blocking the anoxic depolarization protects without functional ...

Articles in PresS. J Neurophysiol (September 29, 2004). doi:10.1152/jn.00654.2004

BLOCKING THE ANOXIC DEPOLARIZATION PROTECTS WITHOUT FUNCTIONAL COMPROMISE FOLLOWING SIMULATED STROKE IN CORTICAL BRAIN SLICES

TRENT R. ANDERSON, CATHRYN R. JARVIS, ALYSON J. BEIDERMANN, CHRISTINE MOLNAR and R. DAVID ANDREW Department of Anatomy and Cell Biology Queen`s University Kingston, Ontario, Canada K7L 3N6

Address for correspondence:

Dr. R. David Andrew Department of Anatomy and Cell Biology Queen’s University Kingston, Ontario, Canada K7L 3N6

Copyright © 2004 by the American Physiological Society.

1 ABSTRACT

Within two minutes of stroke onset, neurons and glia in brain regions most deprived of blood (the ischemic core) undergo a sudden and profound loss of membrane potential caused by failure of the Na+/K+ ATPase pump. This anoxic depolarization (AD) represents a collapse in membrane ion selectivity that causes acute neuronal injury because neurons simply cannot survive the energy demands of repolarization while deprived of oxygen and glucose. In vivo and in live brain slices, the AD resists blockade by antagonists of neurotransmitter receptors (including glutamate) or by ion channel blockers. Our neuroprotective strategy is to identify AD blockers that minimally affect neuronal function. If the conductance underlying AD is not normally active then its selective blockade should not alter neuronal excitability. Imaging changes in light transmittance in live neocortical and hippocampal slices reveals AD onset, propagation and subsequent dendritic damage. Here we identify several sigma-1 receptor ligands that block the AD in slices that are pretreated with 10-30 uM of ligand. Blockade prevents subsequent cell swelling, dendritic damage and loss of evoked field potentials recorded in layers II/III of neocortex and in the CA1 region of hippocampus. Even when AD onset is merely delayed, electrophysiological recovery is markedly improved. With ligand treatment, evoked axonal conduction and synaptic transmission remain intact. The large non-selective conductance that drives AD is still unidentified but represents a prime upstream target for suppressing acute neuronal damage arising during the first critical minutes of stroke. Sigma receptor ligands provide insight to better define the properties of the channel responsible for anoxic depolarization.

Running Head: Neuroprotection following anoxic depolarization Key words: anoxia, hypoxia, peri-infarct depolarizations, stroke, ischemia, intrinsic optical signals

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2 INTRODUCTION

Neurons and glia suddenly depolarize within two minutes of stroke onset where cerebral blood flow falls to ifenprodil > DTG > loperamide. Likewise the order for reduction of high voltage-activated Ca2+ channel activity (Church and Fletcher, 1995) differed from this ranking. Therefore a R-mediated inhibition of AD appears more likely than NMDA receptor antagonism or Ca2+ channel blockade. The potency of

1R

ligands to block the AD improves when pre-incubation times are increased

from 15-20 min to 30-35 minutes. Secondary messenger pathways may be involved, but a slow diffusion of the ligands into the slice cannot be ruled out. The maintenance of ATP levels through action upon mitochondrial function (Klouz et al., 2003) seems unlikely because induced AD where ATP levels are not compromised.

1R

ligands can block ouabain-

22 AD Blockade through Numerous

1R

1R

Mediation

ligands have been identified based on their binding affinity to

1

receptors,

including the non-prescription antitussives dextromethorphan (DM) and carbetapentane (CP). At least two subtypes of the

receptor exist (Quiron et al., 1992; Booth et al., 1993) with selective binding of

