Elevated Synaptic Activity Preconditions Neurons against an in Vitro Model of Ischemia
2008; Elsevier BV; Volume: 283; Issue: 50 Linguagem: Inglês
10.1074/jbc.m805624200
ISSN1083-351X
AutoresJoseph S. Tauskela, Hung Fang, Melissa Hewitt, Eric Brunette, Tarun Ahuja, Jean‐Philippe Thivierge, Tanya Comas, Geoffrey Mealing,
Tópico(s)Neuroinflammation and Neurodegeneration Mechanisms
ResumoTolerance to otherwise lethal cerebral ischemia in vivo or to oxygen-glucose deprivation (OGD) in vitro can be induced by prior transient exposure to N-methyl-d-aspartic acid (NMDA): preconditioning in this manner activates extrasynaptic and synaptic NMDA receptors and can require bringing neurons to the "brink of death." We considered if this stressful requirement could be minimized by the stimulation of primarily synaptic NMDA receptors. Subjecting cultured cortical neurons to prolonged elevations in electrical activity induced tolerance to OGD. Specifically, exposing cultures to a K+-channel blocker, 4-aminopyridine (20–2500 μm), and a GABAA receptor antagonist, bicuculline (50 μm) (4-AP/bic), for 1–2 days resulted in potent tolerance to normally lethal OGD applied up to 3 days later. Preconditioning induced phosphorylation of ERK1/2 and CREB which, along with Ca2+ spiking and OGD tolerance, was eliminated by tetrodotoxin. Antagonists of NMDA receptors or l-type voltage-gated Ca2+ channels (L-VGCCs) applied during preconditioning decreased Ca2+ spiking, phosphorylation of ERK1/2 and CREB, and OGD tolerance more effectively when combined, particularly at the lowest 4-AP concentration. Inhibiting ERK1/2 or Ca2+/calmodulin-dependent protein kinases (CaMKs) also reduced Ca2+ spiking and OGD tolerance. Preconditioning resulted in altered neuronal excitability for up to 3 days following 4-AP/bic washout, based on field potential recordings obtained from neurons cultured on 64-channel multielectrode arrays. Taken together, the data are consistent with action potential-driven co-activation of primarily synaptic NMDA receptors and L-VGCCs, resulting in parallel phosphorylation of ERK1/2 and CREB and involvement of CaMKs, culminating in a potent, prolonged but reversible, OGD-tolerant phenotype. Tolerance to otherwise lethal cerebral ischemia in vivo or to oxygen-glucose deprivation (OGD) in vitro can be induced by prior transient exposure to N-methyl-d-aspartic acid (NMDA): preconditioning in this manner activates extrasynaptic and synaptic NMDA receptors and can require bringing neurons to the "brink of death." We considered if this stressful requirement could be minimized by the stimulation of primarily synaptic NMDA receptors. Subjecting cultured cortical neurons to prolonged elevations in electrical activity induced tolerance to OGD. Specifically, exposing cultures to a K+-channel blocker, 4-aminopyridine (20–2500 μm), and a GABAA receptor antagonist, bicuculline (50 μm) (4-AP/bic), for 1–2 days resulted in potent tolerance to normally lethal OGD applied up to 3 days later. Preconditioning induced phosphorylation of ERK1/2 and CREB which, along with Ca2+ spiking and OGD tolerance, was eliminated by tetrodotoxin. Antagonists of NMDA receptors or l-type voltage-gated Ca2+ channels (L-VGCCs) applied during preconditioning decreased Ca2+ spiking, phosphorylation of ERK1/2 and CREB, and OGD tolerance more effectively when combined, particularly at the lowest 4-AP concentration. Inhibiting ERK1/2 or Ca2+/calmodulin-dependent protein kinases (CaMKs) also reduced Ca2+ spiking and OGD tolerance. Preconditioning resulted in altered neuronal excitability for up to 3 days following 4-AP/bic washout, based on field potential recordings obtained from neurons cultured on 64-channel multielectrode arrays. Taken together, the data are consistent with action potential-driven co-activation of primarily synaptic NMDA receptors and L-VGCCs, resulting in parallel phosphorylation of ERK1/2 and CREB and involvement of CaMKs, culminating in a potent, prolonged but reversible, OGD-tolerant phenotype. It is well established that the brain can be prepared to withstand an ischemic insult by a process known as preconditioning (1.Kirino T. J. Cereb. Blood Flow Metab. 