Organization, control and function of extrasynaptic NMDA receptors

1. Introduction

N-methyl d-aspartate receptors (NMDARs) have long been of major interest to neuroscientists owing to their multiple implications in neuronal physiology during development and adulthood. Following the seminal discovery of long-lasting synaptic plasticity by Bliss & Lomo [1], NMDARs have been known for their key role in long-term potentiation (LTP) and long-term depression (LTD) of synaptic transmission [2]. However, because these glutamate-gated ion channels are highly permeable to calcium, they not only relay a physiological signal into neurons, but can also trigger intracellular signalling cascades that can ultimately lead to cell death. In fact, the phenomenon of excitotoxicity [3] and its causal link with excessive glutamate release [4] were described years before the existence of synaptic plasticity was established, but NMDARs were only demonstrated to be the main source of calcium responsible for glutamate-induced excitotoxicity in the late 1980s [5–7]. Ever since, these receptors have been renowned for their duality. They are both essential and potentially harmful receptors whose activity needs to be finely and tightly controlled. Such regulation is achieved by many intracellular and extracellular factors, including intracellular partners, magnesium block, endogenous allosteric modulators, as well as agonist and co-agonist binding. Interestingly, many of these regulatory actions are highly sensitive to the subunit composition of the receptor [8,9]. Along with the development of subunit-specific pharmacological antagonists, evidence for specific roles of NMDAR subtypes has thus accumulated over the past decades, with particular attention on the GluN2B-containing heterodimers [8,9]. In 1995, like other transmitter-gated ion channels, NMDARs were also found to be present in significant proportions at extrasynaptic sites [10–13]. Relationships between receptor location, their GluN2B content and their roles in the direction of synaptic plasticity and in cell death were soon assumed [14,15]. This generated a new craze for NMDARs and laid the groundwork for a meaningful understanding of how these receptors impact cellular physiology and pathology. More recently, NMDARs have gained a new interest, as d-serine, a potential gliotransmitter released by astrocytes ([16], but see [17]) was found to act as an endogenous co-agonist on their glycine-binding site [16,18–21]. This discovery has made glia a possible regulator of NMDAR function and marked the emergence of an exciting cross-talk between the field of NMDARs and the expending domain of glial cells. In this review, we discuss the latest findings on the organization, the endogenous control and the functions of NMDARs at extrasynaptic sites, and what role glia may play in the activity of these receptors.

2. Defining the extrasynaptic space

Making sharp delineation of synaptic and extrasynaptic spaces is challenging, especially when one tries to reconcile data from microscopy and electrophysiology. This difficulty arises from the fact that what one considers as extrasynaptic based on morphological clues can still be part of the synaptic space as far as signal transmission is concerned. From the morphological point of view, receptors are often considered extrasynaptic if they lie more than 100 nm away from the post-synaptic density (PSD) [22,23]. However, this morphological definition does not take into account glutamate spillover during synaptic transmission, which probably activates receptors located on the neck of dendritic spines (i.e. perisynaptic receptors) or on the dendritic shaft. From the electrophysiological point of view, a convention of synaptic physiologists defines synaptic NMDARs as those recruited during afferent stimulation at low frequency (less than 0.05 Hz), or in response to spontaneous glutamate release producing miniature excitatory responses [24–29]. Extrasynaptic NMDARs correspond to receptors that are not activated during such conditions [24–29]. This electrophysiological definition of synaptic NMDARs may encapsulate receptors located not only at synapses but also at peri- or extrasynaptic locations that possibly contribute, even mildly, to low-frequency synaptic transmission. Because it is difficult to determine to what extent glutamate spills over during basal synaptic transmission, it is unclear to what degree the morphological and electrophysiological definition of the synaptic and extrasynaptic spaces overlap. In fact, the precise delineation of the synaptic–extrasynaptic frontier seems specific to the parameter that one considers, and it is likely that an all-encompassing definition of this boundary cannot be found.

