Mice
The use of mice in this study was approved by the Yale Animal Resources Center according to internationally recognized guidelines. All mice were housed with a 12-hour light/12-hour dark cycle and fed ad libitum. Coinjected congenic APPswe/PSEN1dE9 transgenic mice [13] on a pure C57BL/6J background were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). 3xTg-AD mice were a gift from Dr Frank LaFerla (UC Irvine, CA, USA) and were obtained via Dr Paul Lombroso (Yale University). They express the mutated knockin gene PS1M146V, as well as APPswe and tauP301L, at the same locus, both under control of the mouse Thy1.2 regulatory element [14]. 3xTg-AD mice were on a mixed C57BL/6J × 129/Sv background as described elsewhere [14]. For chronic drug experiments, mouse groups were sex-matched, with 40% to 60% of each sex in different cohorts.
Behavioral studies
For experiments correlating epileptiform activity, animals underwent EEG monitoring prior to behavioral testing. Animals tested for behavior during treatment with AEDs did not have EEG electrodes implanted. Animals were randomized, and the experimenter was blinded to genotype for the duration of behavioral testing. Morris water maze testing [15] based on previously described methods [12] was performed over the course of 3 days. Each swim was performed at room temperature in an open-water pool approximately 1.3 m in diameter, utilizing a submerged, nonvisible escape platform located in the center of one of the pool’s four quadrants. This location remained constant for the 3 days of testing. Over the course of each testing day, an animal swam a total of eight times—four times in the morning, constituting one “block” of swims, and four times in an afternoon block. The interval between blocks was approximately 2 hours. For each block, the mice would begin their swim in one of four distinct locations around the wall of the pool and were timed for its latency and path length to reach the escape platform for a maximum time of 1 minute. If the mouse did not find the submerged platform by 1 minute, it was placed on the platform for approximately 10 seconds before being removed from the pool. The water maze probe trial was performed 48 hours following the third and last day of the memory acquisition phase and in the same 1.3-m pool described above. For the purposes of the probe trial, the platform was removed from the pool. All mice were started from a location opposite to the platform location and allowed to swim for 1 minute. To ensure that all mice were equal in terms of swim speed, motivation and visual acuity, a block of five swims to a visible platform was conducted after the probe trial. Mice were excluded from the study if the latency to the visible platform exceeded 3 standard deviations above the average latency for control mice, as previously described [16]. By this criterion, one APP/PS1 mouse with SWDs was excluded. Latency to the platform, swim speed, path length and resting time were automatically recorded using Panlab SMART video tracking and analysis program, v2.5 (Panlab, Cornellà de Llobregat, Spain).
Brain tissue collection
Mice were deeply anesthetized with isoflurane and immediately perfused with ice-cold 0.9% NaCl for 2 minutes. Their brains were then dissected out and placed in ice-cold 0.9% NaCl. For biochemical analysis, the right hemibrain was weighed and immediately frozen in liquid nitrogen, followed by storage at −80°C. To extract the soluble cytosolic fraction, the brains were homogenized in 3 volumes (w/v) of 50 mM Tris-HCl, 150 mM NaCl, pH 7.6 (TBS), containing a protease inhibitor cocktail (cOmplete Protease Inhibitor Cocktail, catalog number 10745000; Roche Diagnostics, Mannheim, Germany), 1 mM sodium orthovanadate and 50 mM sodium fluoride. Tissue was homogenized using an ultrasonic cell disruptor (Branson Ultrasonics Corporation, Danbury, CT, USA) and ultracentrifuged at 100,000 × g for 20 minutes at 4°C. The pellet was then resuspended to the same volume as the original homogenate in TBS with 2% Triton X-100 (AmericanBio, Natick, MA, USA), 0.1% SDS (AmericanBio), a protease inhibitor cocktail (cOmplete Protease Inhibitor Cocktail), 1 mM sodium orthovanadate and 50 mM sodium fluoride. Tissue was homogenized and ultracentrifuged at 100,000 × g for 20 minutes. The supernatant was mixed with 4× SDS-PAGE loading buffer, boiled for 5 minutes and stored for subsequent analysis.