2002; 22: 1283-1296Crossref PubMed Google Scholar). Preconditioning can be achieved by subjecting the brain to transient ischemia in vivo or cultured neurons to oxygen and glucose deprivation (OGD) 2The abbreviations used are: OGD, oxygen-glucose deprivation; 4-AP/bic, 4-aminopyridine/bicuculline; CaMK, Ca2+/calmodulin-dependent protein kinase; 8-CPT, 8-cyclopenthyltheophylline; L-VGCCs, L-type voltage-gated Ca2+ channels; pERK1/2, ERK1/2 phosphorylation; CREB, cAMP-response element-binding protein; pCREB, CREB phosphorylation; PKA, protein kinase A; PI, propidium iodide; TTX, tetrodotoxin; MEA, multielectrode array; NMDA, N-methyl-d-aspartic acid; GABAA, γ-aminobutyric acid, type A; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/ERK kinase; MK-801, (5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate; ANOVA, analysis of variance. in vitro, resulting in delayed, potent, and reversible tolerance to otherwise lethal ischemia/OGD. Neurons can also be preconditioned by exposure to numerous environmental or chemical stimuli that generate "cross-tolerance" to ischemia (2.Tauskela J.S. Gendron T. Morley P. Schaller B. Cerebral Ischemic Tolerance. Nova Science Publishers, Inc., Hauppauge2004: 45-94Google Scholar). A key role for activation of the NMDA type of glutamate receptor during preconditioning by ischemia or OGD is well established (3.Kato H. Liu Y. Araki T. Kogure K. Neurosci. Lett. 1992; 139: 118-121Crossref PubMed Scopus (142) Google Scholar, 4.Bond A. Lodge D. Hicks C.A. Ward M.A. O'Neill M.J. Eur. J. Pharmacol. 1999; 380: 91-99Crossref PubMed Scopus (100) Google Scholar, 5.Mabuchi T. Kitagawa K. Kuwabara K. Takasawa K. Ohtsuki T. Xia Z. Storm D. Yanagihara T. Hori M. Matsumoto M. J. Neurosci. 2001; 21: 9204-9213Crossref PubMed Google Scholar, 6.Grabb M.C. Choi D.W. J. Neurosci. 1999; 19: 1657-1662Crossref PubMed Google Scholar, 7.Tauskela J.S. Chakravarthy B.R. Murray C.L. Wang Y. Comas T. Hogan M. 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Zhang Q.G. Zhang G.Y. J. Biol. Chem. 2005; 280: 21693-21699Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) or to OGD (1.Kirino T. J. Cereb. Blood Flow Metab. 2002; 22: 1283-1296Crossref PubMed Google Scholar, 6.Grabb M.C. Choi D.W. J. Neurosci. 1999; 19: 1657-1662Crossref PubMed Google Scholar, 14.Tauskela J.S. Comas T. Hewitt K. Monette R. Paris J. Hogan M. Morley P. Neuroscience. 2001; 107: 571-584Crossref PubMed Scopus (35) Google Scholar). To date, preconditioning achieved either by transient ischemia OGD or NMDA can require bringing neurons to the "brink of death" (1.Kirino T. J. Cereb. Blood Flow Metab. 2002; 22: 1283-1296Crossref PubMed Google Scholar, 14.Tauskela J.S. Comas T. Hewitt K. Monette R. Paris J. Hogan M. Morley P. Neuroscience. 2001; 107: 571-584Crossref PubMed Scopus (35) Google Scholar, 15.Dirnagl U. Simon R.P. Hallenbeck J.M. Trends Neurosci. 2003; 26: 248-254Abstract Full Text Full Text PDF PubMed Scopus (721) Google Scholar), a trait shared with some other preconditioning paradigms (2.Tauskela J.S. Gendron T. Morley P. Schaller B. Cerebral Ischemic Tolerance. Nova Science Publishers, Inc., Hauppauge2004: 45-94Google Scholar). Induction of a state resistant to ischemia or OGD by brink of death preconditioning may be a "byproduct" of neurons responding to the intense stressor, a state that persists only until normal baseline homeostasis is restored. Can NMDA receptors be engaged in preconditioning neurons against ischemia without approaching the threshold required for death? Synaptic plasticity describes a process whereby the strength of synapses, as well as the efficiency of communication between synapses, is modified by normal (as well as pathological) activity. Neuronal fate can differ depending on the location of NMDA receptors on neurons, with survival or demise linked with synaptic or extrasynaptic NMDA receptor activation, respectively. Perhaps synaptic NMDA receptor activation associated with synaptic plasticity may circumvent the brink of death requirement of preconditioning, a constraint that may be more associated with activation of extrasynaptic NMDA receptors (14.Tauskela J.S. Comas T. Hewitt K. Monette R. Paris J. Hogan M. Morley P. Neuroscience. 