Questioning Glutamate Excitotoxicity in Acute Brain Damage: The Importance of Spreading Depolarization

Neurocrit Care (2022) 37:S11–S30 https://doi.org/10.1007/s12028-021-01429-4 SPREADING CORTICAL DEPOLARIZATION Questioning Glutamate Excitotoxicity inAcute Brain Damage: The Importance ofSpreading Depolarization R. David Andrew1* , Eszter Farkas2, Jed A. Hartings3, K. C. Brennan4, Oscar Herreras5, Michael Müller6, Sergei. A. Kirov7, Cenk Ayata8, Nikita Ollen‑Bittle9, Clemens Reiffurth10,11, Omer Revah12, R. Meldrum Robertson1, Ken D. Dawson‑Scully13, Ghanim Ullah14 and Jens P. Dreier10,11,15,16,17,18,19,20 © 2022 The Author(s) Abstract Background: Within 2 min of severe ischemia, spreading depolarization (SD) propagates like a wave through compromised gray matter of the higher brain. More SDs arise over hours in adjacent tissue, expanding the neuronal damage. This period represents a therapeutic window to inhibit SD and so reduce impending tissue injury. Yet most neuroscientists assume that the course of early brain injury can be explained by glutamate excitotoxicity, the con‑ cept that immediate glutamate release promotes early and downstream brain injury. There are many problems with glutamate release being the unseen culprit, the most practical being that the concept has yielded zero therapeutics over the past 30 years. But the basic science is also flawed, arising from dubious foundational observations beginning in the 1950s Methods: Literature pertaining to excitotoxicity and to SD over the past 60 years is critiqued. Results: Excitotoxicity theory centers on the immediate and excessive release of glutamate with resulting neuronal hyperexcitation. This instigates poststroke cascades with subsequent secondary neuronal injury. By contrast, SD theory argues that although SD evokes some brief glutamate release, acute neuronal damage and the subsequent cascade of injury to neurons are elicited by the metabolic stress of SD, not by excessive glutamate release. The chal‑ lenge we present here is to find new clinical targets based on more informed basic science. This is motivated by the continuing failure by neuroscientists and by industry to develop drugs that can reduce brain injury following ischemic stroke, traumatic brain injury, or sudden cardiac arrest. One important step is to recognize that SD plays a central role in promoting early neuronal damage. We argue that uncovering the molecular biology of SD initiation and propaga‑ tion is essential because ischemic neurons are usually not acutely injured unless SD propagates through them. The role of glutamate excitotoxicity theory and how it has shaped SD research is then addressed, followed by a critique of its fading relevance to the study of brain injury. Conclusions: Spreading depolarizations better account for the acute neuronal injury arising from brain ischemia than does the early and excessive release of glutamate. *Correspondence: andrew[email protected]; [email protected] 1 Present Address: Queen’s University, Kingston, ON, Canada Full list of author information is available at the end of the article This article belongs to the Collection: Spreading Cortical Depolarization. S12 Introduction Spreading Depolarizations andBrain Ischemia Year after year, published reviews imply that the basic cellular mechanisms underlying acute neuronal death following brain ischemia are reasonably well established based on glutamate excitotoxicity theory. But in fact, the concept that glutamate release by overexcited neurons leads to shortand long-term brain cell death is neither well supported by basic science nor well supported by clinical evidence. In contrast, the role of spreading depolarization (SD) in evoking acute brain damage is more compelling despite SD being underestimated or ignored for years by most researchers of brain ischemia. In this review, we briefly outline the importance of SD in early brain injury, with evidence presented in more detail in the accompanying reviews published in the current issue of Neurocritical Care [1]. We then address the glutamate excitotoxicity theory and compare it with the SD theory in terms of interpreting acute brain injury. SD is the principal mechanism of electrochemical membrane disruption and neuronal swelling [2–5] in gray matter of the higher brain. Severe ischemia depletes the adenosine triphosphate (ATP) pool, leading to Na+/ K+ pump failure, which generates a front of cellular depolarization that propagates at 1–9mm/min through the ischemic gray matter and even