C57BL/6 mice were used to generate mice carrying a Ube3a mutation at Nagasaki University and were shipped to Hokkaido University Graduate School of Medicine or Hamamatsu University School of Medicine for use in all experimental procedures. Ube3am−/p+ mice were obtained by crossing a heterogeneous female mouse lacking paternal Ube3a with a wild-type (WT) male mouse. Genotyping was performed using polymerase chain reaction of mouse tail DNA, as described previously. After weaning, 3–5 mice were housed in ventilated cages with water and feed provided ad libitum. Mice were housed uner a 12-h light/ 12-h dark cycle (lights on at 7:00 a.m.), with temperature and humidity maintained at 23–25°C and 45–55%, respectively. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the Hamamatsu University School of Medicine and Hokkaido University, and followed the National Institutes of Health guidelines and ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines for the care and use of laboratory animals.
The hippocampal regions were dissected from the collected brains and homogenized in ice-cold lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid, and 1% Triton X-100) containing protease inhibitors (Roche, #1697498). The samples were centrifuged for 10 min at 12,000 × g at 4°C, and the supernatants were mixed with Laemmli sample buffer and heated at 100°C for 5 min. The protein concentrations of the samples were determined using the Bio-Rad DC protein assay (Bio-Rad). Equal amounts of proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (7.5% acrylamide gel) and transferred to polyvinylidene fluoride membranes. The membranes were then blocked in 1% bovine serum albumin and incubated overnight with antibodies against KCC2 (1:1,000; Millipore, #07-432), NKCC1 (1:1,000; Millipore, #MABS1237), and β-actin (1:5,000; Sigma-Aldrich, #A5441) at 4°C. The blots were then incubated with a horseradish peroxidase-conjugated secondary antibody (GE Healthcare) for 1 h at room temperature (23–25°C). Bands were visualized with ECL prime or ECL select western blot detection reagent (Cytiva) and imaged using a ChemiDoc MP imaging system (Bio-Rad). Quantification was performed using Image Lab software (version 6.0, Bio-Rad). The KCC2 and NKCC1 band intensities were normalized to β-actin band intensity.
Experiments were performed on acute hippocampal slices prepared from P25–28 Ube3am−/p+ or WT littermate mice. Both male and female mice were included in the electrophysiological experiments. We recorded each genotype on consecutive days, whenever possible. Mice were killed by decapitation under deep anesthesia using halothane or isoflurane, and brain coronal slices containing the hippocampus (350 µm thick) were cut on a microslicer (VT-1000S, Leica Microsystems; or VF-300-0Z, Precisionary) in ice-cold modified artificial cerebrospinal fluid (ACSF) containing (in mM): 220 sucrose, 2.5 KCl, 1.25 NaH2PO4, 12.0 Mg2SO4, 0.5 CaCl2, 26.0 NaHCO3, and 30.0 glucose, pH 7.4 when gassed with 95% O2/5% CO2. The slices were kept in standard ACSF solution consisting of (in mM) 126 NaCl, 2.5 KCl, 1.25 NaH2PO4, 2.0 MgSO4, 2.0 CaCl2, 26.0 NaHCO3, and 20.0 glucose, pH 7.4 when gassed with 95% O2/5% CO2, at room temperature for over 1 h before experiments.
Slices were then transferred to a recording chamber on the stage of a microscope (BX61, Olympus, or Axioskop2, Zeiss) and continuously perfused with oxygenated ACSF at a flow rate of 2 ml/min at 30°C. CA1 pyramidal neurons were visually identified on a monitor using a 40× water immersion objective lens with an infrared differential interference contrast filter. The patch electrodes were pulled from borosilicate capillary tubing with a 1.5 mm diameter (GD-1.5; Narishige) with a horizontal puller P-97 (Sutter Instruments). The electrode resistance ranged from 4 to 6 MΩ for conventional whole-cell patch-clamp recordings and from 3 to 5 MΩ for gramicidin-perforated patch-clamp recordings. Signals were recorded using a MultiClamp 700 B amplifier or Axopatch 200 B (Molecular Devices), low-pass filtered at 2 kHz, and digitized at 6–10 kHz using a Digidata 1332A data acquisition system (Molecular Devices).