2001; 107: 571-584Crossref PubMed Scopus (35) Google Scholar). Here we investigate if increasing synaptic input is sufficient to induce tolerance to OGD in neurons. Elevations in electrical activity, achieved by exposing neurons to a GABAA receptor antagonist, bicuculline, sometimes combined with a K+ channel blocker, 4-aminopyridine (4-AP/bic treatment), or by treatment with low concentrations of NMDA, induce tolerance in neuronal culture to apoptosis induced by serum deprivation or by exposure to chemical agents, or to excitotoxicity induced in apoptotic and non-apoptotic manners, or oxidative stress (16.Hardingham G.E. Fukunaga Y. Bading H. Nat. Neurosci. 2002; 5: 405-414Crossref PubMed Scopus (1368) Google Scholar, 17.Papadia S. Stevenson P. Hardingham N.R. Bading H. Hardingham G.E. J. Neurosci. 2005; 25: 4279-4287Crossref PubMed Scopus (241) Google Scholar, 18.Lee B. Butcher G.Q. Hoyt K.R. Impey S. Obrietan K. J. Neurosci. 2005; 25: 1137-1148Crossref PubMed Scopus (146) Google Scholar, 19.Soriano F.X. Papadia S. Hofmann F. Hardingham N.R. Bading H. Hardingham G.E. J. 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Neurochem. 2002; 82: 1097-1105Crossref PubMed Scopus (105) Google Scholar, 24.Ivanov A. Pellegrino C. Rama S. Dumalska I. Salyha Y. Ben Ari Y. Medina I. J. Physiol. 2006; 572: 789-798Crossref PubMed Scopus (261) Google Scholar, 25.Mulholland P.J. Luong N.T. Woodward J.J. Chandler L.J. Neuroscience. 2008; 151: 419-427Crossref PubMed Scopus (24) Google Scholar), although the role of ERK1/2 in neuroprotection induced by electrical activity has not yet been examined. In contrast, extrasynaptic NMDA receptor activation can induce an ERK1/2 and CREB phosphorylation "shut-off" pathway. We considered if 4-AP/bic treatment of cultured cortical neurons induced a synaptic NMDA receptor-mediated signaling pathway, which culminated in tolerance to OGD, thereby accomplishing preconditioning in a manner more aligned with neuronal signaling associated with synaptic plasticity, rather than with a brink of death stress. Materials—Tissue culture dishes and plates were purchased from either Du Pont-Invitrogen (Burlington, Ontario, Canada) or VWR Canlab (Mississauga, Ontario, Canada). Glass coverslips were purchased from Bellco Glass, Inc. (Vineland, NJ). Fetal bovine serum was bought from Gemini Bio (Woodland, CA). Minimal essential medium was obtained from Wisent Canadian Laboratories (St-Bruno, Quebec, Canada). Horse serum was acquired from HyClone Laboratories (Logan, UT). Fluo-4-AM was bought from Molecular Probes (Eugene, OR). H89 was bought from Axxora (San Diego, CA). 4-AP, 5,7-dichlorothiokynurenic acid, 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX), (5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801), KN-62, and KN-93 were purchased from Tocris Bioscience (Ellisville, MO). Bicuculline, 8-cyclopenthyltheophylline, glybenclamide, memantine, nifedipine, N-methyl-d-aspartate (NMDA), propidium iodide (PI), tetrodotoxin (TTX), U0126, KT 5720, tolbutamide, PKA inhibitory fragment 14–22 (myristoylated), staurosporine, a protease mixture inhibitor, and all other reagents were purchased from Sigma. The serum-free protein block and Antibody Diluting Buffer were purchased from DakoCytomation (Mississauga, Ontario, Canada). Preparation of Cultures—Cultures of E18 rat cortical neurons were prepared as described previously, with experiments performed on cultures grown 14–18 days in vitro (26.Tauskela J.S. Brunette E. O'Reilly N. Mealing G. Comas T. Gendron T.F. Monette R. Morley P. FASEB J. 2005; 19: 1734-1736Crossref PubMed Scopus (30) Google Scholar). Experimental Treatment of Cultures—For preconditioning experiments, each 12-well plate was subjected to 4 conditions in culture