into healthy tissue [6–8]. The SD wave is a sudden loss of membrane potential to near-zero millivolts that occurs over seconds. SD can be evoked by various noxious electrical, chemical, thermal, or mechanical disturbances of gray matter that stress the Na+/K+ pump. Thus, SD is associated with a range of diseases and conditions, including migraineassociated aura, concussion, traumatic brain injury (TBI), subarachnoid hemorrhage, intracerebral hemorrhage, ischemic stroke, circulatory arrest, and brain death prior to circulatory collapse [1, 9–14]. SD can also invade gray matter that has not been metabolically compromised, inducing a loss of neuronal activity termed “spreading depression” [15]. In the ischemic core, neurons will die under a maintained depolarization that typically lasts 20–30min or more [16–19]. However, if the ischemic core is reperfused within ~ 15 min, all neurons will survive, even though persistently depolarized for about 15 min [18, 20]. In contrast, perfusion completely ceases after cardiac arrest, and so neurons start to die after about 5min [21]. In milder invivo models, there is no terminal SD (those with no recovery), but typically a cluster of recurrent moderately prolonged SDs occurs superimposed on a relatively shallow negative ultraslow potential. Yet cell death also develops. Clustered SD events pose particularly high metabolic challenge for recovery. The delayed nature of penumbral SDs presents a potential therapeutic window, whereby their inhibition could improve neurological outcome [22]. These SDs appear to arise as a consequence of energy supply–demand mismatch [23, 24]. The cumulative effect of many secondary SDs is a progressive deterioration of metabolic status and lesion expansion. This occurs because of cytotoxic membrane failure as well as SD evoking microvascular constriction in injured tissue with impaired neurovascular coupling. Known as “spreading ischemia” [25], this inverse hemodynamic or initially vasoconstrictive response to SD promotes prolongation of the cellular depolarization and thus cell death [7, 9, 24, 26–28]. In brain trauma as well as ischemic and hemorrhage stroke, 50–90% of patients exhibit neocortical SDs, and many show continuous, repetitive events lasting several days or even weeks after injury, with total counts of 50 to 100 or more. Even terminal SDs have been observed as the correlate of newly developing focal infarcts and of brain death at end-of-life [10, 11, 18, 29]. The full continuum from the normal hyperemic to the inverse ischemic response to SD has been found in patients with aneurysmal subarachnoid hemorrhage [18, 30, 31], TBI [32], and malignant hemispheric stroke [8]. The Na+/K+ ATPase is the main transporter regulating transmembrane cationic gradients. Its compromise leads to SD. The pump exchanges three cytosolic Na+ for two extracellular K+ via hydrolysis of ATP [33]. In mammalian gray matter, the Na+/K+ pump is responsible for ~ 50% of ATP hydrolysis [34]. Lack of blood oxygen and glucose inhibits ATP production, with pump failure evoking sudden SD, driven by the opening of a cryptic Na+/K+ current [35, 36]. Neurons in live slices undergo SD in response to oxygen–glucose deprivation (OGD) [1, 37–39] (Fig.1). Similarly SD is imaged and recorded Keywords: Stroke, Traumatic brain injury, Sudden cardiac arrest, Concussion, Modeling, Migraine, Ischemia, Na+/ K+ pump, Huntington disease, Alzheimer disease, Amyotrophic lateral sclerosis, Ketamine, Penumbra, Persistent vegetative state, Dendritic beading, Brain swelling S13 invivo under ischemia [1, 40–42] (Fig.2). In both cases, it is the twin stresses of Na+/K+ pump failure combined with the energy-demanding requirement to repolarize that can kill or injure neurons. Yet the central role of the Na+/K+ ATPase is rarely considered in explaining excitotoxic mechanisms. Rather, it is accepted by many researchers that the glutamate excitotoxicity theory essentially accounts for the acute neuronal swelling and brain injury that follows ischemia. Simply stated, on reduced blood flow to the brain, nerve cells suffer overexcitation, swelling, and death when the neurotransmitter glutamate is released or is not retaken up. Pathologically high glutamate levels are proposed to overstimulate its receptors, inducing further excitation in a vicious cycle of release and excitation. This neuronal firing leads to high levels of intraneuronal Ca2+, which activates enzymes (phospholipases, endonucleases, and proteases), thereby damaging the cytoskeleton, membrane, and