Title: Bcl-w Protects Hippocampus during Experimental Status Epilepticus
Abstract: Experimentally evoked seizures can activate the intrinsic mitochondrial cell death pathway, components of which are modulated in the hippocampus of patients with temporal lobe epilepsy. Bcl-2 family proteins are critical regulators of mitochondrial dysfunction, but their significance in this setting remains primarily untested. Presently, we investigated the mitochondrial pathway and role of anti-apoptotic Bcl-2 proteins using a mouse model of seizure-induced neuronal death. Status epilepticus was evoked in mice by intra-amygdala kainic acid, causing cytochrome c release, processing of caspases 9 and 7, and death of ipsilateral hippocampal pyramidal neurons. Seizures caused a rapid decline in hippocampal Bcl-w levels not seen for either Bcl-2 or Bcl-xl. To test whether endogenous Bcl-w was functionally significant for neuronal survival, we investigated hippocampal injury after seizures in Bcl-w-deficient mice. Seizures induced significantly more hippocampal CA3 neuronal loss and DNA fragmentation in Bcl-w-deficient mice compared with wild-type mice. Quantitative electroencephalography analysis also revealed that Bcl-w-deficient mice display a neurophysiological phenotype whereby there was earlier polyspike seizure onset. Finally, we detected higher levels of Bcl-w in hippocampus from temporal lobe epilepsy patients compared with autopsy controls. These data identify Bcl-w as an endogenous neuroprotectant that may have seizure-suppressive functions. Experimentally evoked seizures can activate the intrinsic mitochondrial cell death pathway, components of which are modulated in the hippocampus of patients with temporal lobe epilepsy. Bcl-2 family proteins are critical regulators of mitochondrial dysfunction, but their significance in this setting remains primarily untested. Presently, we investigated the mitochondrial pathway and role of anti-apoptotic Bcl-2 proteins using a mouse model of seizure-induced neuronal death. Status epilepticus was evoked in mice by intra-amygdala kainic acid, causing cytochrome c release, processing of caspases 9 and 7, and death of ipsilateral hippocampal pyramidal neurons. Seizures caused a rapid decline in hippocampal Bcl-w levels not seen for either Bcl-2 or Bcl-xl. To test whether endogenous Bcl-w was functionally significant for neuronal survival, we investigated hippocampal injury after seizures in Bcl-w-deficient mice. Seizures induced significantly more hippocampal CA3 neuronal loss and DNA fragmentation in Bcl-w-deficient mice compared with wild-type mice. Quantitative electroencephalography analysis also revealed that Bcl-w-deficient mice display a neurophysiological phenotype whereby there was earlier polyspike seizure onset. Finally, we detected higher levels of Bcl-w in hippocampus from temporal lobe epilepsy patients compared with autopsy controls. These data identify Bcl-w as an endogenous neuroprotectant that may have seizure-suppressive functions. Seizures, particularly when prolonged, are capable of causing neuronal loss within vulnerable brain structures such as the hippocampus. Delineating the mechanisms of such cell death may offer approaches to neuroprotection and perhaps anti-epileptogenesis.1Pitkänen A Drug-mediated neuroprotection and anti-epileptogenesis.Neurology. 2002; 59: S27-S33Crossref PubMed Google Scholar The cell and molecular mechanisms of seizure-induced neuronal death originate in part from prolonged glutamate receptor activation resulting in elevations of intracellular sodium and calcium, culminating in activation of proteases, cell and organelle swelling, and necrotic cell death.2Meldrum BS Implications for neuroprotective treatments.Prog Brain Res. 2002; 135: 487-495Crossref PubMed Scopus (68) Google Scholar, 3Fujikawa DG Prolonged seizures and cellular injury: understanding the connection.Epilepsy Behav. 2005; 7: S3-S11Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar However, cell and molecular hallmarks of apoptosis signaling have been reported in some experimental models of seizure-induced neuronal death and in temporal lobe material from patients with intractable epilepsy.4Liou AKF Clark RS Henshall DC Yin XM Chen J To die or not to die for neurons in ischemia, traumatic brain injury and epilepsy: a review on the stress-activated signaling pathways and apoptotic pathways.Prog Neurobiol. 