Experiments were performed on male Sprague-Dawley rats (Taconic Farms, Rockville, MD), 5–6 weeks old, weighing 170–200 g at the start of the experiments. Animals were individually housed in an animal facility approved by the Association for Assessment and Accreditation of Laboratory Animal Care, in an environmentally controlled room (20–23 °C, 12-h light/12-h dark cycle, lights on 07:00 a.m.), with food and water available ad libitum. All animal use procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and were approved by the Animal Care and Use Committees of the National Institute of Neurological Disorders and Stroke and Uniformed Services University of the Health Sciences.
Following 5 days of acclimatization, the rats were stereotaxically implanted with 5 cortical stainless steel screw electrodes under general anesthesia [ketamine 60 mg/kg intraperitonally (i.p.) and medetomidine 0.5 mg/kg i.p.], using the following coordinates, in mm, after Paxinos and Watson (1998)
: Two frontal electrodes AP = +1.5, ML = ±2.5 from bregma; two parietal electrodes AP = −5.0, ML = ±4 from bregma; and a single cerebellar reference electrode midline, AP = −1.0 from lambda. Each screw electrode with socket (E363/20; Plastics ONE Inc., Roanoke, VA) was placed in a plastic pedestal (MS363; Plastics ONE Inc., Roanoke, VA) and attached to the skull with dental acrylic cement. Anesthesia was reversed by i.p. injection of 1 mg/kg atipamezol.
Induction of status epilepticus
After 1 week of recovery, 25 rats were injected (i.p.) with KA to induce SE (“KA-SE rats”). Fifteen implanted rats were injected with corresponding amounts of saline at corresponding times as the KA-treated rats; this group of rats served as controls (“sham control rats”). SE was induced following a modified titration protocol after Hellier et al. (1998)
. In order to maximize consistency of the SE expression between rats, EEG seizure activity rather than the behavioral seizure correlate was used to determine the necessity of additional KA injections. Rats were initially treated with 7.5 mg/kg KA dissolved in 0.1 M phosphate buffered normal saline (PBS; 5mg/ml), followed by additional 5 mg/kg doses every 60 min, until SE began, defined by 5 min of continuous generalized electrographic seizure activity (all four cortical electrodes involved; no breaks longer than 10 sec). Following this protocol, rats received between 12.5 and 32.5 mg/kg KA. SE was allowed to continue without intervention for 180 min, and then 25–30 mg/kg of diazepam was injected i.p. for termination of SE. Fluid was substituted with 6 ml lactated Ringer’s solution injected subcutaneously (s.c.) after resolution of continuous convulsive seizures, usually less than 10 min after the diazepam injection. The sham control rats also received diazepam injections. For three days after SE, rats were offered fruits and soft chow. Additional lactated Ringer’s solution was administered by s.c. injection if dehydration was apparent. The wooden bedding was changed to iso-PADs™
(Harlan Teklad, Madison, WI) to avoid aspiration in case of frequent seizures.
EEG recordings and analysis
Recordings were performed in freely moving rats using a commercially available Stellate® EEG monitoring system modified for the use in rodents (Respitech Medical Inc., Lancaster, PA; sampling rate, 200 Hz). EEG recordings were visually analyzed offline with filter settings set to 0.3 Hz low frequency filter, 60 Hz notch filter, and 70 Hz high frequency filter, using the Harmonie Viewer 6.1c from Stellate® (Montreal, Quebec, Canada). An electrographic seizure was defined as a period of EEG changes marked by an abrupt starting and ending, that included a minimum of 10 sec of consistent, repetitive discharges at least double the amplitude of the background activity and not less than 1 Hz frequency. A single graphoelement was classified as sharp wave when it was clearly distinguishable from movement artifacts, had twice the amplitude of the background EEG, and was less than 200 ms in duration. Animals were recorded starting in the early morning, throughout SE, and continuing for at least two hours after the diazepam injection (7 to 9 hours total recording time per animal). The EEG seizure activity had stopped at the end of the recording period in 20 of the 25 KA-treated rats, with four mortalities. Post-SE EEG monitoring for detection of spontaneous sharp waves and electrographic seizure activity was performed either for 24 hours, 6 to 9 days after status epileptics (n = 16 rats) or on days 6 and 7 for 8 hours per day (n = 5 rats). The non-occurrence or occurrence of spontaneous sharp waves, spikes and seizures was noted during each recording period.