3. NMDAR organization at extrasynaptic sites

Despite some occasional observations of NMDARs at extrasynaptic locations [10–13,30–36], the first study to directly and extensively investigate the organization of post-synaptic NMDARs at extrasynaptic sites was published in 2010 [22]. Interestingly, it seems that extrasynaptic NMDARs are organized as clusters, with a discrete distribution along the perisynaptic zone (45%) and dendritic shaft (55%) [22]. In particular, these clusters often correspond to contact area between a dendrite and adjacent cell processes, including glial (approx. 25%), axonal-like (approx. 50%) or dendritic (approx. 25%) processes. These extrasynaptic sites spread to an average width of less than 100 nm, which means they are substantially narrower than synaptic sites that have an estimated width of 195 nm in the CA1 region [37]. Interestingly, extrasynaptic NMDAR clusters seem to colocalize with intercellular adhesion molecules such as β-catenins and some scaffolding proteins such as SAP102 and PSD95 in culture [22], although it is not clear in this work whether any specific MAGUKs are preferentially interacting with extrasynaptic NMDARs. These observations strongly suggest that extrasynaptic NMDARs are clustered and organized at the plasma membrane in a similar manner to their synaptic homologues. NMDARs are neither evenly nor randomly distributed along the dendritic shaft, and probably are not as free to diffuse as demonstrated in cultures [38–40]. This challenges the common idea that extrasynaptic NMDARs are essentially a reserve pool of receptors, waiting to be recruited to synapses. In fact, the finding that these receptors are clustered to very specific cell contact areas suggests that they may serve a distinct signalling function, independent from their synaptic homologues. The work from Harris & Pettit [26] already supported this idea by demonstrating that synaptic and extrasynaptic NMDARs represent distinct and stable pools of receptors at the surface of CA1 neurons in hippocampal slices.

4. GluN2B-NMDARs at extrasynaptic sites

It is generally believed that extrasynaptic NMDARs are enriched in GluN2B-containing heterodimers (GluN2B-NMDARs). This would fit with the idea that extrasynaptic NMDARs constitute a distinct population, serving a specific function. This idea is also quite satisfying when one considers that GluN2B-NMDARs have a higher affinity for glutamate, whose concentration is lower in the extrasynaptic space [8,9,41,42]. Finally, this localization of GluN2B-NMDARs would also make sense regarding the interaction of NMDARs with their intracellular partners. Indeed, the C-terminal domain (CTD) of GluN2 subunits dictates the scaffolding protein the receptor can interact with and, thus, strongly influences the surface localization of NMDAR subtypes (as reviewed in great detail in [8,9]). It was proposed that GluN2A-NMDARs associate preferentially with PSD95, which is believed to limit their field of action to the PSD and increase their half-life time before recycling [38,43–47]. Because they tend to interact with SAP102 rather than with PSD95, GluN2B-NMDARs are thought to be more mobile, widespread and not confined to synapses in adults [45,47]. In fact, the expression of GluN2A and GluN2B subunits closely follows the expression of PSD95 and SAP102, respectively, during development [47], and evidence suggests that (i) neonatal GluN2B-NMDARs are held at synapses through their interaction with SAP102 during early stages of development; (ii) the expression of PSD95 increases throughout postnatal development and competes with SAP102 for insertion into the PSD; and (iii) this displaces SAP102 and GluN2B subunits outside of synapses, whereas PSD95 and GluN2A become predominant at synaptic sites [22,45,48,49]. Along with the fact that neurons of the visual cortex and superior colliculus still show significant expression of SAP102 and GluN2B subunits in adulthood [45,47], these observations together strongly fuel the idea that GluN2B-NMDARs exist at extrasynaptic sites more abundantly than GluN2A-NMDARs, which are preferentially found at synapses.

