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 () (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 ( and ) 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 (). 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 (). 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 (). 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.