medium: 4-AP/bic (preconditioned), control wash, 4-AP/bic plus antagonist/inhibitor, or antagonist/inhibitor alone, followed by washing and replacement with stored culture medium. OGD was performed as previously described (26.Tauskela J.S. Brunette E. O'Reilly N. Mealing G. Comas T. Gendron T.F. Monette R. Morley P. FASEB J. 2005; 19: 1734-1736Crossref PubMed Scopus (30) Google Scholar) by placing 12-well plates containing cortical cultures exposed to a basic salt solution (which had the following composition (n mm): 140 NaCl, 5 KCl, 2 CaCl2, 20 HEPES, 0.03 glycine, pH 7.4) in a 37 °C incubator housed in an anaerobic glovebox (Forma Scientific, Marjetta, OH). Assessment of Neuronal Injury—Neuronal injury was assessed 24 h following treatments as described previously using PI, with fluorescence quantitated using a Cytofluor 2350 fluorescence platereader (Millipore Corp., Bedford, MA) (26.Tauskela J.S. Brunette E. O'Reilly N. Mealing G. Comas T. Gendron T.F. Monette R. Morley P. FASEB J. 2005; 19: 1734-1736Crossref PubMed Scopus (30) Google Scholar). Briefly, cultures were exposed to basic salt solution containing 3 mm glucose with 33 μg/ml PI for 30 min at 22 °C, followed by measurement of fluorescence intensities from four locations within each well (Ex = 520 ± 20 nm; Em = 645 ± 20 nm). For normalization purposes, PI was added to sister cultures that were untreated (0% PI-uptake) or exposed to 100 μm NMDA for 15 min to kill all neurons (100% PI-uptake). Intracellular Ca2+ Spiking—To monitor intracellular Ca2+ spiking, fluorescence images were acquired every 2 s for 120 s from cultures previously loaded with 4.5 μm Fluo-4-AM (26.Tauskela J.S. Brunette E. O'Reilly N. Mealing G. Comas T. Gendron T.F. Monette R. Morley P. FASEB J. 2005; 19: 1734-1736Crossref PubMed Scopus (30) Google Scholar). Excitation was delivered through a Lambda DG-5 shutter instrument (Sutter Instrument Co, Novato, CA) housing a Zeiss Axiovert 200 inverted microscope (Ex 480 ± 15 nm; Em 535 ± 20 nm) equipped with a Fluar 40×/1.3 numerical aperture oil immersion objective, with images captured using a low light-sensitive Retiga Exi camera (QImaging, Burnaby, BC, Canada). Due to variability between coverslips in 4-AP/bic-induced Ca2+-spiking profiles, each coverslip served as its own internal control. A minimum of two sets of 120-s runs was acquired for 4-AP/bic alone, and then for 4-AP/bic with a drug, with a different region of interest chosen for each spike train. For each drug evaluated, images were acquired from a minimum of two coverslips from a minimum of two different platings. Plots of Ca2+ spike trains represent average intensities calculated from 12–15 neuronal soma. Immunoblotting—The effect of preconditioning on levels of CREB (phosphorylated and non-phosphorylated), ERK1/2 (phosphorylated), or actin was determined by immunoblotting. Cultures were washed with basic salt solution and exposed to radioimmune precipitation assay buffer (50 mm Tris, 150 mm NaCl, 2 mm EDTA, 0.1% SDS, 1% deoxycholate, 1% Triton X-100, phenylmethylsulfonyl fluoride, and protease mixture inhibitor), manually homogenized over ice, vortexed briefly and stored at –80 °C. Upon re-thawing, samples were vortexed for 30 s and centrifuged at 5000 × g for 5 min at 4 °C. The protein concentration of supernatants was determined using a Bio-Rad DC protein assay kit. Protein homogenates were loaded (15 μg) onto 10% SDS-PAGE gels and subsequently transferred to polyvinylidene difluoride membranes (NEN Life Science Products, Boston, MA). Membranes were blocked with 5% skim milk-TBST (200 mm Tris, 140 mm NaCl, 1% Tween-20 (v/v), pH 7.60) for 1 h at room temperature, and then for 4 h at 4 °C. Immunoblotting was performed by overnight incubation (4 °C) with either anti-phospho-Ser133 CREB (1:2000 in 5% milk-TBST, rabbit polyclonal, Upstate (Millipore), Lake Placid, NY) or anti-phospho-ERK1/2 (1:1000 in 5% milk-TBST, mouse monoclonal (E10), Cell Signaling Technology, Beverly, MA). Membranes were washed 3 × for 5 min each and placed into the