However, only a few studies have functionally investigated this aspect in brain slices, and very often indirectly. Harris & Pettit [26], who specifically tackled that question, did not find a particular enrichment of GluN2B-NMDARs at an extrasynaptic location compared with synapses, in the CA1 region of rat hippocampus, as they identified large amounts of GluN2B-NMDARs at both locations. By contrast, Fellin et al. [50], who performed pioneering work on extrasynaptic NMDARs activated by glia-derived glutamate, observed a relative enrichment of GluN2B-NMDARs outside of synapses, in line with similar observations [51–53]. Unfortunately, these studies were all performed in slices obtained from young rodents (P10–P22), at a time during development when the switch from GluN2B- to GluN2A-subunit-containing NMDARs is still occurring at synapses [9]. For instance, Harris and Pettit found, as expected, that GluN2B-NMDARs disappear from synapses throughout the first month of age, but regrettably carried out the comparison of synaptic versus extrasynaptic composition in juvenile animals (P15) [26] and concluded there was no difference in the GluN2B content. Additionally, it is not known whether extrasynaptic NMDARs also undergo a developmental change of their subunit composition. Hence, because it has been mostly conducted in slices from immature animals or in cultures, the study of NMDAR subunit composition at synaptic and extrasynaptic sites has, so far, essentially led to contradictory results and confusion.

Even when tackled in adult rodents, the situation does not seem to be any clearer. In hippocampal CA1 pyramidal neurons of adult rats, GluN2B-NMDARs were shown to be absent at synapses, but could still be found at extrasynaptic locations [19]. However, the replacement of GluN2B- with GluN2A-NMDARs at synapses is not ubiquitous and complete, and GluN2B-NMDARs still represent a major portion of synaptic NMDARs in many other regions of the adult central nervous system (CNS) [16,54] as reviewed in [9]. Therefore, even though it is broadly accepted, the idea that extrasynaptic receptors are essentially GluN2B-NMDARs, or are at least enriched in GluN2B-NMDARs compared with their synaptic counterpart, is still open for debate.

5. Agonist control of NMDAR at extrasynaptic sites

In 1989, the existence of a tonic current mediated by NMDARs was reported in principal neurons of the hippocampus under mild depolarization [55]. Similar to what is known for GABAR-mediated tonic currents [56], NMDAR-mediated tonic current seems to be allowed by sufficient concentrations of ambient agonist [42,55] and generated by receptors located outside of synapses [55,57,58]. Indeed, NMDAR-mediated tonic current persists when synaptic activity is suppressed with tetrodotoxin, and conversely synaptic NMDAR activity remains unaffected after NMDARs mediating tonic current are blocked with MK801 [58], which establishes those receptors as distinct, and somehow distant, from synaptic receptors. However, the origin of the so-called ambient glutamate that allows such tonic activation is still unclear, and little evidence for the source exists, aside from the observation that it is of non-synaptic origin [58,59]. Interestingly, the amplitude of NMDAR-mediated tonic current is strongly enhanced either by blocking glutamate reuptake through the glutamate transporter 1 (GLT1) [58,60], which is predominantly present on glial cells and mediates 95% of glutamate transport, or when slices are challenged with glial toxins [60]. Additionally, a recent study showed that the extent of NMDAR-mediated tonic current depends on glial coverage in the supraoptic nucleus of the hypothalamus [60]. Hence, the activity of NMDARs that mediate tonic current probably relies on the regulation of extrasynaptic glutamate tone by glia. The corollary of these observations is that this subset of extrasynaptic NMDARs is constitutively activated. However, extrasynaptic NMDARs can also be recruited experimentally by exogenous applications of NMDA or glutamate. This indicates that another subpopulation of extrasynaptic NMDARs exists (but see below) that can be activated by phasic, ‘ectopic’ release of glutamate from non-active zone sites. The main manifestation of such phasic activity of extrasynaptic NMDARs is slow (decay time on the order of seconds), infrequent (approx. 0.05 Hz) inward currents of large amplitude (hundreds of pA), generated in principal neurons of the CA1 region of the hippocampus [50,51,53] and in the superficial layers of the dorsal horn [52,61]. Such currents, termed slow inward currents (SICs), persist in the absence of neuronal and/or synaptic activity [51] and are completely suppressed by glial inhibitors [52]. Evidence points to the fact that such currents are caused by glutamate release from glia onto extrasynaptic NMDARs [33,41,50,62,63]. However, the exact pathway involved in this process is still under investigation. It has been shown that SIC frequency and amplitude increase upon activation of astrocytic G-protein coupled receptors (GPCRs) in the hippocampus [50,51], such as PAR1 receptors [53]. Concomitantly, stimulating the PAR1 receptor on astrocytes was recently shown to trigger calcium-dependent release of glutamate through Best1 channels in the hippocampus [64–66]. Thus, SICs could be caused by astrocyte-derived glutamate release through Best1 channels onto extrasynaptic NMDARs, upon activation of PAR1. Although this appealing theory would fit with earlier observations that calcium activity in the astrocytes is needed for SICs to occur [50,53,61], it would be in striking contradiction to the idea that vesicular loading from astrocytes is required [50]. Additionally, whether endogenous PAR1 activity can trigger SICs without exogenous experimental stimulation is unclear. Other pathways could thus be at play, involving metabotropic glutamate receptors, prostaglandin receptors or other astrocytic GPCRs, and relying on vesicular release [50,51].