DNA. At least, that is the original textbook story, and numerous modifications have ensued to shore up the concept. However, there are many problems with glutamate being the culprit in brain swelling and neuronal death [4], the most practical being that the concept has yielded zero therapeutics despite decades of work. Moreover, the basic science related to the glutamate excitotoxicity theory must also be reexamined, as well as the assumption that this theory can be extended to yield insights to other central nervous system (CNS) disorders. The purpose of this critique is to compare SD theory, briefly outlined above and described in detail in an accompanying review [1], with glutamate excitotoxicity theory. Additionally, we explore whether these two concepts might have some common ground. We start with briefly examining the historical basis for the acceptance of elevated glutamate levels being the trigger for activating a cascade of biochemical events that kills neurons. How did Excessive Glutamate Release Gain Traction asa Major Cause ofBrain Injury? Glutamate release caused by ischemia has been de facto accepted as the cause of stroke injury for more than 35years. This developed from a series of four foundational observations beginning in the 1950s. The first finding was that excess glutamate injection injured the mouse CNS. But to produce a severe retinal lesion, Lucas and Newhouse [43] in 1957 showed that a parenteral dosage of “a little less than lethal was needed.” In 1969 Olney [44] administered a near-lethal single dose of glutamate subcutaneously, which caused massive brain lesions, later showing that glutamate analogues at high concentration were also effective. A second observation was that ischemia caused glutamate release, an unsurprising finding given that neurons depolarize and that about 70% of CNS neurons are glutamatergic. It is important to note that the extracellular increase was short lived and included other neurotransmitters [45, 46]. For example, OGD-evoked dopamine release is particularly high in striatal slices [47], as is gamma aminobutryic acid (GABA) release in hippocampal slices [48]. The third finding by Choi etal. [49] in 1987 was that glutamate receptor (gluR) antagonists protected cultured neurons from glutamate toxicity. However, note that cultured neurons, being derived from immature tissue Fig. 1 In brain slices, SD is well underway before the extracellular glutamate concentration climbs significantly. A The intrinsic optical signal (IOS) change (top trace) was temporally correlated with the DC negative shift (bottom trace). Both signals denote SD onset (shaded region). The glutamate efflux transient is shown in the middle trace. The horizontal bar indicates [K+]o elevation to 40 mM for 80 s, thereby inducing SD. From Dr. N. Zhou’s doctoral thesis [161]. B More recent recordings using a finer glutamate biosensor also implicated SD as preceding glutamate release. C During SD, glutamate release took ~ 35 s to peak. In mice where the glutamate transporter GLT‑1 is knocked out, glutamate uptake is slowed. B and C from [83]; B is digitally stretched horizontally from the original. In such multi‑recording studies, it is important that sensors are closely placed to precisely determine signal onset times, as further demonstrated in Fig. 2 S14 and having adapted to living in a dish, can tolerate long periods of OGD and do not undergo the incisive event of early stroke ischemic SD. The fourth observation was that N-methyl-d-aspartate receptor (NMDAR) antagonists were reported to reduce ischemia-induced neuronal damage in rodents invivo. But hundreds of compounds unrelated to gluRs showed efficacy, most reported in rodent models of ischemia over the past 30years [50]. Yet no drug has proven to be protective in clinical trials of ischemia. Twenty-eight suspected inhibitors of excitotoxicity were tested preclinically in patients with acute stroke in 1993–2001 [50], but none proved to be neuroprotective. Two other potentially useful drugs, ketamine (“Ketamine and SD Inhibition” section) and NA-1 (“Therapeutic Hope for Combating Glutamate Excitotoxicity?” section), antagonize NMDARs but may also have broader actions. The many rationalizations for these negative results have included claims of too broad patient selection criteria, problems with delayed intervention, poor tolerability by patients, publication bias in favor of positive results (perhaps for commercial or other reasons), quality of the molecules (pharmacokinetic