801 when synaptically activated. Although our estimates of synaptic and extrasynaptic receptor percentages agree with previous estimates, it is likely too simplistic to dichotomize receptor populations. The literature’s operational definition can be affected by factors that promote transmitter spillover, for instance, and that variably recruit perisynaptic receptors. There is also growing realization that complex trafficking pathways can dynamically affect exchange between these two populations of surface receptors (Gladding and Raymond, 2011).
We were surprised that toxicity in response to 50 µM glutamate was also strongly reduced by synaptic NMDAR block (Figure 5E). This protection could be explained in at least two ways. First, synaptic receptors constitute the majority of NMDARs (Rosenmund et al., 1995; Harris and Pettit, 2007). Thus, synaptic receptor block may have protected by simply blocking a large number of receptors. A second possibility is that exogenous agonist, perhaps acting preferentially at extrasynaptic receptors (Sinor et al., 2000; Hardingham et al., 2002) evoked secondary synaptic glutamate release from the network, contributing to the observed death (Monyer et al., 1992). By this explanation, extrasynaptic receptors act indirectly, as a conduit to trigger synaptic NMDAR toxicity. MEA data showed a significant increase in overall network activity during prolonged glutamate exposure, consistent with the idea that synaptic activity is increased by exogenous glutamate. However, toxicity from network disinhibition with bicuculline was milder and required longer than glutamate treatment required. Thus, while repetitive synaptic activity can be toxic, the effects of exogenous glutamate are unlikely to be explained by the increase in network synaptic activity alone. Rather, direct activation of synaptic receptors by glutamate probably mediates most of the effect of synaptic receptor preblock protection. Regardless of whether synaptic NMDAR activation is direct from exogenous glutamate or indirect from synaptic glutamate, our data offer strong evidence that synaptic receptors can be neurotoxic in multiple circumstances, including energy deprivation and insults induced by exogenous agonist.
Our results are in broad agreement with a previous paper that selectively disrupted synaptic NMDAR function with actin depolymerization and observed neuroprotection from glutamate released during oxygen/glucose deprivation (Sattler et al., 2000). However, manipulations of actin polymerization can also have presynaptic effects which could confound interpretations (Morales et al., 2000; Yao et al., 2006; Andrade and Rossi, 2010). Our approach avoided this potential complication. The previous study showed that toxicity to exogenous toxin was not prevented by actin depolymerization, apparently implicating extrasynaptic receptors (Sattler et al., 2000). This is in apparent conflict with our studies in which synaptic receptor blockade prevented exogenous excitotoxicity (Figure 5E). This discrepancy is likely explained by the fact that actin depolymerization did not disrupt the total number of surface receptors; rather, it relocated synaptic receptors to non-synaptic membrane. Therefore, our approach reduced overall toxic Ca2+ load in response to exogenous glutamate, whereas the Sattler et al. approach did not.
Excitotoxicity is a complex phenomenon that is mediated in part by over activation of NMDARs. The extrasynaptic NMDAR hypothesis of excitotoxicity has been one of the most exciting proposals in the glutamate toxicity field in recent years, in part because of the clinical implications. However, our observations substantially undermine two arms of the extrasynaptic NMDAR hypothesis. First, we find that memantine at neuroprotective concentrations significantly blocks synaptic receptors during sustained or repetitive stimulation as likely experienced during excitoxicity-triggering insults. Second, we demonstrate that synaptic receptor activation, proposed to be uniquely protective, is toxic during at least some endogenous and exogenous excitotoxic insults. Our findings point towards a need to consider synaptic receptor contributions when developing therapeutics for disorders with excitotoxic components.
This work was supported by the National Institute of Health grants GM08151 (C.M.W), DA07261 (J.H.), MH78823 (S.M.), NS52823 (S.M). We would like to thank Ann Benz and Amanda Taylor for assistance with cultures and lab members for advice and discussion.