Besides their mechanistic relevance, these observations are interesting because they open the possibility that extrasynaptic NMDARs may exist in two functionally distinct pools: (i) tonically activated receptors, which face suprathreshold amounts of glutamate and co-agonist, and (ii) overall silent extrasynaptic NMDARs, which can be acutely recruited by exogenous or endogenous (non-synaptic) glutamate release. If true, this could mean that glutamate availability is not homogeneous outside of synapses and could be spatially regulated, giving rise to different subcompartments. Another interpretation, however, could be that tonic and phasic extrasynaptic NMDAR responses are mediated by the same receptor population: tonic current could be carried out by these receptors under partial occupancy of their glutamate binding site, and additional (i.e. phasic) glutamate release would cause transient saturation and recruitment of supplementary receptors. Although this is purely speculative, we here propose that it could be relevant to consider the extrasynaptic space, at least from the point of view of glutamate, as comprising separate domains instead of one large homogeneous volume (figure 1). Understanding the rules that govern glutamate release, uptake and diffusion in the extracellular space would certainly shed light onto this situation and onto the control and roles of extrasynaptic NMDARs.

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6. Co-agonist control of extrasynaptic NMDARs

As demonstrated more than 25 years ago, NMDARs have the unique property of requiring not one but two agonists to be activated [67]. The identity of the second agonist, termed co-agonist, was elusive until recently, even though glycine was originally shown to bind to the co-agonist site [67–69]. In the CA1 region of the adult rat hippocampus, it was demonstrated recently that d-serine is the endogenous co-agonist of synaptic NMDARs, but that glycine gates the extrasynaptic NMDARs that mediate the tonic current and contribute to the response induced by exogenous application of NMDA [19]. SICs were not investigated in this study and whether NMDARs that mediate SICs are also gated by glycine, or by d-serine, is still unknown. This is potentially of great interest, because there is no actual demonstration that SICs are caused by the release of glutamate. Instead, SICs could be generated by the release of d-serine or glycine by astrocytes, on NMDARs that face suprathreshold levels of ambient glutamate. This hypothesis, which has never been considered, fits with the body of data already available on SICs (but see below considerations about glycine concentrations in the extracellular space) and would explain why blocking glutamate transporters does not necessarily increase SIC amplitude or frequency, as if SIC-mediating NMDARs were already facing saturating levels of glutamate [50].

