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Department of Anatomy and Cell Biology, Queen’s University, Kingston, Ontario, Canada
Submitted 29 January 2004; accepted in final form 21 September 2004
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ABSTRACT |
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INTRODUCTION |
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). This collapse in membrane function results from failure of the Na+/K+ ATPase pump as ATP is depleted. It was originally described as a spreading "anemic depolarization," a wave of electrical silence propagating across the neocortex (Leao 1947). Without immediate reperfusion, this "ischemic" or "anoxic" depolarization (AD) leads to acute neuronal death and represents the most reliable determinant of ensuing brain damage (Kaminogo et al. 1998). For 20 yr, excessive glutamate release has been considered the primary initiator of acute neuronal death following stroke. However AD generation does not require glutamate release or glutamate receptor activation (Lipton 1999; Obeidat et al. 2000; Obrenovitch 2001; Obrenovitch and Urenjak 1997).
Spreading depression (SD) generates migraine aura (Anderson and Andrew 2002; Hadjikhani et al. 2001; Leao 1944; Somjen 2001) and, as with AD, is a mass depolarization of neurons and glia that initiates focally and propagates at 2–4 mm/min across gray matter (Joshi and Andrew 2001; Leao 1944, 1947; Obeidat and Andrew 1998). However unlike SD, AD arises in a severe energy crisis, resisting any single ion channel blocker or neurotransmitter receptor antagonist in vivo (Hernandez-Caceres et al. 1987; Lipton 1999; Marrannes et al. 1988; Nellgard and Wieloch 1992; Xie et al. 1995) or in brain slices (Aitken et al. 1991; Obeidat et al. 2000; Somjen et al. 1992; Tanaka et al. 1997; Taylor et al. 1999). While a cocktail of several such compounds can block AD in brain slices of immature rats (Rossi et al. 2000), a comparable therapeutic treatment would be immediately lethal.
On onset of focal ischemia, AD is generated in the ischemic core, where it is unremitting unless blood flow is quickly re-established. For the next 3 h, recurrent AD-like events [peri-infarct depolarizations (PIDs)] initiate and propagate outward from the border of this core, further degrading the rim of surrounding tissue, the penumbra (Back 1998; Back et al. 1996; Obrenovitch 1995). PIDs recruit penumbral tissue into the infarct core throughout the period of infarct maturation during the 24 h following stroke (Hartings et al. 2003). As PIDs course into uncompromised gray matter, they repolarize more quickly (within 1–2 min) and so are indistinguishable from SD. We have proposed that the AD, PID, and SD represent variations of a common depolarizing and propagating process, the particular version depending on the degree of local metabolic compromise (Andrew et al. 2002).
Despite the hundreds of neuroprotective compounds reported to work in animal models and neuronal cultures, no drug for stroke treatment has yet made it to market (Palmer 2001). Damage in the expanding penumbra (from recurrent PIDs) is generated during the 3 h following stroke onset, the window when thrombolytic treatment has some therapeutic benefit (Grotta 2003). A useful anti-stroke drug would have the ability to block AD and PIDs without depressing CNS function. Here we use the neocortical brain slice to model the ischemic core and show the remarkable ability of several sigma-1 receptor (
1R) ligands to block or delay the anoxic depolarization, thereby increasing neuronal survival at concentrations that do not overtly alter neuronal excitability.
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METHODS |
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Male Sprague-Dawley rats, 21–28 days old (Charles River, St. Constant, Canada) were cared for in accordance with the Canadian Council on Animal Care. They were housed in a controlled environment (22 ± 1°C, 12-h light:12-h dark) with Purina rat chow and water supplied ad libitum. A rat was placed in a rodent restrainer (DecapiCone, Braintree Scientific) and guillotined. The brain was excised and placed in ice-cold oxygenated (95% O2-5% CO2) artificial cerebrospinal fluid (ACSF). Coronal slices (400 µm) were taken from the frontal and parietal regions of the neocortex using a vibrating blade microtome (Leica VT1000S). Five to seven slices were transferred to a net submerged in a beaker of ACSF gassed with O2/CO2 at 22°C. The slices were slowly warmed over 1 h to 32 ± 1°C prior to experimentation at 33–34°C.