carbetapentane and dextromethorphan at

1R

(Musacchio et al., 1989). The

1R

has been sequenced and

cloned but it has no homology to known mammalian proteins (Hanner et al., 1996). It includes two transmembrane domains and can modulate K+ channels (Aydar et al., 2002) but its physiological role is unknown but is implicated in immunosuppressant, antipsychotic and neuroprotective effects (Moebius, 1997). Sigma-1 receptors have a regional distribution within the CNS with the highest concentrations in hindbrain and intermediate densities in neocortex and hippocampus (Tortella et al., 1989; Leitner et al., 1994). They are found primarily in neurons and significant levels occur in cortical pyramidal cells (Gundlach et al., 1986). Dextromethorphan is neuroprotective in rodent models of stroke (George et al. 1988; Prince and Feeser 1988) attributed to its moderate antagonism of NMDA-induced responses (Choi 1987; Fletcher et al. 1995; Klette 1997; Thurgur and Church 1998; Palmer 2001). Other R ligands can potentiate NMDAinduced responses (Bergeron 1996; 1997; Debonnel 1996; Couture et al. 1998; Gronier et al. 1999). Whittemore et al. (1997) showed that 4-IBP was essentially inactive against all NMDA receptor subunit combinations up to its solubility limit in saline of ~ 30 µM. It was also inactive at the PCP binding site of the NMDA receptor. Since we found that 4-IBP could block AD, it is unlikely that the NMDA receptor is involved. Also carbetapentane has a much lower affinity for NMDA receptors than DM yet we found CP to be more the more potent AD blocker. The inability of NMDAR antagonists (both competitive and non-competitive) to inhibit AD onset is strong evidence that AD blockade by R ligands is independent of NMDA receptors (Obeidat et al. 2000; Jarvis et al. 2002; Joshi and Andrew 2001). The compounds tested that blocked or delayed AD onset in the current study (i.e. DM, CP and 4IBP) each have high inhibited by the

1R

affinity and are designated

1R antagonists

1R

agonists. Their ability to block AD at 100 µM is

BD-1063 or (+) 3-PPP at 100 µM (Musacchio et al., 1989; Matsumoto et

al., 1995; Whittemore et al., 1997). However, a competitive antagonist would only be expected to block agonist actions if present at a significantly higher multiple of binding affinity than the agonist. But note that a minimum of 15-20 minutes of agonist pretreatment is required to block AD. The antagonist preexposure for 20 minutes and then co-exposure with agonist (Fig. 4C,D) is apparently enough to slow agonist binding and so permit AD onset. We have found that several other R agonists block or delay ouabain-induced AD at 30-50 µM, including (-)SKF 10,047, DTG, ifenprodil and haloperidol (Jarvis et al., 2002). While each has some cross-reactivity with other neurotransmitter receptors, their only common binding is at the

receptor. In

23 contrast, spiperone shares structural features of R ligands with only low binding to Rs, (Monnet et al., 1992) and so did not prevent AD or antagonize the blockade of AD by DM. 1R

Ligand Effects Upon Evoked Field Potentials At the lowest concentration tested (0.1 µ ), we found that the three R ligands enhanced the

evoked response at the Schaffer collateral/CA1 synapse.

This AMPA receptor-mediated event is

similarly enhanced by the R ligand SR31742A (Liang and Wang 1998). DM, CP and 4-IBP reversibly increased the amplitude of the population spike evoked orthdromically but not antidromically. Moreover this enhancement was lost in the presence of the response is likely mediated by

R antagonist (+) 3-PPP so the increased synaptic

receptors. At 1.0 µM, DM or CP had no effect probably because the dose

was intermediate between excitatory at 0.1µM (above) and inhibitory at 10-100 µM (below). At 10-30 µM, DM or CP blocked or delayed AD but with no effect on the evoked response from CA1 or of neocortical layers II/III. It is therefore unlikely that AD blockade is through action upon glutamate receptors or on Na+ and Ca2+ channels that regulate normal neuronal excitability. This is an important finding that suggests that AD and AD-like events can be blocked without functional compromise to neocortex. At 100 µM, DM or CP produced a profound inhibition of both the orthodromic and antidromic field potential that was slowly but reversibly induced. Other R ligands such as haloperidol, (-)SKF 10,047, DTG and ifenprodil behave similarly at 100 µM (Jarvis et al., 2002). Likewise Ishihara et al. (1999) found that the evoked orthodromic response was reduced by 100 µM of the

1R

ligand OPC-

24439. We found that the antidromic and orthodromic response were suppressed by the ligands at 100 µM, suggesting that axonal conduction is impeded at this higher concentration. This may be a nonspecific effect of sigma receptor ligands at this high concentration. Limited data from other intracellular studies have shown no change in CA1 membrane potential, cell input resistance or action potential threshold as recorded at the cell body during 50 min of exposure to 100 µM DM (Wong et al., 1988; Church, personal communication) or to the

R ligand OPC-24439 (Ishihara et al., 1999).