Interestingly, the difference in the co-agonist used by NMDARs at synaptic and extrasynaptic sites appears primarily to be based on the fact that d-serine is released at synapses and that the activity of glial glycine transporter 1 (GlyT1) prevents glycine from accessing the synaptic cleft [19]. Thus, the delineation between synaptic and extrasynaptic space, with regard to the co-agonist control of NMDARs, seems to be defined by d-serine and glycine abundance. It is unclear, however, whether the domains delineated by the co-agonists match the domains defined by glutamate spillover (see section ‘Defining the extrasynaptic space’).

Even more unclear is the origin of glycine available to extrasynaptic NMDARs. In structures where glycinergic innervation is abundant, such as in the retina and the spinal cord, glycine that serves as an endogenous co-agonist at NMDARs originates from glycinergic terminals [70,71]. In those preparations, it was demonstrated that glycine released at inhibitory synapses spills over and diffuses to bind to remote NMDARs. However, in the adult hippocampus, even though the existence of extrasynaptic glycine receptors (GlyRs) has been documented [72,73], the presence of glycinergic terminals has never been established. In fact, the inhibitory transmission is entirely abolished by GABA receptor antagonists in that structure [74], and the expression of functional GlyRs is thought to stop after birth [74–76]. Yet, microdialysis has revealed that the amounts of free glycine in vivo are as high as 10 µM in the hippocampus [77,78]. In vivo, a major source of extracellular glycine in the CNS could be the blood flow. Indeed, blood contains approximately 200 µM of glycine [79] and glycine is able to cross the endothelial wall of capillaries by mean of glycine transporters [80,81]. In slices, however, blood vessels are mostly emptied of their initial contents, narrowing down the source of glycine to the brain parenchyma itself. Because they contain 3–6 mM of glycine [82], glial cells could be a major source of glycine in brain slices. It was shown that potassium stimulates the release of glycine by glial cells in culture, through a calcium-independent mechanism [83–85]. In addition, GlyT1 activity can be reversed in certain conditions, leading to an efflux of glycine into the extracellular space [86,87]. For instance, in the cerebellum, if Bergmann glial cells are infused with 4 mM glycine, which is the estimated concentration in hippocampal astrocytes [82], and submitted to a mild membrane depolarization (approx. −50 mV), GlyT1 becomes responsible for an efflux of glycine. In the hippocampus, another noteworthy source of glycine could be GABA interneurons. Song et al. [88] indeed demonstrated in the CA1 region that GABA interneurons contain glycine and express glycine transporter 2 (GlyT2). They proposed that glycine could be co-released with GABA at inhibitory synapses similar to what occurs in the thalamus, the brainstem, the spinal cord and the cortex [89–94].

Regardless of glycine origin, in vivo microdialysis suggests that the amount of glycine available to extrasynaptic NMDARs should be high enough to be saturating [77,78], especially if those receptors are GluN2B = NMDARs (EC₅₀ < 1 µM). In line with this idea, it was observed that exogenous applications of co-agonist do not enhance the amplitude of NMDAR-mediated tonic current [58]. If true, it means there is little room for regulatory mechanisms and modulation of NMDAR activity through the co-agonist-binding site at extrasynaptic locations, but this has never been addressed for SICs or any other manifestation of extrasynaptic NMDAR activity. This puzzling aspect thus needs further investigation.