Gramicidin perforated patch-clamp recordings were performed as previously described. The pipette solution contained (150 mM): 150 KCl and 10 mM HEPES (pH 7.3, KOH). Gramicidin was dissolved in DMSO (10 mg/ml) and then diluted in the pipette-filling solution to a final concentration of 5–10 µg/ml immediately prior to the experiments. The cells were voltage clamped and stepped into various test potentials. GABA (50 µM) was applied for 10 ms through a patch pipette to the soma of the recorded neuron at each membrane potential. To obtain I–V curves from gramicidin recordings, the membrane potential values were corrected for the voltage drop across the series resistance: Vcorr = Vcom − Iclamp × Rs, where Vcom is the command potential, Iclamp is the clamp current, and Rs is the series resistance. To determine the reversal potential for the GABA-induced current (EGABA), these values were plotted as a function of the series resistance-corrected membrane potential. [Cl−]i was calculated from the determined EGABA according to the Nernst equation.
To evaluate the GABAA receptor-mediated currents, whole-cell voltage-clamp recordings were performed under the presence of 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX; 20 µM), D-(-)-2-Amino-5-phosphonopentanoic acid (D-AP5; 50 µM), and CGP55845 (3 µM). Recorded neurons were voltage-clamped at a holding potential of − 60 mV using a pipette solution consisting of (in mM) 130 CsCl, 1 mM CaCl2, 2 MgCl2,10 HEPES-NaOH, 0.5 EGTA-KOH, 1.5 Mg-ATP, 0.5 mM Na2-GTP, and 2.5 QX314 (pH 7.3). Tonic GABA currents were evaluated as the difference in the mean baseline current devoid of synaptic events (total 3 s) during and before the application of SR95531 (10 µM). To evaluate excitatory postsynaptic currents, whole-cell voltage-clamp recordings were performed in the presence of SR95531 (10 µM) and CGP55845 (3 µM). The voltage was clamped at − 60 mV with the pipette solution consisting of (in mM): 150 K-CH3SO3, 5 KCl, 3 MgCl2, 10 HEPES-NaOH, 0.5 EGTA-KOH, 3 Mg-ATP, 0.4 Na2-GTP (pH 7.3). Resting membrane potentials were recorded using the same pipette solution. The reported values were corrected for liquid junction potentials of + 10.5 mV.
For recordings of miniature inhibitory currents (mIPSCs) or excitatory postsynaptic currents (mEPSCs), tetrodotoxin (1 µM) was additionally applied to the perfusion solution. Individual mIPSCs and mEPSCs were visually identified from 5-min current traces to analyze their frequency, peak amplitude, 10–90% rise time, and decay time using Mini Analysis (Synaptosoft, NJ). The data obtained from each event were averaged. For the analysis of paired-pulse ratios, two consecutive inhibitory or excitatory postsynaptic currents were evoked by electrical stimulation (interval, 50 ms; duration, 200 µs; intensity, 100–400 pA) using a monopolar glass pipette filled with ACSF. The ratios of the peak current amplitudes between the second and first evoked postsynaptic currents were determined.
4.4. Long-term administration of bumetanide
We used micro-osmotic pumps (Model, 2004, Alzet, USA) to allow continuous administration of bumetanide. Pumps were filled with 25.6 mg bumetanide in 200 µl 70% PEG/30% DMSO to deliver approximately 0.8 mg kg− 1 h− 1 for up to 28 days and subcutaneously implanted under anesthesia. All subsequent experiments were performed between 21 and 28 days after implantation.
4.5. Behavioral analysis
All behavioral analyses were conducted during the light cycle using 6-to 8-month-old male mice. Prior to each test, mice were habituated to the testing room for at least 60 min. For the novel object recognition and open field tests, a video tracking system (ANY-maze; Stoelting Co., USA) was used to capture the procedures.
The task procedure of the novel object recognition test consists of three phases: habituation, familiarization, and test. Each mouse was allowed to explore the empty arena (40 × 40 × 40 cm) freely for 5 min, which was followed by exploration of two identical sample objects for 5 min. After a retention interval of 30 min, the mice were returned to the arena for 5 min during the test phase. In this phase, one of the two samples is replaced with a novel object. The number of times a mouse showed exploratory behavior (direct contact or sniffing toward an object within less than 2 cm of the object) was manually counted offline by an examiner who was blinded to the subjects. The discrimination index (DI) was calculated using the following equation:
DI = (NN − NF) / (NN + NF),
where NN and NF represent the number of exploration times for the novel and familiar objects, respectively.