We will

examine in future if R ligands reduce axonal conduction while leaving action potential generation at the soma unaffected. How Do R Ligands Block AD? Following the onset of global or focal ischemia, CA1 neurons can maintain their energy stores to drive ATP-dependent ionic pumps for about 60 seconds (Kristian, 1997). Mitochondrial respiration is inhibited during ischemia by the rapid decline in O2-tension and reduced ATP production leads to failure of the Na+/K+ATPase pump (Lipton, 1999). As [K+]o increases to ~13 mM (Kristian and Siesjo, 1997), a sudden and rapid depolarization of astrocytes and glia (the AD) is measured as an abrupt negative

24 deflection of the extracellular potential when neurons and glia suddenly depolarize (Walz, 1997; Tanaka et al., 1997). Extracellular Na+, Cl- and Ca2+ rush in with water following osmotically, causing cell swelling that shrinks the extracellular space (Hansen, 1981). The return of cell membrane potential depends on the rapid return of cerebral blood flow. Even brief inhibition of the Na+/K+ ATPase pump causes catastrophic depolarization which can arise from: 1) restricted ATP production caused by OGD, 2) metabolic inhibitors such as sodium cyanide or dinitrophenol (Tanaka et al., 1997; Anderson et al., 1998), or 3) direct binding to the pump by ouabain. This specific Na+/K+ ATPase inhibitor induces AD identical to that induced by OGD, sodium cyanide or dinitrophenol when imaged (Obeidat et al., 1998; Jarvis et al., 2001; Anderson et al., 2001) or recorded electrophysiologically (Balestrino, 1999). The ability of R ligands to delay or block AD at 10-30 µM without ostensibly altering synaptic transmission or axonal conduction is not in keeping with an action upon conventional ion channels or neurotransmitter receptors. With one exception, there are no reports of outright AD blockade by reducing K+, Cl- , Ca2+ or nonspecific cationic conductances. Weber and Taylor (1994) noted delay or block of AD in about 40% of hippocampal slices using low levels (0.5- 2.0 µM) of the sodium channel blockers TTX or lidocaine. However, synaptic transmission and axonal conduction were not rescued post-AD. Higher doses of lidocaine or phenytoin (60 - 200 µM) were more protective of synaptic function but still only blocked AD in ~60% of slices. They make the important point that not all Na+ channels are inactivated during AD so Na+ channel blockade may be of some neuroprotective benefit. Aarts et al. (2003) have observed protection from OGD damage in cultured neurons by blocking the TRPM7 channel. However cultured nerve cells are notoriously resistant to the stress of OGD. The induced depolarization slowly builds over two hours, having a slow and graded onset common in cultured neurons and cultured brain slices (Perez-Velaquez et al., 1997). In acute brain slices or in the ischemic core in vivo, AD is sudden (within 1-3 min of OGD onset) and involves a massive conductance increase. Whether the TRPM7 channel has a role in AD needs to be determined. Could a Megachannel Generate the AD? There are no reports of AD block by neurotransmitter antagonists or by lowering intra- or extracellar [Ca2+]. Such manipulations may delay AD onset but will also affect CNS excitability. In contrast, the key player in maintaining the cell resting potential, the Na+/K+ ATPase pump (Lipton, 1999), runs in the background and theoretically could be protected or augmented without obvious electrophysiological consequences. However the catalytic region of the pump protein involved with ATP dephosphorylation (which minimally functions during OGD) is intracellular whereas the region bound by ouabain is extracellular (Habiba et al., 2000). Sigma receptor ligands block AD whether evoked by reduced ATP availability (caused by OGD) or by by ouabain binding to the pump protein thereby preventing ATP dephosphorylation. This R action would seem to be downstream from the pump, probably acting by preventing the large non-selective conductance underlying AD initiation (Tanaka et

25 al., 1997, 1999). We propose that if such a conductance is inactive under normal physiological conditions then its blockade should have minimal electrophysiological consequences. In this way DM and CP can suppress the AD while the responsiveness of the pyramidal neuron remains intact.