7. Role of slow inward currents and tonic current in physiology and pathology

The roles played by extrasynaptic NMDARs are still very elusive, mostly because specific and reliable tools to investigate them have been lacking so far. Some studies have reported a role for non-synaptic NMDARs in various functions such as extrasynaptic inhibition [95], or dynamic range compression [96]. But, aside from presynaptic NMDARs [97,98], which are not addressed in this review, most studies have focused on the manifestations of extrasynaptic NMDAR activity such as tonic currents and SICs. It is accepted that the tonic current mediated by extrasynaptic NMDARs plays a role in the excitability of principal neurons and in modulating dendritic inputs [99]. It also becomes increasingly clear that NMDAR-mediated tonic current can be upregulated in pathological conditions, ranging from cocaine addiction [100] to Alzheimer's disease [101]. However, the relevance of such tonic current to neuronal physiology and the impact of its pathological disturbance need further investigation. In contrast, SICs have been quite well characterized (see above) and were shown to occur simultaneously in distinct neurons within 100 µm of each other [51]. This fits with the idea that SICs are generated by glutamate (or co-agonist) release from astrocytes whose anatomical territory spans an area approximately 50–100 µm in diameter. Based on these observations, SICs have been proposed to play a role in neuronal synchrony and network excitability [51,52]. However, they have only been observed in slices so far, often under non-physiological conditions (low magnesium, GLT1 blockers). Whether SICs represent an important feature of neuron–glia signalling, that also occurs in vivo, remains to be established, but it is worth mentioning that SIC amplitude was demonstrated to be enhanced in animal models of inflammation [61].

8. Long-term potentiation and long-term depression: on the importance of subunit composition and/or localization?

Using both genetic and pharmacological approaches, there has been intense speculation and many demonstrations that specific NMDAR subtypes are responsible for selectively inducing LTP or LTD [102–107]. It was proposed that GluN2A-NMDARs would preferentially trigger LTP, whereas GluN2B-NMDARs are preferentially associated with LTD. This dichotomy, however, has been significantly and repeatedly challenged [106,107] (table 1), and the idea that a particular subtype of NMDAR is specifically involved in inducing potentiation or depression at glutamatergic synapses seems over-simplifying, as reviewed in great detail by Paoletti et al. [9]. This raises the question of whether NMDAR location, rather than subunit composition, could be the determining factor to the direction of synaptic plasticity [117]. Unfortunately, very few studies have directly addressed this aspect. Instead, it seems that the role of synaptic and extrasynaptic NMDARs in plasticity have mostly been inferred from results obtained on the role of NMDAR subunit composition (in particular, GluN2B-NMDARs) in LTP and LTD. However, there seems to be no simple relationship between cellular location and subunit composition in particular at ages most often used in those studies (approx. P20–P25, see discussion above). It is thus notoriously hazardous to form conclusions from works studying the involvement of extrasynaptic NMDARs in synaptic plasticity (and, in fact, in any specific functions) based on their GluN2B pharmacological profile. Activating or silencing specifically synaptic or extrasynaptic NMDARs thus requires other types of approaches that do not rely on subunit composition. The main alternative is the use of the open-channel blocker MK801, which allows inactivation of NMDARs that are recruited while leaving the silent/not recruited receptors intact. When combined with low-frequency stimulation of afferent fibres, this method allows the selective blockade of synaptic NMDARs and leaves intact most of their extrasynaptic counterparts [19,26,27,118,119]. Conversely, this approach also allows the blockade of extrasynaptic NMDARs mediating tonic currents, while leaving intact unstimulated synaptic NMDARs [58]. Either way, this method presents the advantage that it only relies on whether receptors are active or not during MK801 application, and therefore circumvents caveats associated with the use of subunit-selective reagents or immature animals. Interestingly, using such an approach, Liu et al. [120] found that selective stimulation of extrasynaptic NMDARs triggers LTD in CA1 neurons. Alternatively, it was recently demonstrated that d-serine and glycine gate synaptic and extrasynaptic NMDARs, respectively [19]. Taking advantage of this segregation, the authors were able to show in adult rat hippocampal slices that synaptic, but not extrasynaptic, NMDARs are required for LTP induction, whereas activation of both types of receptors is required for LTD. These findings fuel the idea, proposed by Rusakov et al. [117], that what matters in LTP and LTD induction may be the location, rather than the subtype, of NMDARs recruited.