Immunohistochemistry
One hemibrain was immersed in fresh 4% paraformaldehyde (PFA) overnight. After the brains were fixed, they were embedded in 10% gelatin and placed in 4% PFA for 20 hours at 4°C. Parasagittal sections (30 μm) were then cut using a Leica VT1000 S vibratome (Leica Biosystems, Buffalo Grove, IL, USA). For immunohistochemistry, sections were blocked in 10% donkey serum for 1 hour, followed by incubation with primary antibody overnight at room temperature. Primary antibodies were diluted in phosphate-buffered saline (PBS) with 0.2% Triton X-100 (AmericanBio). The following antibodies were used: Aβ antibody (catalog number 2454, Cell Signaling Technology, Danvers, MA, USA; and clone 6E10, monoclonal antibody 1560, EMD Millipore, Billerica, MA, USA: both diluted 1:250), rabbit anti-PSD-95 polyclonal antibody (1:250 dilution, catalog number 51-6900; Invitrogen, Camarillo, CA, USA) and anti-calbindin D28 antibody (1:1,000 dilution; Swant, Marly, Switzerland). Following incubation, the sections were washed three times with PBS and incubated in Alexa Fluor fluorescent secondary antibody (donkey anti-rabbit or anti-mouse, all at 1:500 dilution; Invitrogen) for 2 hours at room temperature. The slices were then washed three times and transferred to PBS. Sections were also stained with secondary antibody alone to rule out nonspecific staining. Each free-floating section was mounted on a microscope slide (Fisherbrand Superfrost Plus; Fisher Scientific, Pittsburgh, PA, USA) and coverslipped using VECTASHIELD mounting medium (H-1000; Vector Laboratories, Burlingame, CA, USA).
Imaging and analysis
All images and analyses were generated by personnel who had no knowledge of the mouse genotype. Aβ images were obtained using a Zeiss Axio Imager Z1 fluorescence microscope (Carl Zeiss Microscopy, Jena, Germany) with a 10× lens objective. Mosaic images of the entire cortex and hippocampus of each animal were obtained and analyzed, and plaque burden was calculated using ImageJ software (National Institutes of Health, Bethesda, MD, USA). This was done by isolating the cortex or hippocampus, thresholding to a standard value and calculating the area occupied. Using an UltraVIEW VoX spinning disc confocal microscope (PerkinElmer, Waltham, MA, USA), hippocampal PSD-95 immunoreactive puncta were imaged with a 60× lens objective and digitally magnified to × 100. Two images were obtained in the molecular layer of the dentate gyrus with two slices from each mouse analyzed. Puncta from the dentate gyrus were analyzed and counted using ImageJ, excluding cell somata. Hippocampal calbindin D28 images were obtained using a Zeiss Axio Imager Z1 fluorescence microscope with a 20× lens objective. Mosaic images of the entire hippocampus of each animal were obtained and analyzed. All histologic analyses were done using ImageJ and analyzed statistically by Student’s t-test, or by analysis of variance (ANOVA) with post hoc comparisons as indicated, using SPSS software (IBM, Armonk, NY, USA).