2003; 69: 103-142Crossref PubMed Scopus (265) Google Scholar, 5Henshall DC Simon RP Epilepsy and apoptosis pathways.J Cereb Blood Flow Metab. 2005; 25: 1557-1572Crossref PubMed Scopus (180) Google Scholar Apoptosis is orchestrated via either an intrinsic pathway that originates from within the cell or via extrinsic pathways mediated by activation of surface-expressed death receptors.6Danial NN Korsmeyer SJ Cell death: critical control points.Cell. 2004; 116: 205-219Abstract Full Text Full Text PDF PubMed Scopus (4060) Google Scholar Mitochondria are a critical point of apoptosis control on which a variety of cell death-promoting signals converge including raised intracellular calcium, free radicals, and Bcl-2 family proteins.4Liou AKF Clark RS Henshall DC Yin XM Chen J To die or not to die for neurons in ischemia, traumatic brain injury and epilepsy: a review on the stress-activated signaling pathways and apoptotic pathways.Prog Neurobiol. 2003; 69: 103-142Crossref PubMed Scopus (265) Google Scholar Individual members of the Bcl-2 family can be classified as pro- or anti-apoptotic.6Danial NN Korsmeyer SJ Cell death: critical control points.Cell. 2004; 116: 205-219Abstract Full Text Full Text PDF PubMed Scopus (4060) Google Scholar All members of the family contain at least one of four Bcl-2 homology (BH) domains with two distinct proapoptotic subfamilies; the Bax/Bak subfamily and the BH3-only proteins. Killing by BH3-only proteins requires Bax or Bak,7Lindsten T Thompson CB Cell death in the absence of Bax and Bak.Cell Death Differ. 2006; 13: 1272-1276Crossref PubMed Scopus (49) Google Scholar and mitochondrial permeabilization via Bax/Bak culminates in the release of cytochrome c. In the cytosol, cytochrome c promotes apoptosome formation and activation of executioner caspases.8Li P Nijhawan D Budihardjo I Srinivasula SM Ahmad M Alnemri ES Wang X Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade.Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6261) Google Scholar On activation, caspases cleave critical structural and functional proteins,9Fischer U Janicke RU Schulze-Osthoff K Many cuts to ruin: a comprehensive update of caspase substrates.Cell Death Differ. 2003; 10: 76-100Crossref PubMed Scopus (891) Google Scholar culminating in cell death. In addition to regulating the intrinsic apoptotic pathway, several Bcl-2 proteins possess endogenous roles in intracellular processes, such as calcium signaling. In particular, Bcl-xl and Bak may directly influence neuronal excitability.10Fannjiang Y Kim CH Huganir RL Zou S Lindsten T Thompson CB Mito T Traystman RJ Larsen T Griffin DE Mandir AS Dawson TM Dike S Sappington AL Kerr DA Jonas EA Kaczmarek LK Hardwick JM BAK alters neuronal excitability and can switch from anti- to pro-death function during postnatal development.Dev Cell. 2003; 4: 575-585Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 11Jonas EA Hoit D Hickman JA Brandt TA Polster BM Fannjiang Y McCarthy E Montanez MK Hardwick JM Kaczmarek LK Modulation of synaptic transmission by the BCL-2 family protein BCL-xL.J Neurosci. 2003; 23: 8423-8431Crossref PubMed Google Scholar Modulation of Bcl-2 proteins accordingly may influence neuronal function beyond life and death decisions. Prolonged seizures in rats trigger cytochrome c release,12Henshall DC Chen J Simon RP Involvement of caspase-3-like protease in the mechanism of cell death following focally evoked limbic seizures.J Neurochem. 2000; 74: 1215-1223Crossref PubMed Scopus (128) Google Scholar apoptosome formation,13Henshall DC Bonislawski DP Skradski SL Araki T Lan J-Q Schindler CK Meller R Simon RP Formation of the Apaf-1/cytochrome c complex precedes activation of caspase-9 during seizure-induced neuronal death.Cell Death Differ. 2001; 8: 1169-1181Crossref PubMed Scopus (65) Google Scholar and executioner caspase-3 activation.12Henshall DC Chen J Simon RP Involvement of caspase-3-like protease in the mechanism of cell death following focally evoked limbic seizures.J Neurochem. 