Experimental solutions
The ACSF contained (in mM) 120 NaCl, 3.3 KC1, 26 NaHCO3, 1.3 MgSO4, 1.2 NaH2PO4, 11 D-glucose, and 1.8 CaCl2 (pH 7.3–7.4). A slice was weighted at the edges with silver wire and submerged in ACSF flowing through the imaging/recording chamber at 3–4 ml/min (33–34°C). Maintaining constant flow was critical for consistent results. Low-Mg2+ ACSF solution was similar to control ACSF, except MgSO4 was reduced from 1.3 to 0.3 mM and replaced by equimolar NaCl. Ischemia was simulated by either oxygen/glucose deprivation (OGD) or by addition of the Na+/K+ ATPase inhibitor ouabain (100 µM). For OGD, ACSF glucose was reduced from 11 to 1 mM (replaced with equimolar NaCl) and the 95% O2-5% CO2 mixture gassing the ACSF was replaced with 95% N2-5% CO2. With hippocampal experiments, glucose was completely removed to promote AD onset 1 min or so earlier than in 1 mM glucose. Post-AD damage remained extensive.
Glutamate receptor antagonists tested were kynurenic acid (2 mM), (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801; 100 µM), 6-cyano-7-nitroquinozline-2,3-(1H,4H)-dione (CNQX; 10 µM), and D,L-2-amino-5-phosphonovaleric acid (D,L-AP-5; 100 µM). The active dextro-rotatory enantiomer D-AP-5 of the racemic mixture of AP-5, is 50 µM. The concentrations of glutamate receptor antagonists used here inhibited almost all cell swelling induced by glutamate agonists as reported in previous hippocampal slice experiments (Andrew et al. 1996; Polischuk et al. 1998).
R ligands tested were dextromethorphan (DM; 1–100 µM), carbetapentane (CP; 10–100 µM), 3-(3-hydoxyphenyl)-N-(1-propyl)piperidine [R(+)3-PPP; 100 µM], 1[2-(3,4-dichlorophenyl)ethyl]-4-methylpiperazine (BD-1063; 100 µM), and N-(N-benzylpiperidine-4-yl)-4-iodobenzamide (4-IBP; 30 µM). A stock solution of 4-IBP (2.1 x 10–4 mM in 100% dimethylsulfoxide) was added drop-wise to oxygenated ACSF. The maximum solubility achieved by 4-IBP was 
30 µM, and the final concentration of dimethylsulfoxide (DMSO) was a maximum of 0.02%. It should be noted that R "agonist" or "antagonist" designations are based solely on ligand binding studies. Not known is the endogenous R ligand(s), the functional role of the receptor, or whether the active state of the receptor is bound or unbound. Thus the terms agonist or antagonist vary with the bioassay employed, in this case, AD blockade. Spiperone, also termed 8-(3-[p-fluorobenzoyl]propyl)-1-phenyl-1,3,8-triazaspiro(4.5)decan-4-one (100 µM), was also tested because it has structural similarities to some other R ligands but does not bind Rs (Monnet et al. 1992).
Imaging light transmittance
Broad-band changes in light transmittance (LT; Fig. 1A) are monitored in real-time. A front of cell swelling is imaged as an increase in LT during AD initiation and propagation in cortical brain slices (Basarsky et al. 1998; Obeidat and Andrew 1998). Dendritic damage is imaged as decreased LT in the wake of the AD front that follows the LT increase. This LT reduction is caused by the formation of dendritic beads (Jarvis et al. 1999; Polischuk et al. 1998), which form within minutes of AD onset in brain slices (Obeidat et al. 2000; Tanaka et al. 1999) and effectively scatter light. Similar beading is observed in vivo following focal ischemia (Hori and Carpenter 1994). We have outlined several biophysical principles underlying changes in LT (Andrew et al. 2002; Jarvis et al. 1999).
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Each image of a series was an average of 128 or 256 frames. Averaged images were saved to the hard drive and archived on writable compact discs. The first averaged image in a series served as a control (Tcont), which was subtracted from each subsequent experimental image of that series (Texp). The resulting series of subtracted images revealed changes in LT over time. The change (
T) was expressed as the digital intensity of the subtracted images (Texp – Tcont), but the software does not divide T by Tcont, so the images themselves are not normalized with respect to their regional variations in light transmittance. The gain was set using the AIW software. The change in light transmission was displayed using a pseudocolor intensity scale. Zones of interest were selected to quantify and graphically display the experimental data off-line.