Fixation and tissue processing
Five rats from the KA-SE group and five sham control rats were used for histological studies of the BLA. Six to 9 days after SE, rats were EEG monitored for 24 hours. On the day after monitoring, the animals were deeply anesthetized using ketamine (60 mg/kg i.p.) and medetomidine (0.5 mg/kg i.p.) and transcardially perfused with PBS (100 mL) followed by 4% paraformaldehyde (250 mL). The brains were removed and post-fixed overnight at 4° C, then transferred to a solution of 30% sucrose in PBS for 72 hours, and frozen with dry ice before storage at −80° C until sectioning. A 1-in-6 series of sections containing the rostro-caudal extent of the BLA was cut at 40 μm on a sliding microtome. One series of sections was mounted on slides (Superfrost Plus; Daigger, Vernon Hills, IL) in PBS for Nissl staining with cresyl violet. An adjacent series of sections was also mounted on slides for Fluoro-Jade C staining. The remaining series of sections were placed in a cryoprotectant solution (30% ethylene glycol and 30% glycerol in 0.05 M sodium phosphate buffer) and stored at −20° C until processing for immunohistochemistry.
Glutamic acid decarboxylase-67 (GAD67) immunohistochemistry
To label GAD67-immunoreactive neurons, a l-in-6 series of free-floating sections was collected from the cryoprotectant solution, washed three times for 5 min each in 0.1 M phosphate buffered saline (PBS), then incubated in a blocking solution containing 10% normal goat serum (NGS; Chemicon), and 0.5% Triton X-100 in PBS for one hour at room temperature. The sections were then incubated with mouse anti-GAD67 serum (1:1000, MAB5406; Chemicon), 5% NGS, 0.3% Triton X-100, and 1% bovine serum albumin, overnight at 4° C. After rinsing three times in 0.1% Triton X-100 in PBS, the sections were incubated with Cy3-conjugated goat anti-mouse antibody (1:1000; Jackson ImmoResearch) and 0.0001% DAPI (Sigma, St. Louis, MO) in PBS for one hour at room temperature. After a final rinse in PBS, sections were mounted on slides, air dried for 30 min, then coverslipped with ProLong Gold antifade reagent (Invitrogen).
Fluoro-Jade C(FJ-C) staining
FJ-C (Histo-Chem, Jefferson, AK) was used to identify irreversibly dying neurons in the BLA, on days 7–10 after SE. Mounted sections were air-dried overnight, then immersed in a solution of 1% sodium hydroxide in 80% ethanol for 5 min. The slides were then rinsed for 2 min in 70% ethanol, 2 min in dH20, and incubated in 0.06% potassium permanganate solution for 10 min. After a 2 min rinse in dH20, the slides were transferred a 0.0001% solution of FJ-C dissolved in 0.1% acetic acid for 10 min. Following three 1-min rinses in dH20, the slides were dried on a slide warmer, cleared in xylene for at least 1 min and coverslipped with DPX (Sigma).
Design-based stereology was used to quantify the total number of neurons on Nissl-stained sections and total number of inhibitory neurons on GAD67-stained sections in the BLA of KA-SE and sham control rats. Sections were viewed with a Zeiss Axioplan 2ie (Oberkochen, Germany) fluorescent microscope with a motorized stage, interfaced with a computer running StereoInvestigator 7.5 (MicroBrightField, Williston, VT). The BLA was identified on slide-mounted sections and delineated under a 2.5× objective, based on the atlas of Paxinos and Watson (1998)
. Estimated totals were determined using the optical fractionator probe, and all sampling was done under a 63× oil immersion objective.