2000; 74: 1215-1223Crossref PubMed Scopus (128) Google Scholar, 14Liu XM Pei DS Guan QH Sun YF Wang XT Zhang QX Zhang GY Neuroprotection of Tat-GluR6-9c against neuronal death induced by kainate in rat hippocampus via nuclear and non-nuclear pathways.J Biol Chem. 2006; 281: 17432-17445Crossref PubMed Scopus (56) Google Scholar This pathway may be instigated by BH3-only proteins, of which Bim plays a role in vivo and in vitro via a mechanism involving neutralization of Bcl-w.15Shinoda S Schindler CK Meller R So NK Araki T Yamamoto A Lan JQ Taki W Simon RP Henshall DC Bim regulation may determine hippocampal vulnerability after injurious seizures and in temporal lobe epilepsy.J Clin Invest. 2004; 113: 1059-1068Crossref PubMed Scopus (94) Google Scholar Bim and its transcriptional machinery are suppressed in hippocampi from patients with intractable epilepsy and after experimental brief protection-conferring seizures.15Shinoda S Schindler CK Meller R So NK Araki T Yamamoto A Lan JQ Taki W Simon RP Henshall DC Bim regulation may determine hippocampal vulnerability after injurious seizures and in temporal lobe epilepsy.J Clin Invest. 2004; 113: 1059-1068Crossref PubMed Scopus (94) Google Scholar Pharmacological tools for manipulating these pathways remain inadequate, however, because of cross-interference with necrosis-related signaling.16Knoblach SM Alroy DA Nikolaeva M Cernak I Stoica BA Faden AI Caspase inhibitor z-DEVD-fmk attenuates calpain and necrotic cell death in vitro and after traumatic brain injury.J Cereb Blood Flow Metab. 2004; 24: 1119-1132Crossref PubMed Scopus (105) Google Scholar We recently developed models in mice with which to exploit knockouts for the genes of interest.17Araki T Simon RP Taki W Lan J-Q Henshall DC Characterization of neuronal death induced by focally evoked limbic seizures in the C57BL/6 mouse.J Neurosci Res. 2002; 69: 614-621Crossref PubMed Scopus (69) Google Scholar, 18Shinoda S Araki T Lan JQ Schindler CK Simon RP Taki W Henshall DC Development of a model of seizure-induced hippocampal injury with features of programmed cell death in the BALB/c mouse.J Neurosci Res. 2004; 76: 121-128Crossref PubMed Scopus (47) Google Scholar Presently, we examined the role of Bcl-2 family proteins after seizures in mice and report that anti-apoptotic Bcl-w has dual functions, abrogating seizure-induced neuronal death and polyspike seizure onset time, and find that Bcl-w is up-regulated in patients with intractable temporal lobe epilepsy. All animal procedures were approved by the Legacy Institutional Animal Care and Use Committee, the Research Ethics Committee of the Royal College of Surgeons in Ireland, and the principles outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Studies were performed according to previously described methods.17Araki T Simon RP Taki W Lan J-Q Henshall DC Characterization of neuronal death induced by focally evoked limbic seizures in the C57BL/6 mouse.J Neurosci Res. 2002; 69: 614-621Crossref PubMed Scopus (69) Google Scholar, 18Shinoda S Araki T Lan JQ Schindler CK Simon RP Taki W Henshall DC Development of a model of seizure-induced hippocampal injury with features of programmed cell death in the BALB/c mouse.J Neurosci Res. 2004; 76: 121-128Crossref PubMed Scopus (47) Google Scholar C57BL/6 mice were obtained from commercial sources (Charles River, Wilmington, MA; or Harlan, Bicester, UK). BALB/c wild-type and littermate Bcl-w-deficient mice were provided by Dr. G. Macgregor (Emory University, Atlanta, GA) and bred in-house. Adult male mice (20 to 25 g) underwent seizures induced by unilateral stereotaxic microinjection of kainic acid (KA) into the basolateral amygdala nucleus.19Franklin KBJ Paxinos P The Mouse Brain in Stereotaxic Coordinates. Academic Press, Inc., San Diego1997Google Scholar Briefly, mice were anesthetized (isoflurane), maintained normothermic via a heat pad (Harvard Instruments, Holliston, MA), and a femoral vein was catheterized, and then animals were placed in a mouse-adapted stereotaxic frame (Kopf Instruments, Tujunga, CA). Mice were affixed with three recording electrodes (Plastics One, Inc., Roanoke, VA) and a 26-gauge steel guide cannula over the dura using dental cement (Plastics One Inc.). Anesthesia was discontinued, electroencephalography (EEG) recordings were commenced, and then a 31-gauge internal cannula (Plastics One Inc.) was inserted into the lumen of the guide to inject KA [0.3 μg in 0.2 μl of vehicle; phosphate-buffered saline (PBS), pH adjusted to 7.4] into the amygdala. Nonseizure control animals underwent the same surgical procedure but received intra-amygdala vehicle injection. The EEG was monitored until intravenous lorazepam (6 mg/kg) administration at 40 minutes and then recorded for up to 30 minutes thereafter. Mice were euthanized 0.5, 1, 4, 8, 24, or 72 hours after anti-convulsant, and brains were microdissected on ice for immunoblotting or flash-frozen whole for immunohistochemistry as previously described.15Shinoda S Schindler CK Meller R So NK Araki T Yamamoto A Lan JQ Taki W Simon RP Henshall DC Bim regulation may determine hippocampal vulnerability after injurious seizures and in temporal lobe epilepsy.J Clin Invest. 2004; 113: 1059-1068Crossref PubMed Scopus (94) Google Scholar, 18Shinoda S Araki T Lan JQ Schindler CK Simon RP Taki W Henshall DC Development of a model of seizure-induced hippocampal injury with features of programmed cell death in the BALB/c mouse.J Neurosci Res. 2004; 76: 121-128Crossref PubMed Scopus (47) Google Scholar Brains from additional naïve (noninstrumented) wild-type and Bcl-w-deficient mice were used for the examination of hippocampal and amygdala neuroanatomy and gene expression. Additional C57BL/6 mice were subject to brief seizures delivered via a Woodbury & Davenport electroshock apparatus as previously described.15Shinoda S Schindler CK Meller R So NK Araki T Yamamoto A Lan JQ Taki W Simon RP Henshall DC Bim regulation may determine hippocampal vulnerability after injurious seizures and in temporal lobe epilepsy.J Clin Invest. 2004; 113: 1059-1068Crossref PubMed Scopus (94) Google Scholar Maximal electroshock seizures (MESs) were delivered via orbital (corneal) electrodes, adapted for use in mice, using stimuli of 25 V, 500 mA, and 0.2-second duration. In mice, this paradigm induces a tonic-clonic convulsion that lasts ∼15 to 30 seconds. Mice were subject to one, three, or nine MESs throughout a period of 1 to 3 days and euthanized 24 hours after the last MES. Control mice were handled and placed in contact with the electrodes without current. Bcl-w-deficient mice were originally generated using a retroviral gene-trap system in which lacZ (β-galactosidase) replaced bcl-w.20Ross AJ Waymire KG Moss JE Parlow AF Skinner MK Russell LD MacGregor GR Testicular degeneration in Bclw-deficient mice.Nat Genet. 1998; 18: 251-256Crossref PubMed Scopus (229) Google Scholar Mice have a BALB/c background and had been backcrossed for more than eight generations. Mice of either gender were used, and the endogenous or disrupted gene was detected by polymerase chain reaction analysis of tail DNA extracts using the primer sequences as described previously.20Ross AJ Waymire KG Moss JE Parlow AF Skinner MK Russell LD MacGregor GR Testicular degeneration in Bclw-deficient mice.Nat Genet. 1998; 18: 251-256Crossref PubMed Scopus (229) Google Scholar This study was approved by the Legacy Health System Institutional Review Board, and informed consent was obtained from all patients. Data for controls and epilepsy patients have previously been published.15Shinoda S Schindler CK Meller R So NK Araki T Yamamoto A Lan JQ Taki W Simon RP Henshall DC Bim regulation may determine hippocampal vulnerability after injurious seizures and in temporal lobe epilepsy.J Clin Invest. 2004; 113: 1059-1068Crossref PubMed Scopus (94) Google Scholar In brief, epilepsy patients (n = 10) were between 15 to 57 years of age, included seven females and three males, and were referred for surgical resection of the left or right temporal lobe because each had previously been determined to have medically intractable epilepsy with a history of recurring seizures. The resected hippocampus was flash-frozen in liquid nitrogen and stored at −70°C until use. Coronal slabs of ∼1-mm thickness were prepared for biochemical analysis. Human control tissue (n = 6) was obtained from the Brain and Tissue Bank for Developmental Disorders at the University of Maryland, Baltimore, MD. These people were between 13 to 53 years of age, included five males and one female, and did not have neurological disease. These specimens were also fresh-frozen, en bloc hippocampi. Western blotting was performed as previously described.15Shinoda S Schindler CK Meller R So NK Araki T Yamamoto A Lan JQ Taki W Simon RP Henshall DC Bim regulation may determine hippocampal vulnerability after injurious seizures and in temporal lobe epilepsy.J Clin Invest. 