Graphing and statistical analyses of data were carried out using SigmaPlot for Windows (Jandel Scientific). Images were imported and figures were prepared using CorelDraw. The data from the imaging experiments were analyzed such that changes in LT of a given zone of interest were expressed as percent change in Tcont for that region, taken from the control image. That is
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Evoked field potentials
To record evoked field potentials or the spontaneous negative shift representing the AD, a micropipette (5–10 M
) was pulled from thin-walled capillary glass, filled with 2 M NaCl, and mounted on a three dimensional (3-D) micromanipulator. It was connected by a chlorided silver wire to an amplifier probe, and output was monitored on an on-line oscilloscope. The tip was placed in layers II/III of the neocortex and a concentric bipolar electrode (Rhodes Electronics) placed in layer VI to stimulate the immediately overlying layers. In hippocampal slices, the Schaffer collaterals were stimulated, and the orthodromic field potential was recorded in the CA1 pyramidal layer. An antidromic CA1 field potential was evoked from the alveus. A current pulse (0.1-ms duration; 0.5 Hz) was applied to produce a population spike (PS) at just-maximal amplitude. The amplified signals were digitized, signal-averaged (6–15 sweeps/trace), displayed, and plotted using pCLAMP software (Axon Instruments). The effect of a specific drug on the averaged PS amplitude was measured and normalized as a percent of the control amplitude ± SE. A drug’s ability to maintain or help recover population spike amplitude compared with initial values indicated neuroprotection. Probability values of P < 0.05 using a paired Student’s t-test were considered significant. Low-Mg2+ ACSF exposure for 20 min induced a bursting waveform response whose line length during the 50 ms following stimulus artifact was averaged (10 sweeps) and normalized to 100% (Watson and Andrew 1995). A drug’s ability over 10 min to reduce the line length of the waveform indicated N-methyl-D-aspartate (NMDA) receptor antagonism. Probability values of P < 0.05 using a paired Students t-test were considered significant.
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RESULTS |
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OGD for 10 min or exposure to the Na+-K+ ATPase inhibitor ouabain for 5 or 10 min induces AD, also termed ischemic SD. At AD onset, a dramatic focal increase in LT usually appeared first in the neocortical gray and 1 min or so later in hippocampus. The LT front propagated outward from each focus (Fig. 1B, *) at a rate of 2–3 mm/min. The mean peak LT increase in neocortical layers II/III was 58 ± 7% in the 23 slices tested. All six neocortical layers were recruited, but the underlying white matter was not. In the wake of the front, there followed a steady reduction in LT into negative values over several minutes (Fig. 1B, magenta pseudocolor). The reduction of –32 ± 13% (n = 23 slices) did not recover over time and all evoked electrophysiological activity was lost as detailed below. This sequence was the same whether induced by OGD or by 100 µM ouabain, except that AD onset was 2 min earlier using ouabain (Fig. 1C). Both treatments cause failure of the Na+/K+ ATPase pump, evoking AD and post-AD damage.
1R ligands and AD
Reports that DM is neuroprotective (usually attributed to antagonism at the NMDA receptor) initially prompted us to test this drug’s ability to block AD. In initial experiments, AD was induced by a 5-min ouabain exposure (Figs. 1–4). Subsequent experiments involved 10 min of either ouabain exposure or OGD. AD blockade was defined as 1) absence of a focal LT increase, 2) no propagating wave of elevated LT, and 3) no subsequent decrease in LT as judged by sampling zones of interest in layers II/III comprised of a few hundred pixels. Pretreatment of slices with 1 µM DM for 15–20 min before and during a 5-min period of ouabain exposure did not block the AD, whereas 10 µM DM was effective in about one-half the trials (Fig. 2A). DM pretreatment at 30 or 100 µM blocked AD in all 17 slices tested (Fig. 3, A and B). Like DM, CP is an antitussive and R ligand; CP pretreatment for 15–20 min also blocked AD at 50 or 100 µM in all 17 slices tested. Moreover CP consistently blocked AD at 10 µM (Fig. 3A) in 16 slices, where, as noted above, 10 µM DM blocked AD in about one-half the trials (not plotted).