appropriate horseradish peroxidase-linked anti-mouse/rabbit secondary antibody (1:5000 for anti-pCREB or CREB and 1:2000 for anti-pERK1/2 in 5% milk-TBST, goat anti-rabbit, Amersham Biosciences). After each phosphor-antibody was examined, membranes were subjected to a stripping buffer (62 mm Tris, 69 mm SDS, 0.8% β-mercaptoethanol) for 30 min at 55 °C with constant agitation before being reprobed with anti-CREB (1:1000, rabbit polyclonal, Santa Cruz Biotechnology Inc., Santa Cruz, CA) or anti-actin (1:5000, rabbit polyclonal, Sigma-Aldrich). Membranes were treated with an ECL solution (Amersham Biosciences, Baie d'Urfè, PQ, Canada) and exposed to imaging film (Kodak). Images acquired within the linear range of exposures were quantified by densitometric analyses of background-subtracted bands using Northern Eclipse software (Empix Imaging Inc., Mississauga, Ontario, Canada). Immunoblots are representative of three to five experiments from a minimum of three different platings, with each experiment comprised of two to three wells/condition. Immunofluorescence—Immunofluorescence was performed on cultures to detect cellular pCREB immunoreactivity. Cultures were fixed in 4% paraformaldehyde for 10 min, exposed to 0.25% Triton X-100 in phosphate-buffered saline for 10 min, and, to block nonspecific staining, cultures were exposed to serum-free protein block (DakoCytomation) for 1 h at room temperature. Cultures were then treated with anti-phospho-Ser133 CREB (1:300 in Antibody Diluting Buffer) and anti-MAP2 (1:300 in Antibody Diluting Buffer, mouse monoclonal, Sigma) for 1 h at room temperature, followed by treatment with secondary antibodies (Alexa Fluor 488 goat anti-rabbit and Alexa Fluor 568 goat anti-mouse, Molecular Probes, Eugene, OR) for 1 h at room temperature protected from light. Omitting the primary antibody exposure served as a negative control. Coverslips were mounted on slides using Vectashield Hard Set Mounting Medium for Fluorescence (Vector, Burlingame, CA). Fluorescence imaging was performed using an LSM-410 Zeiss inverted laser scanning microscope (Carl Zeiss, Thornwood, NY). MEA Acquisition and Analysis—Prior to initial use, a multielectrode dish (MEA, Alpha MED Sciences, Tokyo, Japan) was washed with deionized water, sterilized with 70% ethanol and UV radiation, and coated overnight with 0.1% polyethyleneimine in 25 mm borate buffer. Cells were dispensed into a silicon tube placed on an MEA to allow coverage of the 64 electrodes but not the perimeter reference electrodes; this tubing was removed 3 h later after cells had adhered to the MEA. Each electrode had a 20-μm diameter, an inter-electrode distance of 100 μm, an impedance of <22 kΩ (1 kHz, 50 mV applied sinusoidal wave), and an average RMS noise of 4.07 μV. For a recording session, the MEA was removed from the standard incubator and placed into a 34 °C incubator, allowed to recover for 15–30 min, and connected to a MED64 recording system (Alpha MED Sciences), and 60 s of activity was acquired at 20 kHz for each of 64 channels using MEA software. Analyses were performed using Matlab. Data were pre-processed by down-sampling the original resolution of 20 kHz to 1 kHz by taking the mean across samples within each bin time (other techniques such as taking the maximum value provided similar qualitative results). Confidence lines were computed by determining the mean and standard deviation of each channel over the 60-s window, followed by subtracting the standard deviation (S.D.) from the mean of each channel: Confidence-Linec = Meanc–(3 × S.D.c), where c indexes the channels. The same procedure was applied for every experimental condition, except the S.D. used to compute the confidence line was always calculated for the baseline condition. Peaks in each channel were identified by scanning through the 60-s time window and determining when the amplitude crossed the confidence line (i.e. went from a value above the confidence line to a value below it). The temporal distributions of peaks were obtained by computing the temporal