Motor function was analyzed using an accelerating rotarod (4–40 rpm for 5 min; model MK-670, Muromachi, Japan). The mice were trained to stay on the rod at a constant speed (5 rpm for 5 min) prior to data acquisition. Four trials per day were conducted for two consecutive days at 30 min interval. The time spent on the rotarod or the time until the mouse made three consecutive rotations on the rotarod was used in the subsequent statistical analysis.
For the open field test, each mouse was placed in the center of an empty arena (40 × 40 × 40 cm) and allowed to explore freely for 30 min. We analyzed the total distance traveled as well as the relative distance traveled in the center region (25 cm × 25 cm in the middle of the arena) in relation to the total distance traveled.
4.6. Flurothyl inhalation-induced seizures
Seizure susceptibility of 6- to 8-month-old male mice was evaluated by an acute seizure induction paradigm using flurothyl inhalation, as reported previously. Mice were placed in an airtight acrylic cylinder chamber (diameter, 14 cm; height, 20 cm; Asone 1-073-01) 1 min before starting flurothyl administration. A 10% fluorothyl (bis [2,2,2-trifluoroethyl] ether; Sigma-Aldrich) solution was dispensed with 95% ethanol and infused into a gauze pad (3 × 3 cm) at a flow rate of 200 µL/min using a syringe pump suspended 5 cm below the ceiling. We recorded the latency from the beginning of administration to the first presentation of 1) a myoclonic seizure: a sudden, brief muscle contraction of the neck and body, and 2) tonic seizures: sustained loss of posture control (> 2 s) accompanied by trunk rigidity. The experiment was performed in a ventilated safety cabinet to avoid exposing the examiner to a gas containing fluorothyl.
4.7. Electroencephalography (EEG) recording and analysis
Cortical EEGs were recorded using stainless steel screw electrodes (Plastic One, USA) in 6–9-month-old male mice. Cortical recording electrodes (A/P: −3.0 mm, ML: ±3.0 mm, relative to bregma), a reference electrode over the cerebellum, and a grand electrode anterior to bregma were implanted subdurally under anesthesia with isoflurane. All electrodes were inserted into a 6-channel pedestal and connected to a commutator for recording. Mice were allowed at least 7 days of recovery from surgery before recording.
Simultaneous video-EEG recording was performed for 24 h. EEG signals were amplified by a differential AC amplifier (Model 1700, A-M Systems, USA), bandpass-filtered between 0.1 Hz and 500 kHz, and digitized at 2,000 Hz (MP170 and AcqKnowlegde software; Biopack Systems, USA) for storage on a PC. EEG spikes were automatically detected by threshold-based event detection using the Clampfit 10 software (Molecular Devices, USA). The threshold amplitude was set to four times the standard deviation of the baseline EEG activity during non-REM sleep. Waveforms over 200 ms in duration were rejected as artifacts. Events including three consecutive spike trains within a 200-ms interval were counted as epileptic discharges.
Spectral analysis was performed using Darbeliai, a plug-in of EEGLAB running MATLAB (MathWorks, Inc., USA). After visually inspecting the EEG traces and excluding artifacts, 10 EEG epochs (duration 9 s) during the awake state were collected for power spectrum analysis. We calculated the relative EEG powers of the δ (1–4 Hz), θ (4–9 Hz), α (9–13 Hz), and β (13–30 Hz) frequency bands for each epoch. The EEG power of each mouse was determined by averaging over 10 epochs.
4.8. Statistical analysis
Differences in immunoblotting, patch-clamp recording, and EEG data between genotypes were determined using an unpaired t-test. Welch’s correction was applied when the F-test result for the comparison of variance was significant. Alterations in EEG data before and after bumetanide application in Ube3am−/p+ mice were determined using a paired t-test. The effects of bumetanide on behavioral analyses and seizure susceptibility were analyzed using two-way analysis of variance (ANOVA) (for all except the rotarod test) or three-way repeated-measures ANOVA (for the rotarod test) followed by Tukey’s post-hoc analysis. All statistical analyses were performed using Prism 9 (GraphPad Software), and statistical significance was set at P < 0.05.