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Rs (Monnet et al. 1992). This D2 dopamine antagonist displays 1B-adrenergic receptor antagonism and mixed 5-HT2A/5-HT1 antagonism. If R ligand blockade of AD is through non–R-mediated effects, spiperone should block the AD or antagonize AD block by DM. Spiperone (100 µM) was bath-applied for 20 min prior to co-application of ouabain (100 µM) for 5 min. It failed to prevent or to delay the initiation or propagation AD (Fig. 3A) or to alter the associated LT changes (Fig. 3B). Also, five slices were exposed to spiperone (100 µM) for 15 min, prior to co-application with DM (100 µM) for 15 min. Ouabain (100 µM) was then co-applied for 5 min. In all five slices, AD blockade by DM was not antagonized by spiperone (Fig. 3, A and B). Therefore the pretreatment technique using a R-like substance does not itself alter AD onset. Glutamate receptor antagonists and AD
Most R ligands show some low affinity binding with NMDA receptors, so while testing the ability of R ligands to block AD, glutamate receptor antagonists were also tested. Slices were pretreated with 50 µM D-AP-5 (a competitive NMDA receptor antagonist) or 100 µM MK-801 (a noncompetitive NMDA receptor antagonist) for 15–20 min (Fig. 2B). Ouabain was co-applied for 5 min. In all slices tested, AD was induced without any change to onset time (Fig. 3A) or to LT at the AD front (Fig. 3B). These antagonists also failed to reduce the LT decrease following AD, which is caused in part by dendritic beading as neurons become damaged (Fig. 3B). If AD blockade by R ligands was through antagonism of the NMDA receptor, NMDA receptor antagonists should mimic AD blockade by R ligands. D-AP-5 (50 µM) was bath applied for 15–20 min, followed by co-application of 100 µM DM for 15–20 min. Exposure to 100 µM ouabain in combination with AP-5 and DM followed for 5 min. In all 11 slices, DM still blocked AD (Fig. 3, A and B), despite NMDA receptor antagonism.
1R antagonists and AD
The 1R ligands (+)-3-PPP and BD-1063 bind Rs with high affinity and specificity, and both have low affinity for the NMDA receptor (Matsumoto et al. 1995; Whittemore et al. 1997). Both have been proposed as 1R antagonists (although such a designation depends on the bioassay used because R function is unknown). A 15- to 20-min bath application of 100 µM (+)-3-PPP (Fig. 4A) or BD-1063 (Fig. 4B) preceded exposure to 5 min of 100 µM ouabain. Each 1R antagonist proved ineffective in inhibiting AD (n = 14). Specifically, neither altered the latency to AD onset (Fig. 3A), the propagation rate (data not shown), nor the peak LT change during AD (Fig. 3B).
We investigated if either antagonist could inhibit AD blockade by the 1R agonist DM. Antagonist application of 100 µM (+)-3-PPP or BD-1063 for 15 min preceded their co-application with 100 µM DM. In all slices, pretreatment with either 1R antagonist prevented blockade of AD by DM (Figs. 3, A and B, and 4, C and D). Thus DM lost its ability to block the AD. Similar results were found in experiments described next, where preapplication of 100 µM (+)-3-PPP or BD-1063 for 15 min inhibited AD block by 30 µM of the 1R agonist 4-IBP. A minimum of 15–20 min of agonist pretreatment is required to block AD. The antagonist pre-exposure for 20 min and then co-exposure with agonist (Fig. 4, C and D) is apparently enough to slow agonist binding and so permit AD onset.
Extending simulated ischemia to 10 min
To examine if AD onset could be blocked beyond 5 min of simulated ischemia, we extended the exposure time of 100 µM ouabain or OGD to 10 min. Pretreatment with potential blockers was also extended from 15–20 to 30–35 min because the latter yielded more consistent effects. Representative experiments are plotted in Fig. 5. In the presence of 100 µM DM, AD induced by ouabain exposure for 10 min (Fig. 6A) was blocked in 14 of 17 slices, displaying no change in light transmission over 20 min. There was pronounced delay in AD onset in the remaining three slices. DM at 30 µM was effective in blocking ouabain-induced AD in 4 of 13 slices, with the remainder showing significantly delayed onset. DM at 10 µM significantly delayed ouabain-induced AD in all 10 slices tested. At 100 µM, DM blocked OGD-induced AD in 19 of 45 slices (Fig. 5A) and delayed AD onset in the remaining slices. At 30 µM, DM only blocked in one slice but was effective in delaying AD in the remaining 12 slices (Fig. 6B). At 10 µM, DM significantly delayed AD onset induced by OGD in all nine slices (Fig. 6B).