The assumption has been that only cations pass through the hypothetical channels that drive AD but Tanaka et al. (1999) have suggested that a larger porosity channel may conduct molecules of several hundred Daltons. This possibility has gained credence recently with the independent discoveries that ischemia can open two different forms of ‘megachannel’. The mitochondrial transition pore (MTP) conducts compounds up to 1500 Da (Ricchelli et al., 2003). A second megachannel candidate is the normally closed ‘hemichannel’ that forms one of two apposing gap junction channels. During ischemia, certain subtypes of these junctions can open, passing molecules up to 1200 Da (Bennett et al., 2003). A third megachannel candidate is the P2X channel where several channel subtypes can dilate to conduct molecules of several hundred Da (Virginio et al., 1999; Duan et al., 2003). A large non-specific cationic conductance, similar to that driving AD but of shorter duration (1-2 min), is responsible for generating SD (Czeh et al. 1992). As with AD, spreading depression is blocked by DM, CP and 4-IBP (Anderson and Andrew, 2002). We propose that the conductance is the same but energy deprivation prohibits channel closure. With

1R

ligand pretreatment, AD blockade relieves

energy-starved cells from a prolonged depolarized state where the cell membrane is leaky to more than just small cations. Extending pretreatment from 20 to 40 minutes improves efficacy. Our argument that AD is the metabolically stressed equivalent of SD has been strengthened by the recent finding that mutation to the Na+/K+ ATPase pump protein leads to familial hemiplegic migraine which is associated with SD (DeFusco et al., 2003). The recent development of a

1R

knockout mouse (Langa et al., 2003)

could help delineate how these receptors mediate blockade of anoxic depolarization and spreading depression. Most importantly, effective blockers will facilitate characterization of the channel responsible for AD. Peri-infarct depolarizations (PIDs), which we have suggested are intermediate events between AD and SD (Andrew et al. 2002), should likewise be inhibited by

1R

ligands thereby increasing

neuroprotection.

Acknowledgements Funded by the Heart & Stroke Foundation of Ontario (grant no. T-4478) and the Canadian Institutes of Health Research.

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32 FIGURE LEGENDS

FIGURE 1 A)

The equipment for imaging light transmittance consists of a broadband halogen light source filtered

by a near-infrared pass filter. The light is scattered, absorbed, or transmitted. Transmitted light is collected and digitized with a charge-coupled device (CCD) and processed using a frame grabber board controlled by imaging software in a Pentium computer. B)

Digitized, pseudo-colored images demonstrate the percentage of light transmittance (LT) change

( T/T%) in response to oxygen/glucose deprivation (OGD). The anoxic depolarization (AD) was induced in a coronal slice of the rat neocortex and hippocampus. The focal increase in LT representing AD initiated at several points(*). A wave of elevated LT (blue-yellow) propagated through the neocortex (5:18 to 7:10) and then through the CA1 region (7:10 to 9:00) shown by the arrows. Where the AD passed, there was a delayed and irreversible decrease in LT (magenta pseudocoloring) representing damage that results from increased light scatter by beaded dendrites. C)

Time course of LT changes in two neocortical slices, one slice exposed to 100 µM ouabain and the

other to oxygen/glucose deprivation (OGD). Each LT peak represents the AD front passing through a zone of interest comprised of several hundred pixels in neocortical layers II/III. By 10 min, both zones display an irreversible decrease in LT representing damage in the wake of AD.

FIGURE 2 The time course of light transmittance change sampled in layers II/III of neocortex.

A)

Pretreatment with 10 µM DM can block AD induced by 5 min of 100 µM ouabain in half

the slices tested. At 1 µM, DM is not effective in blocking or delaying AD onset. B)

Glutamate receptor antagonists do not block or delay AD whether induced by OGD or ouabain. In

this example, 100 µM MK-801 pretreatment fails to alter ouabain-induced AD onset.

FIGURE 3

Onset of the anoxic depolarization and associated changes in light transmission. Drug

pretreatments were for 15-20 min at 30oC. If the AD is blocked (white bars in A), then the LT values in B remain near baseline. If the AD proceeds (shaded bars in A), then the LT maximum (indicating cell swelling) and valley (indicating cell damage) in B are pronounced. A)

Sigma-1 receptor agonists block AD induced by exposure to 100 µM ouabain for 5 minutes which

normally induces AD in untreated slices. Drug concentrations were 100 µM where not stated. Pretreatment for 10 – 15 min in 30-50 µM DM or CP or 30 µM 4-IBP blocked AD onset. Antagonism of the NMDA receptor by MK-801 or AP-5 did not even delay AD onset nor did AP-5 inhibit DM’s ability to block AD. In contrast, the R antagonists (+)-3PPP and BD-1063 inhibited block by DM and 4-IBP

33 while displaying no blocking ability themselves. Spiperone displayed no blocking ability nor did it affect AD blockade by DM. Error bars represent standard deviation from the mean. B)

The dramatic LT changes accompanying AD (Fig. 4A) were not observed when AD was blocked.