interval between peaks, and averaging across all channels for a given condition. The distributions of amplitude were obtained by computing the difference between the peak amplitude and the confidence line: PeakAmplitudec,t = Amplitudec,t–ConfidenceLinec, where t indexes the timing of a peak, and Amplitudec,t is the raw amplitude of a channel c at peak t. Each MEA was analyzed separately by performing a Student's t test (α = 0.05, with Bonferroni correction for multiple comparisons: 5 conditions × 5 cultures, α = 0.05/25 = 0.002). For each MEA, the average timing and amplitude of peaks for each individual channel was determined, followed by comparisons aimed at determining if there were significant differences in the timing and amplitude of peaks between the baseline and other conditions. Statistical Analyses—Data are presented as the mean ± S.E. Statistical comparisons were made by analysis of variance (ANOVA) or the Student's t test. When significant differences were observed, the Bonferroni test was employed for multiple comparisons. Statistical significance was inferred at p < 0.05, unless a Bonferroni correction was applied (as noted above), in which case p < 0.002. 4-AP/bic Preconditioning Induces OGD Tolerance—Neuronal glutamatergic synaptic activity can be enhanced by 4-aminopyridine (4-AP), which prolongs the duration of neuronal action potentials, and by bicuculline (bic), a GABAA receptor antagonist, which induces network disinhibition, resulting in action potential-dependent intracellular Ca2+ spiking and synaptic release of neurotransmitters such as glutamate (27.Tapia R. Medina-Ceja L. Pena F. Neurochem. Int. 1999; 34: 23-31Crossref PubMed Scopus (66) Google Scholar). To determine if excitatory activity elevated in this manner induced tolerance to otherwise lethal OGD, rat cortical neuron cultures were exposed to 4-AP (2.5 mm) and bicuculline (50 μm) (4-AP/bic preconditioning) for 48 h, followed by washout and a 24-h recovery period, and exposed to 65–80 min OGD. In the absence of preconditioning, OGD resulted in destruction of neuronal morphology and substantial PI uptake and fluorescence; in contrast, OGD did not have these effects on preconditioned cultures, because neuronal morphology was preserved and PI fluorescence was negligible (Fig. 1, A–H). Bicuculline treatment alone was not as effective as 4-AP/bic in providing OGD tolerance (data not shown). To confirm Ca2+ spiking by preconditioning, neuronal intracellular Ca2+ levels were monitored in Fluo-4-loaded cultures. The frequency and amplitude of action-potential-dependent Ca2+ spikes increased above background levels upon exposure to bicuculline, and was further increased with the co-addition of 4-AP (Fig. 1I). Hence, preconditioning by bicuculline alone might have failed due to insufficient elevation of electrical activity. Blocking Na+ channels with TTX eliminated Ca2+ spiking induced by 4-AP/bic (Fig. 1I), confirming a required role for electrically driven Ca2+ spiking (16.Hardingham G.E. Fukunaga Y. Bading H. Nat. Neurosci. 2002; 5: 405-414Crossref PubMed Scopus (1368) Google Scholar). The range of conditions able to provide OGD tolerance was determined by varying the concentration of 4-AP, and the durations of exposure to 4-AP/bic and recovery period following 4-AP/bic washout (Fig. 1, J–P). Concentrations of 4-AP ranging from 20 μm ("low") to 2500 μm ("high") in 4-AP/bic treatments all provided robust OGD tolerance, with 24–48 h representing an optimum length of treatment. The optimum recovery period following 4-AP/bic washout was 0–48 h; a 72-h interval resulted in some loss of OGD tolerance for low 4-AP/bic preconditioning, whereas a 96-h interval resulted in no protection against OGD for any 4-AP concentration. Arguing against protection by any 4-AP/bic remaining after washout, subjecting neurons to 4-AP/bic during OGD was not neuroprotective (data not shown). Exposing neurons to 4-AP/bic for only 4 h did not induce OGD tolerance (Fig. 1P), suggesting the requirement for a minimum duration of exposure. The ability of 4-AP to induce OGD tolerance at concentrations two orders of magnitude lower than a lethal concentration (∼5 mm; data not shown) suggests that preconditioning did not threaten neuronal survival. Because excessive activation of glutamate or NMDA receptors (excitotoxicity) underlies neuronal death induced by OGD, we attempted to confirm tolerance to lethal glutamate or NMDA receptor activation by 4-AP/bic preconditioning (20.Bengtson C.P. Dick O. Bading H. BMC. Neurosci. 