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The R ligand 4-IBP was also tested against AD because it lacks cross-reactivity at NMDA receptor sites (Whittemore et al. 1997). Slices were pretreated with 30 µM 4-IBP (the approximate solubility limit in ACSF) prior to 10 min of exposure to 100 µM ouabain or OGD (Fig. 5B). Pretreatment prevented the initiation of AD and post-AD damage in all slices as judged by the lack of significant change in LT following ouabain exposure (Fig. 6A) or OGD (Fig. 6B). Preapplication of 100 µM (+)-3-PPP or BD-1063 for 15 min inhibited AD block by 30 µM of the 1R agonist 4-IBP. Thus these two 1R antagonists prevented AD blockade by 4-IBP.
Effects of 1R ligands on pyramidal cell excitability
We investigated if 1R ligands affect pyramidal cell excitability because, to have any therapeutic efficacy, an ideal drug would block AD without inhibiting the general excitability of the tissue being protected. Was there a range of drug concentrations where electrophysiological effects were minimal but where AD was blocked? The preceding imaging experiments indicated that concentrations of DM or CP at 0.1 or 1.0 µM did not block nor delay AD or the damage that followed. Nevertheless, we examined the effects of 0.1 and 1.0 µM CP on the evoked population responses in the CA1 region because R ligands have been reported to have excitatory effects at low concentrations. At 0.1 µM, the 1R agonist DM elicited a 145% reversible enhancement of the orthodromic response. The PS amplitude recorded in CA1 pyramidale increased from 1.3 ± 0.1 to 1.9 ± 0.1 mV (Fig. 7A). In contrast, the antidromic response was unaffected (Fig. 7B). Similarly 0.1 µM CP enhanced orthodromic PS amplitude by 143% from 1.5 ± 0.1 to 2.2 ± 0.2 mV (Fig. 7A). Again there was no antidromic effect (Fig. 7B). We tested if this enhancement of the ortho-PS by 0.1 µM CP or DM was 1R mediated. Slices were pretreated in 100 µM of the R antagonist (+)-3-PPP for 20 min prior to co-application with 0.1 µM CP or DM. The (+)-3-PPP itself had no effect on ortho- or antidromic responses (Fig. 7A). However the DM and CP enhancement of the orthodromic PS was reversed in the presence of (+)-3-PPP. The antidromic responses were unchanged (Fig. 7B). In identical experiments, there was no significant effect observed on the ortho- or antidromic response by DM or CP at 1.0 µM (data not shown). These results show that we can detect changes in synaptic strength mediated by 1R.
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R ligands protected brain slices from AD at 10–100 µM, so it was of interest to test pyramidal cell excitability in this range. Remarkably, at 10–30 µM, DM or CP had no detectable effect on evoked CA1 responses even after exposure for 30 min (Fig. 8, A and B). This was also true for evoked responses from layers II/III in the neocortical slice where 10–30 µM DM or CP delayed or blocked AD without altering the evoked synaptic response.
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To examine if this suppression of the evoked PS by 100 µM CP or DM was 1R-mediated, hippocampal slices were pretreated with 100 µM of either of the 1R antagonists (+)-3-PPP or BD-1063 for 20 min. This was followed by co-application of 100 µM DM or CP for 20 min. The ortho- and antidromic evoked responses were recorded every 10 min throughout the experiment. The responses were still lost (data not shown), unlike the excitatory response evoked by 0.1 µM DM or CP described above. This profound suppression of ortho- and antidromic-evoked responses by the higher concentrations of DM or CP (100 µM) suggests a general suppression of spike conduction following longer exposure times (10–40 min).