Neither increases in LT (the AD front) nor subsequent decreases in LT (damage) were moderated by the NMDA receptor antagonists MK-801 or AP-5. Such changes associated with AD are apparently all-ornone. Error bars represent standard error of the mean of LT change within a zone of interest.

FIGURE 4 Pretreatment with 100 µM of the

1R

ouabain-induced AD, as was the

antagonist BD-1063 shown in B. However, both (+)-3PPP (shown

1R

antagonist (+)-3PPP shown in A was ineffective in blocking

in C) and BD-1086 (shown in D) effectively eliminated AD block by 100 µM DM or 100 µM CP. Thus both (+)-3PPP and BD-1086 antagonized AD blockade by DM or CP without inducing AD block themselves.

FIGURE 5 Sigma receptor ligands block the anoxic depolarization induced by ouabain or OGD for 10 min. A) A 15-20 min pretreatment with the

1R

ligand DM (100 µM) blocked AD during a 10 min exposure

to OGD or during exposure to 100 µM ouabain. B) A 15-20 min pretreatment with the

1R

ligand 4-IBP (30 µM) blocked AD during a 10 min exposure

to OGD or exposure to 100 µM ouabain.

FIGURE 6 A) Sigma receptor agonists blocked AD caused by exposure to 100 µM ouabain for 10 min. Drug concentrations were 100 µM unless stated. Pretreatment for 30-35 min at 30oC in 30-50 µM DM, 10-100 CP µM or 30 µM 4-IBP blocked or delayed AD onset. The

R antagonists (+)-3PPP and BD-1063

inhibited block by 4-IBP while displaying no blocking ability themselves. Error bars represent standard deviation from the mean time of onset. B) Sigma-1 receptor agonists block AD caused by exposure to OGD for 10 min. Drug concentrations were 100 µM unless stated. Pretreatment for 30-35 min at 30oC in 10-50 µM DM, 10-50 CP µM or 30 µM 4-IBP blocked or delayed AD onset. Error bars represent standard deviation from the mean.

FIGURE 7 Sigma receptors mediate synaptic excitation by sigma receptor ligands at low concentration. Downward arrow represents the stimulus artifact.

34 A)

At a low concentration (0.1 µM) that had no effect on AD onset, DM and CP demonstrated an

excitatory modulation of the CA1 orthodromic field potential (inset). This enhancement was blocked by 100 µM (+)-3PPP, a B)

1R

antagonist with no effect itself on the orthodromic response.

The CA1 antidromic field potential evoked from the alveus remained unaltered by 0.1 µM DM or

CP (inset). Therefore the effect of DM and CP was confined to the synaptic response as shown in A.

Error bars represent standard deviation from the mean. FIGURE 8 At 100 µM, but not 30 µM, DM and CP suppress evoked CA1 firing. Downward arrow represents the stimulus artifact. A)

At intermediate concentrations 10-30 µM DM or CP had no obvious effect on evoked responses

from the CA1 region (or neocortex, Fig. 11A) even though these concentrations blocked or delayed AD (Fig. 6). At the highest concentration (100 µM) DM and CP each inhibited the orthodromic and antidromic responses, the former displaying better recovery following drug washout. B)

Time course of the orthodromic and antidromic responses noted in A. At 30 µM, DM had no

significant effect on either the CA1 orthodromic response (left) or the antidromic response (right), even after 50 min exposure. In contrast, 100 µM DM or CP completely suppressed the orthodromic and antidromic responses by 30 min of exposure. The orthodromic, but not antidromic, response was fully reversible. The asterisk indicates significant difference from the response at zero time (p‹0.05). Error

bars represent standard deviation from the mean. FIGURE 9

At concentrations that block AD, sigma-1 receptor agonists did not affect the NMDA

receptor mediated the `bursting` component of the orthodromic CA1 field potential. Error bars represent

standard deviation from the mean. A)

The NMDA receptor antagonist AP-5 reversibly blocked the slow bursting component that

developed in low–Mg2+ aCSF but not the initial spike. Downward arrow represents the stimulus artifact. The increased line-length of the evoked waveform represents the 100% response in B) to F). B)

Time course of the change in excitability (shown in A) in response to application of 50 µM AP-5.