Immunoblotting and enzyme-linked immunoassay experiments
Precast 10% Tris-glycine or 10–20% Tris-tricine gels were used (Bio-Rad Laboratories, Hercules, CA, USA). After transfer, the polyvinylidene fluoride membranes (catalog number 162-0174; Bio-Rad Laboratories) were incubated in blocking buffer for 1 hour at room temperature (catalog number 927-4000, Odyssey blocking buffer; LI-COR Biosciences, Lincoln, NE, USA). Membranes were then washed five times in a mixture of Tris-buffered saline and Tween 20 (TBST) and incubated overnight in primary antibodies. The following antibodies were used: clone 6E10 (MAB1560, 1:1,000 dilution; EMD Millipore), clone 22C11 (MAB348, 1:100 dilution; EMD Millipore) and actin (catalog number sc-1616, 1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA). All antibodies were diluted in Odyssey blocking buffer, and membranes were incubated overnight at 4°C. Following primary antibody incubation, the membranes were washed five times with TBST, and secondary antibodies were applied for 1 hour at room temperature (Odyssey donkey anti-mouse or anti-goat IRDye (LI-COR Biosciences) at 680 or 800 nm). Membranes were then washed, and proteins were visualized using a LI-COR Odyssey Infrared imaging system. Blots were analyzed using ImageJ and normalized to actin optical density. Total Aβ enzyme-linked immunosorbent assay (ELISA) experiments on TBS-soluble mouse brain lysates were performed according to the manufacturer’s instructions (Invitrogen).
Continuous electroencephalography video monitoring
For dural electrode implantation, the mice anesthetized and maintained with inhaled isoflurane and mounted in a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA). A midline incision was made, and two bilateral burr holes were manually drilled anterolateral and posterolateral to the bregma. Four presoldered intracranial screw electrodes (catalog number 8403; Pinnacle Technology, Lawrence, KS, USA) or a prefabricated headmount (catalog number 8201; Pinnacle Technology) was inserted and secured with a layer of dental cement (catalog number 526000; A-M Systems, Sequim, WA, USA). In the case of presoldered screw electrodes, the electrode wires were soldered onto a six-pin surface mount connector (catalog number 8235-SM; Pinnacle Technology) and secured by a final layer of dental cement. All mice were allowed to recover for 7 days prior to chronic EEG recordings.
Mice were video-recorded using an in vivo EEG video monitoring system (8200-K1-SE3, 8236; Pinnacle Systems). EEGs were sampled at 400 Hz with 100× preamplifier gain and filtered at 30 Hz. Each mouse underwent 72 hours of continuous EEG video recording and was maintained on a regular 12-hour light/12-hour dark cycle with full access to food and water. EEG traces were scored manually by an investigator, blinded to genotype, using Pinnacle Technology software. A convulsive seizure was defined as an abrupt onset of evolving SWDs lasting >30 seconds, associated with tonic-clonic activity by synchronized video analysis, and followed by postictal attenuation of cerebral EEG rhythms. A SWD was defined as a burst of sharp-wave discharges, with an amplitude of at least twice the background amplitude and 1 to 2 seconds in duration. A single spike was defined as a sharp discharge at least twice the background amplitude and <100 milliseconds in duration. SWDs were manually correlated with synchronized video analysis and scored as with or without behavioral arrest. Twenty-four-hour EEGs were manually scored for single spikes. A full 72 hours of EEG were manually screened for SWDs for each mouse, comprising 24 hours before drug delivery and 48 hours afterward. Epileptiform discharges were analyzed using Student’s t-test.
Drug administration
Each mouse received a single intraperitoneal (IP) injection of drug as indicated. All drugs were dissolved in normal saline. Each mouse underwent a 1-week washout with verification of a return to baseline SWD frequency prior to subsequent drug injection. Each mouse first received an IP injection of levetiracetam, followed by ethosuximide, phenytoin and brivaracetam. For long-term drug delivery, ethosuximide was delivered in the drinking water at a concentration of 30 mg/ml. Brivaracetam was continuously administered IP for 28 days using an osmotic minipump (ALZET Osmotic Pumps, Cupertino, CA, USA) at a rate of 8.5 mg/kg/day. Owing to the short half-life of ethosuximide in mice (1 hour) [17], periodic injections were not feasible. Minipump infusions could not be used, because the amount required exceeded the solubility of ethosuximide. Therefore, drinking water was chosen as the route of administration for ethosuximide. For brivaracetam, an osmotic minipump is the most reliable route of administration for continuous dosing.