For Nissl-stained neurons in the BLA of both KA-SE and sham control rats, a 1-in-6 series of sections was analyzed (on average, 6 sections). The counting frame was 35 × 35 μm, the counting grid was 190 × 190 μm, and the disector height was 12 μm. Nuclei were counted when the top of the nucleus came into focus within the disector which was placed 2 μm below the section surface. Section thickness was measured at every counting site, and the average mounted section thickness was 19 μm. An average of 288 Nissl-stained neurons per rat was counted.
For GAD67-stained neurons in BLA, of both KA-SE and sham control rats, a 1-in-6 series of sections was analyzed (on average, 6 sections), the counting frame was 60 × 60 μm. The counting grid was 100 × 100 μm for KA-SE rats and 110 × 110 μm for sham control rats. The disector height was 12 μm (n = 1), 16 μm (n = 5), or 20 μm (n = 4), depending on extent of antibody penetration in the tissue. Cells were counted if they came into focus within the disector, which was placed 2 μm below the section surface. Section thickness was measured at every fifth counting site, and the average mounted section thickness was 40 μm. An average of 207 GAD67-stained neurons per rat was counted. The coefficient of error (CE) for the estimated total of Nissl-stained and GAD67-stained neurons in the BLA was calculated using both the Gunderson (m = 1; Gundersen et al., 1999
) and Schmitz-Hof (2nd Estimation; Schmitz and Hof, 2000
Quantification of FJ-C stained neurons
Tracings of the BLA from an adjacent series of Nissl-stained sections were superimposed on the sections stained with FJ-C. FJ-C positive cells were counted at 20×, and recorded as number of cells per section from, on average, 6 sections containing the BLA of sham control rats and KA-SE rats.
Six rats from the KA-SE group and three sham controls were used for measuring the levels of the GluK1 subunit, the GABA synthesizing enzyme GAD65/67, and the α1 subunit of the GABAA receptor, in the BLA. The CA3 subfield of the hippocampus and the ventral posteromedial and ventral posterolateral (VPM/VPL) thalamic nuclei were also included in the Western blot analysis, for comparison. Six to nine days after SE, rats were video-EEG monitored for 24 hours. On the day after monitoring, rats were anesthetized with CO2 and then decapitated. Discrete brain regions were micro-dissected from individual 800 μm-thick brain sections cut with a Vibratome (Technical Products International). The tissues were sonicated in lysis buffer (1% NP-40, 20 mM Tris, pH 8.0, 137 nM NaCl, 10% Glycerol, Tyr &Ser/Thr Phosphatase Inhibitor Cocktails; Upstate, Temecula, CA). After removal of cellular debris by centrifugation, protein levels in the lysates were measured by the Bradford Coomassie Blue colorimetric assay (Bio-Rad Laboratories, Hercules, CA) and equalized accordingly. Aliquots (60 μg) were boiled for 5 minutes in the presence of loading buffer (NuPAGE LDS Sample Buffer (4×), Invitrogen, Carlsbad, CA), then placed on ice for 1 minute. Each brain region was loaded in triplicate and proteins were separated on a 7.5% SDS-PAGE under reducing conditions using the Bio-Rad Mini-Protean3 cell system. Proteins were transferred to nitrocellulose membranes (0.45μm; Invitrogen). After blocking with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) at room temperature for 1 hour, blots were incubated overnight at 4°C with specific primary antibodies: anti-GluK1, 1:500 (Tocris Bioscience, Ellisville, Missouri), anti-GAD65/67 (1:10,000; Chemicon), and anti-GABAA α1 (1:200, Chemicon), prepared in 5% bovine serum albumin (BSA) in TBS-T. After washing in TBS-T, membranes were incubated with peroxidase-conjugated goat anti-rabbit (1:10,000; Jackson Immuno Research, West Grove, PA) prepared in 5% BSA in TBS-T for 2 hours, at room temperature. Anti-β-actin (1:500; Cell Signaling Technology, Danvers, MA) was used as a loading control in all experiments. After washing in TBS-T, blots were developed using enhanced chemiluminescence detection according to the manufacture’s recommendation (Pierce, Rockford, IL) and exposed to BioMax MR Film (Kodak Biomax, Rochester, NY) under non-saturating conditions. The blots were stripped with Restore Western blot Stripping Buffer (Pierce) and then incubated with subsequent antibodies (see above). Absorbance values of bands for GluK1, GAD65/67, and GABAA α1 were analyzed by densitometry using Image J analysis systems (NIH, Bethesda, MD), and normalized relative to the β-actin absorbance value.