Lack of 1R ligand action on the NMDA receptor-mediated synaptic component
Since neuroprotective effects by 1R ligands are commonly interpreted as acting through NMDA receptor antagonism, 1R ligand effects were tested on the NMDA-receptor mediated component of the orthodromic PS. This component was revealed in the absence of ACSF Mg2+ because increased bursting activity following the initial PS. It was blocked reversibly by 50 µM of the competitive NMDA receptor antagonist D-AP-5 (Fig. 9, A and B). Effects of 1R ligands were tested on this component at the minimal time period required for AD block by a 1R ligand (10 min). For experiments in low-Mg2+ ACSF, the orthodromic PS was evoked at 3 min (the time required for inhibition induced by AP-5) and at 10 min (the time required to block AD). The line length in low-Mg2+ was designated as the 100% bursting response, so the initial control response in regular ACSF was 75–85% (Fig. 9, B–F). The D-AP-5 significantly decreased line length after 3 and 10 min to 85.3 ± 1.9 and 80.6 ± 2.5%, respectively (Fig. 9, A and B). DM (100 µM) also significantly decreased line length to 89.3 ± 3.8% (P = 0.037) after a 10-min exposure (Fig. 9D). However, 30 µM DM, 30 µM 4-IBP, and 100 µM CP did not significantly reduce line length in low-Mg2+ ACSF (Fig. 9, C, E, and F). Thus at a concentration (30 µM) and minimal exposure time (10 min) that blocks or delays the AD, the 1R ligands DM, 4-IBP, and CP did not significantly reduce the NMDA receptor-mediated component of the CA1 orthodromic response.
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1R ligand protectionBased on the above findings, we focused on the intermediate dosages (10–30 µM) to confirm electrophysiologically that DM or CP protected from AD without reducing electrophysiological responsiveness in the CA1 region and neocortex. In both preparations, AD onset was recorded as a negative voltage shift (Fig. 10A). In CA1 pyramidale, OGD quickly eliminated the synaptic response in both untreated and CP-treated slices (Fig. 10B). This loss occurred earlier than and independent of AD onset (Fig. 10C). There was no recovery 60 min after AD in untreated slices, whereas 10 µM CP pretreatment resulted in full recovery. Figure 10C shows that this is because the untreated slices go through AD, whereas the CP slices do not. The time course is shown in more detail in Fig. 10D. Synaptic transmission fails within 3 min in both untreated and CP slices, whereas AD onset at 7.9 ± 0.9 min is observed only in the six untreated slices. Figure 10E compares these untreated slices that go through AD with a subgroup of three slices where AD onset was delayed to 10 ± 0.4 min with substantial recovery over the ensuing hour. These slices would have spent little time depolarized from AD while also experiencing OGD.
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3 min of ouabain washout. The response recovered to about 70% over the next 20 min (Fig. 12B). Note in Fig. 12, A and B, there was minimal reduction in the orthodromic response prior to AD onset. Indeed synaptic failure followed ouabain-induced AD, whereas it preceded OGD-induced AD, so ouabain exposure does not exactly mimic the effects of OGD.
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DISCUSSION |
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1R agonists at 10–30 µM prevent or delay the AD with dramatic protection of cortical neurons independent of glutamate receptor mediation. Second, these ligands do not alter cortical responsiveness evoked in CA1 pyramidale or neocortical layers II/III. This argues against inhibition through those standard channels and neurotransmitter receptors that underlie normal CNS function and that have been the preferred targets for AD blockade. Our results implicate an abnormal conductance driving AD because normal CNS function can be retained while AD is blocked. Third, simply delaying AD onset greatly enhances recovery because the tissue spends less time recovering from AD while energy-deprived.
Leao (1947) originally described AD induced by global ischemia as a "slow variation" of SD. Both AD and SD generate a negative voltage shift representing sudden neuron/glia depolarization that propagates across gray matter. Imaging altered LT reveals similarities. One or more foci expand as a front of elevated LT that engages neocortical gray, striatum, or hippocampus and propagates at 2–3 mm/min (Anderson and Andrew 2002; Jarvis et al. 2001; Joshi and Andrew 2001). Unlike SD, AD seriously damages neurons in slices deprived of O2 and glucose for only a few minutes, as evidenced by permanent loss of the evoked field potential (Jarvis et al. 2001; Obeidat and Andrew 1998; Taylor et al. 1999; Weber and Taylor 1994) and by dendritic beading and swelling of pyramidal neurons (Jarvis et al. 1999; Obeidat et al. 2000; Tanaka et al. 1999). Thus there is little point in extending OGD beyond 10 min because the damage is done once AD propagates.
As outlined in the Introduction, glutamate receptors do not play a major role in instigating or preventing AD induced by ischemia. In this study, 1R ligands, but not NMDA receptor antagonists, inhibited the onset of OGD- and ouabain-induced AD, protecting slices from subsequent damage as seen previously (Jarvis et al. 2002; Joshi and Andrew 2001; Obeidat et al. 2000). The fact that extracellular glutamate begins to accumulate only after the AD further argues against a major role for glutamate in AD initiation and propagation (Obrenovitch 2001; Obrenovitch and Urenjak 1997; Obrenovitch et al. 2000).