2008; 9: 11Crossref PubMed Scopus (38) Google Scholar, 28.Hardingham G.E. Arnold F.J. Bading H. Nat. Neurosci. 2001; 4: 261-267Crossref PubMed Scopus (439) Google Scholar), but were not successful (supplemental Fig. S1, A and B, respectively). In view of this discrepancy, we evaluated if apoptotic tolerance could be confirmed. Tolerance to staurosporine during 4-AP/bic treatment (acute) was not observed (supplemental Fig. S2, A and B), but tolerance to this apoptotic agent added after 4-AP/bic washout (delayed) was verified (supplemental Fig. S2C) (16.Hardingham G.E. Fukunaga Y. Bading H. Nat. Neurosci. 2002; 5: 405-414Crossref PubMed Scopus (1368) Google Scholar, 20.Bengtson C.P. Dick O. Bading H. BMC. Neurosci. 2008; 9: 11Crossref PubMed Scopus (38) Google Scholar). Overall, the different neurotoxicity profiles might be attributed to different neuron culture systems (hippocampals elsewhere versus corticals). Receptor Dependence of Preconditioning—Receptors activated during preconditioning were identified by evaluating whether including a receptor antagonist during preconditioning suppressed OGD tolerance; both the highest (2500 μm, Fig. 2A) and lowest (20 μm, Fig. 2B) 4-AP concentrations were evaluated, because additional receptors can be activated at millimolar 4-AP concentrations (29.Rogawski M.A. Barker J.L. Brain Res. 1983; 280: 180-185Crossref PubMed Scopus (87) Google Scholar). A Na+ channel blocker, TTX, reversed OGD tolerance produced by both high and low 4-AP/bic (although TTX was moderately protective on its own for an unknown reason). Thus, action potential-dependent neurotransmitter release was required for development of OGD tolerance. Protection against OGD was significantly suppressed by the presence of memantine (12.5 μm) during exposure to low (Fig. 2B) but not high (Fig. 2A) 4-AP/bic treatment. Memantine may not fully block rapid activation of the NMDA receptor during an action potential (30.Chen H.S. Pellegrini J.W. Aggarwal S.K. Lei S.Z. Warach S. Jensen F.E. Lipton S.A. J. Neurosci. 1992; 12: 4427-4436Crossref PubMed Google Scholar), so we examined a higher affinity antagonist, MK-801. However, in a control experiment, exposing cultures to MK-801 alone induced tolerance to subsequent OGD (supplemental Fig. S3), possibly due to the presence of residual antagonist and/or induction of a preconditioning stress response (31.Tremblay R. Chakravarthy B. Hewitt K. Tauskela J. Morley P. Atkinson T. Durkin J.P. J. Neurosci. 2000; 20: 7183-7192Crossref PubMed Google Scholar). This protective effect likely accounted for why MK-801 did not reverse 4-AP/bic induced tolerance to OGD. Summarizing, a role for NMDA receptors was established using memantine. Blocking l-type voltage-gated Ca2+ channel (L-VGCCs) with nifedipine or AMPA receptors with NBQX during low 4-AP/bic preconditioning partially suppressed OGD tolerance (Fig. 2B), but neither of these antagonists had any effect on high 4-AP/bic preconditioning (Fig. 2A). Combining memantine with nifedipine more effectively suppressed OGD tolerance than by either antagonist alone for both high (Fig. 2A) or low (Fig. 2B) 4-AP/bic preconditioning, suggesting that co-activation of NMDA receptors and L-VGCCs contributes to the generation of OGD tolerance. Preconditioning accomplished by inducing epileptiform activity by kainic acid administration in vivo induces activation of adenosine A1 receptors and KATP channels (32.Plamondon H. Blondeau N. Heurteaux C. Lazdunski M. J. Cereb. Blood Flow Metab. 1999;
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