deficiencies), inability to reach effective concentrations in the penumbra, inappropriate neuroprotective time window, insufficient receptor subunit selectivity, high drug toxicity in humans, inequivalent doses compared to rodents, development of tolerance (eg, upregulation of NMDARs), and finally side effects blocking normal synaptic N-methyld-aspartate (NMDA) activity that would have promoted neuronal survival [51]. Some of our authors feel that it is still worthwhile to search for clinical applications for gluR antagonists. Nevertheless, it is surprising that this litany of excuses has rarely led to questioning the basic science that has portrayed glutamate release as the great destroyer of the CNS. A handful of publications provide exceptions [52–55]. Glutamate Release andSD Use ofAntagonists toBlock SD inLive Brain Slices Glutamate was originally considered a contender as an SD mediator because of its progressive release into the interstitial space during brain ischemia [56]. There is a long history of studies using live brain slices with the goal of identifying how SD is initiated and sustained. A common presumption has been that glutamate release must have a central role. Various inhibitors of channel opening, of transmitter receptors, or of transporters have been bath-applied to test which mechanisms elicit SD (Table1). Na+ Channel Blockers The Na+ channel blocker tetrodotoxin (which silences all action potential activity) does not block either hypoxiaor OGD-induced SD (Fig.3b, d), indicating that spikeactivated synaptic release of neurotransmitters is not a requirement for SD. Likewise, Na+ channel blockers composing the caine family (dibucaine, lidocaine, procaine, etc.) silence action potential firing but merely delay OGD–SD onset [46, 57–59]. And invivo, lidocaine only delays SD and is not neuroprotective [21]. Although not necessary for SD generation, V-sensitive Na+ channel opening supports SD onset [60]. NMDAR Antagonists NMDAR antagonists tested individually could indeed inhibit the propagation of SD in nonmetabolically stressed brain slices [61]. Under hypoxia in slices, NMDAR antagonists generally delay but cannot block SD [62]; however two hypoxia studies did report blockade (Table1), so the results are inconsistent. NMDAR antagonists did not prevent SD evoked by lowering both oxygen and glucose levels [39, 63, 64]. Non‑NMDAR Antagonists Tested individually, these inhibitors of α-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kynurenate receptor binding could delay but, again, not block OGD-evoked SD [39, 64] (Table1). Antagonist Cocktails Slice researchers have also combined blockers (Table1). Early slice studies showed that gluR antagonists can delay or stop the onset of hypoxic SD [62] but only delay SD evoked by OGD [36, 39, 64, 65], findings supported in many but not all subsequent slice studies (Table1, Fig.3b, d). Commonly the cocktails include antagonists of gluRs, of glutamate transport, and/or of cation channels to (See figure on next page.) Fig. 2 Dynamics of extracellular glutamate and K+ during SD in vivo. The SD was evoked at a distance by applying a droplet of 1 mM KCl to the surface of the mouse brain. A iGluSnFR, a fluorescent probe, monitors the extracellular glutamate level. B Speed of the extracellular glutamate wave. C Average fluorescence traces with 95% confidence interval, amplitude, and duration of the glutamate transient. D Glutamate trace (blue) aligned to the DC potential below. E Latency between negative DC deflection and glutamate increase. F Relationship between [K+]o, DC potential, and neuronal [Ca2+]i. The latency between 0.25 mM [K+]o rise (arrow over K+ trace) and increase in fluorescence (red vertical line) is indicated to the right. Dashed line indicates start of the negative DC potential shift. G As in F but with [Ca2+]i in astrocytes instead of neurons. H As in F but with [glutamate]e instead of neuronal [Ca2+]i. Images in F–H show positions of electrodes (stippled lines) and sampled regions (white circles). Sampled regions were picked along the front edge of the SD wave as it hit the K+‑sensitive microelectrode. Scale bars: 50 μm; error bars, SEM. From [85] S15 Fig. 2 (See legend on previous page.) S16 Table 1 Studies using live brain slices totest one or more drugs thatmight block SD underthe energy compromise imparted byhypoxia or OGD Study Stimulus Glutamate recep‑ tor blockers Na+ channel blockers GABAA receptor blockers Ca2+ channel blockers Other channels blocked Recording mode SD blocked? (in some