2004; 113: 1059-1068Crossref PubMed Scopus (94) Google Scholar Protein concentration was determined using Bradford reagent spectrophotometrically at A595 nm, and 50-μg samples were boiled in gel-loading buffer and separated on 12 to 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. Proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA) and then incubated with antibodies against the following: α-tubulin, actin, Bax, and Bcl-2 (Santa Cruz Biotechnology, Santa Cruz, CA); Bcl-w, Bim, and manganese superoxide dismutase (MnSOD) (Stressgen, Victoria, BC, Canada); Bcl-xl and cytochrome c (BD Transduction Laboratories, Lexington, KY); cleaved caspase-3, caspase-7, and caspase-9 (Cell Signaling Technology, Beverly, MA); and GluR5-7 (Chemicon, Temecula, CA). Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (1:2000 dilution) followed by chemiluminescence detection (NEN Life Science Products, Boston, MA), and then exposed to Kodak Biomax film (Kodak, Rochester, NY). Mouse hippocampal tissue was fractionated to obtain the mitochondria according to previous methods.21Schindler CK Shinoda S Simon RP Henshall DC Subcellular distribution of Bcl-2 family proteins and 14-3-3 within the rat hippocampus during seizure-induced neuronal death in the rat.Neurosci Lett. 2004; 356: 163-166Crossref PubMed Scopus (38) Google Scholar In brief, samples (pools of four hippocampi) were homogenized in a mannitol/sucrose buffer containing a protease inhibitor cocktail and then centrifuged twice at 1200 × g for 10 minutes. The postnuclear supernatant was then centrifuged twice at 10,000 × g for 15 minutes, and the resulting mitochondrial pellet was resuspended in a sucrose buffer and purified through a Percoll bilayer by centrifugation at 41,000 × g for 30 minutes. For immunoprecipitation studies,15Shinoda S Schindler CK Meller R So NK Araki T Yamamoto A Lan JQ Taki W Simon RP Henshall DC Bim regulation may determine hippocampal vulnerability after injurious seizures and in temporal lobe epilepsy.J Clin Invest. 2004; 113: 1059-1068Crossref PubMed Scopus (94) Google Scholar hippocampal protein samples (0.5 mg) were first incubated overnight at 4°C with 5 μg of anti-Bcl-w and then incubated with protein A/G agarose beads (Santa Cruz Biotechnology) for 2 hours at 4°C. The protein-bead complex was collected by centrifugation and boiled, and samples were subject to Western blotting with anti-Bim. Nonspecific binding was assessed by omission of the primary (immunoprecipitation) antibody, and whole cell lysates were run concurrently to confirm molecular weights. To determine whether KA receptor (GluR5-7) levels were equivalent between wild-type and Bcl-w-deficient mice, bilateral tissue cores (∼1 mm wide by 2 mm deep) of the amygdala nuclei (including the basolateral amygdala) were extracted from a selection of naïve wild-type or knockout mice. Tissue samples were then processed for Western blotting as described above. Coronal mouse brain sections (12 μm) were air-dried and postfixed in 100% methanol (Bcl-w immunostaining) or 10% formalin (histopathology and NeuN immunostaining) for 30 minutes, followed by washes in PBS. For histopathology, sections were stained with Cresyl violet. For immunohistochemistry, sections were first blocked with 5% goat serum and then incubated overnight at 4°C with antibodies against Bcl-w (Stressgen) or NeuN (Chemicon). Sections were washed in PBS and then incubated for 2 hours at room temperature in a 1:1000 dilution of goat anti-mouse or 1:500 dilution of goat anti-rabbit Alexa Fluor 488 or 568 (Molecular Probes, Eugene, OR). After additional washes sections were counterstained with 4′,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA), to assess nuclear morphology. Counts of NeuN-stained hippocampal CA1 and CA3 and amygdala neurons from naïve