Thurgur and Church (1998) ascribed the anticonvulsant properties of several R ligands to NMDA receptor antagonism and to Ca2+ channel blockade at 50–100 µM levels, and this can help explain the loss of evoked CA1 responses at these concentrations. However, their rank order of potency for NMDA receptor antagonism or Ca2+ channel blockade bears no resemblance to that described here and by Jarvis et al. (2002) for ouabain-induced AD blockade, i.e., carbetapentane > dextromethorphan > haloperidol = 4-IBP > 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 seems more likely than NMDA receptor antagonism or Ca2+ channel blockade. The potency of 1R ligands to block the AD improves when preincubation times are increased from 15–20 to 30–35 min. 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 on mitochondrial function (Klouz et al. 2003) seems unlikely because 1R ligands can block ouabain-induced AD, a situation where ATP levels are not compromised.
AD blockade through 1R mediation
Numerous 1R ligands have been identified based on their binding affinity to 1Rs, including the nonprescription antitussives DM and CP. At least two subtypes of the R exist (Booth et al. 1993; Quiron et al. 1992), with selective binding of CP and DM 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. It is implicated in immunosuppressant, antipsychotic, and neuroprotective effects (Moebius et al. 1997). 1Rs have a regional distribution within the CNS, with the highest concentrations in hindbrain and intermediate densities in neocortex and hippocampus (Leitner et al. 1994; Tortella et al. 1989). They are found primarily in neurons, and significant levels occur in cortical pyramidal cells (Gundlach et al. 1986).
DM 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; Palmer 2001; Thurgur and Church 1998). Other R ligands can potentiate NMDA-induced responses (Bergeron and Debonnel 1997; Bergeron et al. 1996; Couture and Debonnel 1998; Debonnel and de Montigny 1996; Gronier and Debonnel 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 CP has a much lower affinity for NMDA receptors than DM, yet we found CP to be the more potent AD blocker. The inability of NMDA receptor antagonists (both competitive and noncompetitive) to inhibit AD onset is strong evidence that AD blockade by R ligands is independent of NMDA receptors (Jarvis et al. 2002; Joshi and Andrew 2001; Obeidat et al. 2000).
The compounds tested that blocked or delayed AD onset in this study (i.e., DM, CP, and 4-IBP) each have high 1R affinity and are designated 1R agonists. Their ability to block AD at 100 µM is inhibited by the 1R antagonists BD-1063 or (+)3-PPP at 100 µM (Matsumoto et al. 1995; Musacchio et al. 1989; 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. A minimum of 15–20 min of agonist pretreatment is required to block AD. The antagonist pre-exposure for 20 min and then co-exposure with agonist (Fig. 4, C and 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 R. In 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 on evoked field potentials
At the lowest concentration tested (0.1 µM), 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 R antagonist (+)3-PPP, so the increased synaptic response is likely mediated by Rs. At 1.0 µM, DM or CP had no effect, probably because the dose was intermediate between excitatory at 0.1 µM (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 on 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 R 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; personal communication) or to the R ligand OPC-24439 (Ishihara et al. 1999). In the future, we will examine 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 s (Kristian and Siesjo 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 deflection of the extracellular potential when neurons and glia suddenly depolarize (Tanaka et al. 1997; Walz 1997). Extracellular Na+, Cl–, and Ca2+ rush in, with water following osmotically, causing cell swelling that shrinks the extracellular space (Hansen and Zeuthen 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), 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 (Jarvis et al. 2001; Obeidat et al. 1998) 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 on 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 is 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 2 h, 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). R 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 nonselective conductance underlying AD initiation (Tanaka et al. 1997, 1999). We propose that if such a conductance is inactive under normal physiological conditions, 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 "megachannels." The mitochondrial transition pore (MTP) conducts compounds 
1,500 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 1,200 Da (Bennett et al. 2003). A third megachannel candidate is the P2X channel, where two channel subtypes can dilate to conduct molecules of several hundred Daltons (Duan et al. 2003; Virginio et al. 1999).
A large nonspecific 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 min 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. 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.
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Address for reprint requests and other correspondence: R. D. Andrew, Dept. of Anatomy and Cell Biology, Queen’s Univ., Kingston, Ontario K7L 3N6, Canada (E-mail: andrewd{at}post.queensu.ca)
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