9. Is excitotoxicity mediated by extrasynaptic NMDARs?

Since the discovery that they are responsible for glutamate-induced cell death [5–7], NMDARs have been studied intensively for their role in excitotoxicity. In 2002, Hardingham et al. [15], published the first compelling evidence that synaptic and extrasynaptic NMDARs may serve distinct roles in neuronal survival/death. Ever since, the prevailing theory suggests that synaptic and extrasynaptic NMDAR activation have opposing effects on cell fate. The activity of synaptic NMDARs is thought to favour neuronal survival, via the phosphorylation of intracellular factors such as CREB or Erk1/2. In contrast, cell death is mainly mediated by the activation of extrasynaptic NMDARs (mostly GluN2B-NMDARs) which inhibits the above pathways in addition to promoting pro-death signalling, through the expression of caspase-3 for instance [23]. This view has gained additional attention, because extrasynaptic NMDAR activity has been associated with neuronal death in neurodegenerative disorders such as Huntington's [121] or Alzheimer's disease [101,122,123]. However, several lines of evidence point to the limitation of this overall theory.

First, there are multiple causes and pathways that could lead a system to malfunction. As such, excitotoxicity is unlikely to arise from one precise sequence of events, even though the ultimate result is cell death. Indeed, the word excitotoxicity has been used to describe cell death originating from any number of failures to maintain neurons in a healthy excitation/inhibition balance. However, artificially challenging neurons with bath-applied glutamate or NMDA, pharmacologically blocking glutamate uptake, artificially depolarizing the cell membrane, rendering the network epileptic, transiently depriving oxygen and glucose supply or directly compromising cell integrity with toxins certainly do not translate into identical stress at the cellular level and are unlikely to recapitulate endogenous mechanisms at play in neurodegenerative diseases. Each of these experimental procedures leads to various degrees of neuronal hyperactivity, metabolic stress, network activity, cell-to-cell miscommunication or direct cell damage, which, in turn, translates into as many corruptions of intracellular calcium signalling. It is therefore very unlikely that one unique pathway or one unique receptor subclass (location or composition) is responsible for transducing each of these insults into excitotoxicity.

Second, the idea that glutamate-induced excitotoxicity is essentially mediated by NMDARs located at extrasynaptic sites, in neuron cultures, is severely challenged by several studies (table 1). In the most recent of these publications [114–116], the authors fail to replicate the canonical dichotomy between synaptic and extrasynaptic NMDARs in terms of cell survival/death, even though they follow the protocols established by Hardingham et al. [15]. Instead, they demonstrate that synaptic NMDARs mediate hypoxia-induced neurotoxicity almost entirely, whereas blocking extrasynaptic NMDARs provides no protection [114]. Zhou et al. [115], on the other hand, show that NMDARs regardless of their location do not promote excitotoxicity when activated separately. They propose that excitotoxicity depends on the magnitude and duration of synaptic and extrasynaptic NMDAR co-activation, regardless of the subtype.

Additionally, the finding that extrasynaptic NMDARs promote neuronal death while synaptic NMDARs are pro-survival has not commonly been reproduced in brain slices or in vivo. In fact, several experiments point to a role for synaptic NMDARs in cell death during NMDA application in acute slices or oxygen glucose deprivation (OGD) in vivo (table 1). Although this discrepancy can be explained by differences between culture, brain slices and intact brain, it could also be that no simple rule relates synaptic or extrasynaptic NMDARs to excitotoxicity. The idea that excitotoxicity may not be caused solely by extrasynaptic receptors is fuelled by recent and thorough insights into the mechanism of the use-dependent NMDAR blocker memantine [114,124]. Memantine was proposed to preferentially block extrasynaptic receptors over synaptic ones, owing to its fast off-rate, low affinity, voltage-dependent binding and uncompetitive nature [23,114,125]. The fact that memantine can successfully prevent neuronal death in vitro or in pathological conditions [23,126,127] has thus strongly contributed to the extrasynaptic hypothesis of excitotoxicity. However, the group of Steven Mennerick demonstrates, in two recent studies, that memantine has a strong and fast inhibitory effect on synaptic NMDARs (80% in 5 min) under basal low-frequency stimulation (0.04 Hz) and, more intriguingly, that the neuroprotective effect of memantine is mediated by the blockade of synaptic, rather than extrasynaptic, NMDARs [114,124]. These new insights, together with the controversy described above, strongly question the prevalent role of extrasynaptic NMDARs in triggering neuronal death during excitotoxic conditions.