wild-type and Bcl-w-deficient mice were the mean of five ×60 lens fields from two adjacent sections at the level of bregma −1.7 mm.19Franklin KBJ Paxinos P The Mouse Brain in Stereotaxic Coordinates. Academic Press, Inc., San Diego1997Google Scholar Analysis of cells exhibiting DNA fragmentation was performed using a fluorescein-based terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) technique (Promega, Madison, WI) as previously described.18Shinoda S Araki T Lan JQ Schindler CK Simon RP Taki W Henshall DC Development of a model of seizure-induced hippocampal injury with features of programmed cell death in the BALB/c mouse.J Neurosci Res. 2004; 76: 121-128Crossref PubMed Scopus (47) Google Scholar Images were visualized using a Hamamatsu Orca 285 camera (Hamamatsu Corporation, Bridgewater, NJ) attached to a Nikon 2000s epifluorescence microscope (Melville, NY) under Ex/Em wavelengths of 330 to 380/420 nm (blue), 472/520 nm (green), and 540 to 580/600 to 660 nm (red). TAT-conjugated proteins were produced according to previously described techniques.22Cao G Pei W Ge H Liang Q Luo Y Sharp FR Lu A Ran R Graham SH Chen J In vivo delivery of a bcl-xl fusion protein containing the TAT protein transduction domain protects against ischemic brain injury and neuronal apoptosis.J Neurosci. 2002; 22: 5423-5431Crossref PubMed Google Scholar cDNAs encoding the human bcl-w (99% identical to mouse sequence) or green fluorescent protein (GFP; control) open reading frame were inserted into the NcoI and EcoRI sites of the pTAT-HA vector (a gift from S. Dowdy, Washington University School of Medicine, St. Louis, MO), amplified, purified, and subjected to sequence analysis as described by Vocero-Akbani and colleagues.23Vocero-Akbani A Lissy NA Dowdy SF Transduction of full-length Tat fusion proteins directly into mammalian cells: analysis of T cell receptor activation-induced cell death.Methods Enzymol. 2000; 322: 508-521Crossref PubMed Google Scholar The pTAT-HA plasmids were transformed into high-expressing BL21 (DE3)LysS (Novagen, Madison, WI) bacteria and amplified, and expression of the recombinant protein analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Recombinant proteins were denatured with 8 mol/L urea buffer, purified on a Ni-NTA column (Qiagen, Chatsworth, CA), and eluted. Purified proteins were applied to G-25 Sephadex desalting columns equilibrated in phosphate buffer to remove urea. Eluted fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fractions containing the TAT fusion proteins frozen in 10% glycerol and stored at −80°C until use. TAT-proteins were applied to mouse cortical neuron cultures (10 μmol/L) 4 hours before recordings. Patch-clamp recordings of γ-amino butyric acid (GABA)-mediated currents were performed according to previously described methods.24Jia Z Agopyan N Miu P Xiong Z Henderson J Gerlai R Taverna FA Velumian A MacDonald J Carlen P Abramow-Newerly W Roder J Enhanced LTP in mice deficient in the AMPA receptor GluR2.Neuron. 1996; 17: 945-956Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar, 25Xiong ZG Pelkey KA Lu WY Lu YM Roder JC MacDonald JF Salter MW Src potentiation of NMDA receptors in hippocampal and spinal neurons is not mediated by reducing zinc inhibition.J Neurosci. 1999; 19: RC37PubMed Google Scholar Patch electrodes had a final diameter of 1 to 2 μm and resistance between 3 to 5 mol/LΩ. Membrane potentials were recorded in current-clamp mode using Axopatch 1-D amplifiers (Axon Instruments, Foster City, CA). Data were filtered at 2 kHz and digitized on-line using Digidata 1320A DAC units (Axon Instruments) with acquisition by pClamp software (version 8.0; Axon Instruments). All electrophysiological experiments were done at room temperature (22 to 24°C). A multibarrel perfusion system (SF-77B; Warner Instrument Co., Hamden, CT) was used to achieve a rapid exchange of GABA (5 μmol/L). Data are presented as mean ± SEM. Protein bands from Western blots were semiquantified using gel-scanning integrated optical density software (AlphaeaseFC, version 4.0; Bioquant, Nashville, TN) and were corrected to the intensity reading for the control sample in each independent experiment. Data were analyzed using analysis of variance