slices) SD imaged? Radek and Giardina [75]Hypoxia 100 µM AP‑5 Yes No Tanaka et al. [39]‑ drugs tested separately OGD 50–250 µM AP‑5 or 10–20 µM CNQX 20 µM bicuculline Intracellular No No Yamamoto et al. [64]‑drugs tested separately OGD 50–250 µM AP‑5 or 10–20 µM CNQX Procaine (0.3–1 mM) or TTX (0.3 μM) 2 mM Co2+ 2 mM Ni2+ or 10 μM nifedipine K+: (20 mM TEA) Intracellular No No Müller and Somjen [70]Hypoxia 10 µM CPP or 10 µM DNQX 1 µM TTX 2 mM Ni2+Field recording or imaging Partly Yes Müller and Somjen [164]Hypoxia 10 µM CPP or 10 µM DNQX 1 µM TTX Field recording or intracellular No in 50% of neurons No Rossi et al. [88] Chemical ischemia + OGD 50 µM D‑AP5, 50 µM MK‑801, 25 µM NBQX, 100 µM 7‑chlo‑ rokynurenate 100 µM bicucul‑ line Intracellular No, but yes in a few slices when glutamate transport also inhibited No Jarvis et al. [163] (rat) Joshi and Andrew [38] (mouse) OGD 50 μM AP‑5 + 10 μM CNQX or 2 mM kynurenate Field recording or intracellular Delayed Yes Anderson et al. [37]OGD Sigma receptor‑ activated Imaging Yes Yes Madry et al. [162] Chemical ischemia + OGD 50 µM D‑AP5, 50 µM MK‑801, 25 µM NBQX, 100 µM 7‑chlo‑ rokynurenate 100 µM bicucul‑ line Pannexin block had no effect Intracellular Yes, but residual current No Douglas et al. [57] OGD 1–10 µM dibu‑ caine or other caines Imaging Yes Yes Revah et al. [71] Hypoxia 50 μM APV, 50 μM MK‑801, 50 μM DNQX, 25 µM NBQX 25 μM bicuculline Pannexin block had no effect Intracellular Yes No Gagolewicz et al. [165]OGD 1 mM kynurenic acid 1 μM TTX 100 μM picrotoxin 10 μM nifedipine K+: (10 mM TEA); pannexin block had no effect Intracellular No Yes S17 isolate conductances required for SD generation and propagation. There are variations in cocktail constituents, in the mode of SD induction, and in the degree of SD inhibition. Additionally, there is no reported drug reversibility because of high antagonist concentration, tight chemical binding, or both. The role of gluRs in SD generation remains a contentious issue for several reasons. Normoxic and hypoxic SDs are more easily inhibited than ischemic SD both invivo and in slices. Finally, note in Table1 that cocktails may contain one or more non-NMDAR antagonists (CNQX (6-cyano-7-nitroquinoxaline2,3-dione), DNQX (6,7-dinitroquinoxaline-2,3-dione), NBQX (2,3-dioxo6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide)) at concentrations between 25 and 100μM. Yet these drugs are approximately equipotent at the same AMPA receptors and are fully potent in hippocampal slices at only 3 to 5μM based on several standard electrophysiological paradigms [66]. Likewise, the NMDAR antagonist MK-801 applied at 50μM nevertheless fully blocks evoked field potentials in slices at 1 to 2μM [67, 68]. This is also an issue with the wide-spectrum gluR antagonist, kynurenate. It selectively blocks responses to NMDA at 200 μM [69] yet is incapable of blocking OGD–SD at 1 to 2mM (Fig.3b, d, Table1). So gluR antagonists are commonly bath-applied at much higher concentrations than the effective dose required to block the receptors during normal synaptic transmission. These gluR blocker levels may represent pharmacological overkill. This is an important point because Tanaka et al. [39] showed that individually increasing AP-5 stepwise from 50 to 100μM, and then to 200μM, significantly delayed OGD–SD onset longer at each step. Likewise, individually increasing CNQX from 10 to 20μM significantly delayed OGD–SD onset further. This suggests that including enough gluR blockers in a cocktail at concentrations that exceed their effective physiological thresholds for synaptic blockade can cause cumulative and nonspecific effects. Most importantly, this includes a nonreversible silencing of the gray matter, making it appear that SD has been blocked [70]. This issue of the potentially toxic effects by gluR antagonists was discussed by Revah etal. [71]. They noted that the high levels used in slice studies had confounded their use clinically. Whether lower levels of gluR antagonists might prove therapeutically useful is worth pursuing. False SD Blockade Another reason why potential SD inhibitors may appear to block SD (but do not) is the issue of “false positives in SD suppression” [72]. When SD is imaged invivo [73] or in slices, SD often does not invade the entire field under view. If a neuron is recorded without imaging of Table 1 (continued) Study Stimulus Glutamate recep‑ tor blockers Na+ channel blockers GABAA receptor blockers Ca2+ channel blockers Other channels blocked Recording mode SD blocked? (in