Finally, subunit-specific approaches were also largely used in the study of excitotoxicity to assess a preferential involvement of specific NMDAR subtypes, and they often highlighted a role of GluN2B-NMDAR in causing neurotoxicity. But, here again, this idea is facing contradictory results (table 1). For example, even though they followed the protocols previously published in the field [15], Zhou et al. [116] were recently unable to observe any preferential involvement of GluN2A- or GluN2B-NMDARs in excitotoxicity. By contrast, the group of Giles Hardingham demonstrates in a recent study that the CTD of NMDARs, rather than the channel's properties, is crucial in determining how they influence excitotoxicity [128]. They show in particular that the CTD of GluN2B-NMDARs enhances NMDA-induced excitotoxicity in cultures compared with the CTD of GluN2A-NMDARs, and that this selectivity is overcome when strong stimulation (100 µM NMDA) is used. Because the CTD can dictate NMDAR cellular location, these data corroborate the idea that both location and subunit composition can influence excitotoxicity and may cooperate under certain circumstances. But this study too is in contradiction with another recent finding reporting that neither extrasynaptic NMDARs nor GluN2B-NMDARs favour cell death during mild excitotoxic events [19]. Indeed, in this work [19], it was shown that synaptic NMDARs, which are essentially GluN2A-NMDARs, are responsible for neuronal death under NMDA-induced excitotoxicity in slices, in line with other work (table 1). Thus, the role of specific NMDAR subtypes or location in causing excitotoxicity remains a debated issue. Contradictory results obtained across the different preparations, along with discrepancies found when using the same models and protocols, strongly advocate that no simple rule directly relates synaptic or extrasynaptic NMDARs to excitotoxicity.

10. Conclusion

Extrasynaptic NMDARs have attracted a great deal of attention and have become interesting targets for therapeutic strategies. Yet, still very little is known about the organization and the physiological functions of these receptors. For instance, it is unclear whether extrasynaptic NMDARs that face glial, dendritic or axonal processes serve particular aspects of extrasynaptic NMDAR-mediated communication, such as SICs or tonic currents. Additionally, the classical view of extrasynaptic NMDARs being pro-apoptotic is being challenged and would greatly benefit from a unified methodology. Although the use of various preparations is difficult to circumvent, agreeing on a conventional, relevant and reliable way to induce excitotoxicity would provide more consistency and reproducibility across studies performed in this field. It is also crucial to understand that the use of subunit-selective agents, in order to target synaptic or extrasynaptic NMDARs, is unreliable if not carried out with great caution. Indeed, no clear association exists between receptor location and subunit composition in most cases, in particular in young animals.

It seems clear that studying extrasynaptic receptors requires a better understanding of the extrasynaptic space. We here propose that the latter could be seen as highly organized, compartmentalized and regulated, similar to synapses. In fact, the etymology of the word ‘synapse’ refers to two tightly apposed cellular elements that together participate in the transmission of information. In that respect, the close appositions of astrocytic processes onto dendritic shafts (where some extrasynaptic NMDARs are found) are, per se, astrocyte–neuron synapses. As such, they might be regarded as organized and complex structures (as the presence of β-catenins and SAP102/PSD95 suggests) designed to transmit information, in a similar way to conventional neuron–neuron synapses (figure 1).

Funding statement

Work performed in the authors' laboratories was supported by HFSP, Inserm, FRM, Conseil Regional d'Aquitaine, LABEX BRAIN ANR-10-LABX-43 and Agence National de la Recherche.