with post hoc Fisher's PLSD test or for two group comparisons Student's t-test (StatView software; SAS Institute, Inc., Cary, NC). For the human data analysis, a Huber-White robust variance estimates was undertaken. Additional specialized statistical testing was undertaken for EEG analysis where specified in the results (see Supplemental Table 1 at http://ajp.amjpathol.org). Significance was accepted at P < 0.05. Status epilepticus (40 minutes) evoked in C57BL/6 mice by intra-amygdala KA resulted in unilateral hippocampal neuronal death (Figure 1B). Twenty-four hours after seizure termination, extensive DNA fragmentation was notable within ipsilateral CA3 neurons (Figure 1C) with smaller numbers of CA1 neurons (Figure 1D) also degenerated. Higher magnification images of TUNEL-positive cells are shown in Figure 1, E–H. Damage was not found contralaterally (Figure 1A), and no TUNEL-positive cells were detected in the ipsilateral hippocampus of mice that received intracerebral vehicle injection (data not shown). We next examined cytochrome c release as a key marker of intrinsic pathway induction and mitochondrial dysfunction. Cytochrome c was exclusively present within the mitochondrial fraction at both 1 hour and 4 hours within contralateral hippocampus (Figure 1I). In contrast, cytochrome c release was detected within the ipsilateral hippocampus at 4 hours (but not 1 hour) as evidenced by its presence within the cytoplasmic fraction (Figure 1I). Caspase-9, which is activated via apoptosome formation downstream of cytochrome c release, was constitutively expressed in mouse hippocampus (Figure 1J). Cleaved caspase-9 could be detected ∼4 hours after seizure termination with fragment levels peaking at 72 hours (Figure 1J). Quantification of the cleaved caspase-9 levels revealed a statistically significant difference between the cleavage products evident at 72 hours compared with controls (P < 0.01) and to 0.5 and 4 hours (P < 0.05). Activated caspase-9 is capable of cleaving executioner caspases such as caspase-3 and -7. Although cleaved caspase-3 is detected in this model,17Araki T Simon RP Taki W Lan J-Q Henshall DC Characterization of neuronal death induced by focally evoked limbic seizures in the C57BL/6 mouse.J Neurosci Res. 2002; 69: 614-621Crossref PubMed Scopus (69) Google Scholar caspase-7 has not been investigated. Western blotting detected procaspase-7 at low levels in control mouse hippocampus (Figure 1J). Cleaved caspase-7 could be detected ∼4 hours after seizure termination through 72 hours (Figure 1J). Again quantification of the levels of cleaved caspase-7 revealed a statistically significant difference between 24 hours and control (P < 0.05) and between 72 hours and control, 0.5, 4, and 8 hours (P < 0.01). Because Bcl-2 family proteins regulate events upstream of mitochondrial dysfunction in the intrinsic pathway, we next examined expression of key members in our model. Bax, Bcl-xl, and Bcl-2 were all constitutively expressed in mouse hippocampus, but levels were primarily unchanged during seizure-induced neuronal death (Figure 2A). Remarkably, seizures induced a rapid decline in anti-apoptotic Bcl-w levels, which began at 0.5 and peaked 4 hours (P < 0.05) after seizures, followed subsequently by recovery of expression at later time points up to 24 hours (Figure 2, A and B). A tendency to lower Bcl-w levels at 72 hours was noted but was variable and did not reach statistical significance. To evaluate more completely the responsiveness of Bcl-w to seizures, we subjected mice to one or more brief, nondamaging electroshock seizures, which we and others have shown to be associated with generating a tolerant and/or protected state.15Shinoda S Schindler CK Meller R So NK Araki T Yamamoto A Lan JQ Taki W Simon RP Henshall DC Bim regulation may determine hippocampal vulnerability after injurious seizures and in temporal lobe epilepsy.J Clin Invest. 2004; 113: 1059-1068Crossref PubMed Scopus (94) Google Scholar, 26Kondratyev A Sahibzada N Gale K Electroconvulsive shock exposure prevents neuronal apoptosis after kainic acid-evoked status epilepticus.Brain Res Mol Brain Res. 2001; 91: 1-13Crossref PubMed Scopus (79) Google Scholar Western blotting revealed a dose-related