some slices) SD imaged? Gagolewicz et al. [165]OGD Block of ASIC, P27X, and TRPM7 chan‑ nels + gluta‑ mate transport inhibition had no effect Intracellular No Yes GluR antagonists, applied either separately or as a cocktail, do not block SD in the majority of studies of OGD. Being less metabolically demanding, hypoxic SD is more easily inhibited. Where SD is reported as blocked, a caveat is that SD may have occurred outside the recorded cell’s area, unless confirmed with imaging. Invariably, none of these studies block SD in all slices tested N-methyl-d-aspartate (NMDA) receptor antagonists inhibit NMDA receptors: AP5 or APV DL-2-amino-5-phosphono-valerate, CPP (±)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid, ketamine 2-(2-chlorophenyl)-2- (methylamino)cyclohexan-1-one, MK-801 (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine Non-NMDA receptor antagonists inhibit AMPA/kainate receptors: CNQX 6-cyano-7-nitroquinoxaline2,3-dione, DNQX 6,7-dinitroquinoxaline-2,3-dione, NBQX 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7sulfonamide. TEA tetraethylammonium, TTX tetrodotoxin. For additional treatments that do not block OGD–SD, see [63, 64] Kynurenate 3-Anthraniloyl-l-alanine, broadly inhibits glutamate receptors 1 Some sigma receptor ligands dramatically inhibit SD onset [37], but the function of sigma receptors remains unclear 2 Two studies, [57, 58], showed neuronal protection from SD initiation and injury by 1–10mM dibucaine, the most potent Na+ channel blocker of the caine group 3 OGD-induced intracellular Ca2+ increases are mediated by Ca2+ influx through NMDARs, VGCCs and TRPC channels as well as by Ca2+ release from RyRs and IP3Rs. This impairs mitochondria, facilitating SD generation [63] S18 the entire slice, it is uncertain if the SD front has actually reached the recorded cell. If not, SD would appear to be blocked. Likewise, the lack of a negative direct current (DC) shift recorded near the recorded neuron may represent a true blockade of SD. Alternately, SD simply did not invade the locale of the field recording. Therefore, in studies in which complete SD blockade is reported (Table1), either imaging or multiple electrode recordings would confirm that SD was indeed blocked throughout a recorded slice. Despite variation in slice study findings, several points of consensus stand out: 1. Inhibiting OGD-induced SD is difficult; hence, an unidentified channel (or channels) responsible for generating SD is likely. One proposal being pursued is that the Na+/K+ pump converts to an open channel [74]. 2. The opening of standard Na+, Ca2+, or K+ channels is not necessary for SD generation. 3. High concentrations of toxic drugs are less effective in inhibiting SD than simply lowering slice temperature, which protects neurons from hypoxia [75] or OGD [38]. Nonsynaptic release ofextracellular glutamate duringSD In hippocampal slices, the orthodromically evoked CA1 field potential is typically lost prior to hypoxiaand OGD-induced SD onset [76] (Fig.10c in Ref. [37]). The effect is less obvious in neocortical slices, where SD onset has a shorter latency (Fig.11b in Ref. [37]). The loss of the synaptic response in CA1 prior to SD may be an early effect of lowered ATP levels because it is not seen prior to SD evoked by ouabain or high K+ levels, in which ATP production is not compromised as with OGD. Additionally, an early release of the presynaptic blocker adenosine has also been proposed [77]. However, the OGD-evoked loss of the synaptic response raises the question as to how theextracellularglutamate concentration ([glu]o)begins to climb because of SD onset. There are several proposed mechanisms to account for this increase, including (1) reversal of glutamate transporters [78], (2) release by volume-activated channels in astrocytes [79], (3) release via presynaptic NMDAR activation [80], (4) release of a small but readily releasable pool (RRP) of glutamate [71, 81], (5) Ca2+-dependent vesicular glutamate release from astrocytes (although this process has not been directly linked to SD), and (6) a major source of extrasynaptic glutamate (the cystine/glutamate antiporter) stimulated by ischemia [82]. Specifically, Revah etal. [71] showed that hypoxia can elicit a small RRP of glutamate from axon terminals in slices. The RRP is not linked to action potential invasion of the terminal, which means that there is then less available glutamate for regular synaptic release. This could potentially contribute to the eventual synaptic failure that