Amygdala slice electrophysiology
Coronal slices containing the amygdala were prepared from rats, on days 7–10 after SE. The rats were anesthetized with CO2 and then decapitated. The brain was rapidly removed and placed in ice-cold artificial cerebrospinal fluid (ACSF) composed of (in mM): 125 NaCl, 2.5 KCl, 2.0 CaCl2, 2.0 MgCl2, 25 NaHCO3, 1.25 NaH2PO4, and 22 glucose, bubbled with 95% O2 and 5% CO2 to maintain a pH of 7.4. A block containing the amygdala region was prepared, and 400 μm slices were cut with a Vibratome (Series 1000; Technical Products International, St. Louis, MO). Slices were kept in a holding chamber containing oxygenated ACSF at room temperature, and recordings were initiated ≥1 hr after slice preparation. Slices were transferred to a submersion-type recording chamber, where they were continuously perfused with oxygenated ACSF, at a rate of 3–4 ml/min. Neurons were visualized with an upright microscope (Nikon Eclipse E600fn; Nikon, Tokyo, Japan) using Nomarski-type differential interference optics through a 60× water immersion objective. All experiments were performed at room temperature (28°C). Tight-seal (>1 GΩ) whole-cell recordings were obtained from the cell body of pyramidal-shaped neurons in the BLA region. Patch electrodes were fabricated from borosilicate glass and had a resistance of 1.5–5.0 MΩ when filled with a solution containing (in mM): 135 Cs-gluconate, 10 MgCl2, 0.1 CaCl2, 1 EGTA, 10 HEPES, 2 Na-ATP, 0.2 Na3GTP, pH 7.3 (285–290 mOsm). Neurons were voltage-clamped using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA). IPSCs were pharmacologically isolated and recorded at a -70 mV holding potential. Access resistance (5–24 MΩ) was regularly monitored during recordings, and cells were rejected if it changed by >15% during the experiment. The signals were filtered at 2 kHz, digitized (Digidata 1322A; Axon Instruments), and stored on a computer using pClamp9 software (Axon Instruments). The peak amplitude, 10–90% rise time, and decay time constant of IPSCs were analyzed off-line using pClamp9 software and the Mini Analysis Program (Synaptosoft, Inc., Leonia, NJ). Miniature IPSCs (mIPSCs) were analyzed offline using the Mini Analysis Program and detected by manually setting the mIPSC threshold (~1.5 times the baseline noise amplitude) after visual inspection.
The following drugs were used: kainic acid (Tocris Cookson, Ballwin, MO), diazepam (Hospira Inc., IL), D-APV (Tocris; an NMDA receptor antagonist), (+)-(2S
)-5,5-dimethyl-2-morpholineacetic acid (SCH50911; Tocris; a GABAB
receptor antagonist), ATPA (Sigma; a GluK1R agonist; see Clarke et al., 1997
), GYKI 52466 (Tocris; an AMPA receptor antagonist), and tetrodotoxin (TTX; Sigma; a sodium channel blocker).
All statistical values are presented as mean ± S.E.M. Results from sham control and KA-SE groups were compared using the unpaired Student’s t test; p < 0.05 was considered statistically significant. Sample sizes (n) refer to the number of rats, except for the electrophysiology results where “n” refers to the number of slices or recorded cells.