As expected, the NMDA receptor-mediated bursting component was reversibly blocked (10-20 min) but the non-NMDA receptor-mediated component was not affected (20-40 min). C) – F)

The R ligands had no significant effect on the low-Mg2+ component. The exception was DM

at 100 µM which reduced the line length by about 10%. The asterisk indicates significant difference from the response in low-Mg2+ a CSF (p‹0.05).

35 FIGURE 10

Pretreatment with 10 µM CP for 30-35 minutes blocked or delayed AD in the CA1 region

of the hippocampal slice, permitting recovery of the orthodromic response that was extinguished in untreated tissue. This dosage had no effect on the orthodromic response (Fig. 8B). Error bars represent

standard deviation from the mean. A) In the upper trace, the AD was recorded as a negative voltage shift in response to OGD in CA1 pyramidale, similar to that recorded in response to ouabain and in layers II/III of neocortex (not shown). In the lower trace, slices pretreated with 10 µM CP displayed no negative shift during OGD. B) Averaged evoked orthodromic CA1 response to Schaffer collateral stimulation before, during and after 10 min of OGD, as plotted in the time course experiments shown in C, D and E. Slices pretreated with 10 µM CP substantially recovered by 60 min post-OGD. Arrow represents the stimulus artifact. C) Pretreatment with 10 µM CP blocked AD evoked by OGD in 8 of 11 slices, resulting in dramatic recovery compared to untreated slices. D) More detailed time course of the data shown in C. Pretreatment with CP had no significant effect on the onset of synaptic failure that was complete by 3 min of OGD. E) In the three other slices, pretreatment with 10 µM CP delayed AD from an average of 7.4 +/- 0.9 minutes to 10.0 +/- 0.9 minutes (p‹0.05). The delay notably improved recovery compared to untreated slices.

FIGURE 11

Pretreatment with 10 µM CP for 30-35 min blocked or delayed AD induced by OGD in

layers II/III of the neocortical slice, permitting recovery of the orthodromic response normally lost in untreated tissue. Error bars represent standard deviation from the mean. A) Pretreatment with 10 µM CP for 30-35 min had no effect on the evoked orthodromic response evoked in layers II / III of neocortex. Arrow represents the stimulus artifact. B) All neocortical slices not exposed to R ligand displayed AD within 4 min of OGD. The recovery was variable with most slices being seriously compromised. C) Pretreatment with 10 µM CP significantly delayed AD evoked by OGD in all slices, shortening the time between AD onset and return to oxygenated/normoglycemic conditions. This permitted dramatic recovery compared to untreated slices (Fig. 11B).

FIGURE 12

Pretreatment with 10-30 µM of the sigma-1 ligands CP or DM for 30-35 min blocked or

delayed AD induced by 5 minutes of ouabain exposure as recorded in layers II/III of the neocortical slice. Recovery of the orthodromic response was significant compared to untreated slices. Error bars represent

standard deviation from the mean.

36 A) All neocortical slices not pretreated with CP generated AD within 3 min of ouabain exposure and remained seriously compromised. Pretreatment with 10 µM CP not only blocked AD but also prevented synaptic failure, a feature not seen with OGD. B) Pretreatment with 10-30 µM of DM also prevented synaptic failure caused by ouabain and delayed AD onset, leading to substantial recovery compared to untreated slices. C) Deprivation of oxygen and glucose or ouabain exposure induces anoxic depolarization (AD) by compromising the Na+/K+ ATPase pump. In either case, the current study shows that sigma-1 receptor ligands are remarkably effective in blocking AD so their action is downstream from the pump, inhibiting AD onset possibly through sigma-1 receptor mediation. This greatly reduces metabolic stress that, combined with pump failure, causes acute neuronal damage (Obeidat et al., 2000). We have previously shown that these same ligands inhibit spreading depression (SD) induced by brief exposure to 26 mM KCl (Anderson and Andrew, 2002). Unlike AD, SD can be induced repetitively without damage to neocortical slices because energy stores are intact.