precedes SD initiation under hypoxia or OGD. However, there are, as of yet, no recording techniques sensitive enough to directly detect the small [glu]o increase originating from the RRP. So there are several sources of extracellular glutamate that could support SD ignition evoked either by elevated extracellular potassium concentration ([K+]o)levels or by hypoxia. Nonetheless, with the greater metabolic load of OGD, SD persists in the presence of gluR antagonists in most but not all studies, both invivo and in acute slices (“Use of Antagonists to Block SD in Live Brain Slices” section). Importantly, there is no evidence to date that the initiation of any form of SD onset is preceded by measurable glutamate release. Early invivo studies showed that a [glu]o increase followed local OGD–SD onset, as measured by the initial DC shift or by the slight upswing in [K+]o [55]. A glutamate surge was also detected in slices within seconds of the SD front passing [83, 84] (Fig.1a). More recently, use of genetically encoded optical glutamate sensors allowed improved spatiotemporal resolution of transient glutamate levels near the SD front measured as the negative DC shift [85]. The onset of the DC shift coincided with the first increases in [K+]o, but coregional [glu]o elevation began only after the DC shift, and [K+]o had been increasing for 2–3s (Fig.1b, c). Thus, elevated [glu]o in the locality of SD initiation appears to be a result, rather than a cause, of ischemic or hypoxic SD. This is not to say that elevated [glu]o cannot increase SD propensity in normoxic situations [86, 87]. Glutamate Transporter Studies andOGD‑Induced SD Reduced reuptake of glutamate as a promoter of ischemia-like SD was examined by Rossi etal. [88] using Fig. 3 Unlike O2/glucose deprivation (OGD), bath superfusion of glutamate at pathophysiological concentrations onto a brain slice does not induce SD. A Imaging change in light transmittance (ΔLT) reveals OGD‑induced SD and propagation (arrows) along neocortical gray matter (NC) and through striatum (S) with damage arising in the wake of SD (magenta). w = slice weight. B A cocktail of blockers (Mix‑1, constituents listed in Fig. D) delays OGD‑induced SD onset but not propagation (arrows). By 16.8 min, light scattering in NC caused by dendritic beading indicates acute neuronal damage. C Bath superfusion of glutamate causes slight signal creep from the overlying weight, but no SD. D Superfusion of Mix‑1 signifi‑ cantly (p = 0.002) delayed OGD‑induced SD onset by 46 ± 11.8%. Adding MK‑801 slightly but significantly (p < 0.0001) further delayed SD onset by 52 ± 9.9%. E Percent of slices generating signal creep but no SD in three experimental groups of glutamate application to naïve slices. No SD was observed in slices superfused in aCSF alone (n = 6) or in aCSF + 1 mM glutamate (n = 6) (See figure on next page.) S19 Fig. 3 (See legend on previous page.) S26 Charité – Universitätsmedizin Berlin, Berlin, Germany. 16 Department of Neurology, Corporate Member of Freie Universität Berlin, Berlin, Germany. 17 Department of Neurology, Humboldt‑Universität zu Berlin, Berlin, Germany. 18 Department of Neurology, Berlin Institute of Health, Berlin, Germany. 19 Bernstein Center for Computational Neuroscience Berlin, Berlin, Germany. 20 Einstein Center for Neurosciences Berlin, Berlin, Germany. Author Contributions Much of the manuscript was written by R. David Andrew and Jens Dreier, with important contributions, feedback, editorial comments, and corrections from each of the other authors. Source of Support Grants to RDA from the Canadian Heart & Stroke Foundation, National Sci‑ ence Engineering and Research Council and the New Frontiers in Research Fund, to E.F from the National Research, Development and Innovation Office of Hungary, grant no. K134377; and the EU’s Horizon 2020 research and innovation program under grant agreement No. 739593, and to JPD from the DFG (German research Council) (DFG DR323/5‑1,DFG DR 323/10‑1) BMBF Bundesministerium fuer Bildung und Forschung (Era‑Net Neuron EBio2, with funds from BMBF 01EW2004). Declarations Conflicts of interest The authors declare that they have no conflicts of interest to disclose. Ethical Approval/Informed Consent This article adheres to all ethical guidelines outlined by Neurocritical Care for review articles and aims to provide a balanced discussion of an issue of clinical relevance to the journal. 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. 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