Animals
All experiment procedures for the use and care of all mice were approved by and performed according to the guidelines of the Animal Care and Use Committee of Peking University (Beijing, China). The mice were housed on a 12-h light–dark cycle with food and water ad libitum. The Opcml−/− mice were previously generated by us [25]. For electrophysiological experiments, the sequence in which animals conduct the experiments or be sacrificed is random. Meanwhile, the mice were blinded to the group allocation for the electrophysiological experiments.
Brain slice preparation
Coronary hippocampus slices (250 μm) of mice were prepared and processed as described previously [26]. Briefly, mice were anesthetized with isoflurane and decapitated. The brain was quickly removed and sliced on a vibratome (Leica VT1200s, Wetzlar, Germany) in ice-cold cutting solution consisting of (in mM) 213 sucrose, 3 KCl, 26 NaHCO3, 1 NaH2PO4, 5 MgCl2, 0.5 CaCl2, and 10 glucose (adjusted to pH 7.3–7.4, 300–310 mOsm, saturated with 95% O2 and 5% CO2). The slices were incubated for 30 min at 37 °C, maintained at room temperature (24–25 °C) in a solution consisting of (in mM) 125 NaCl, 5 KCl, 26 NaHCO3, 2 NaH2PO4, 1.3 MgCl2, 2.6 CaCl2, and 10 glucose, bubbled with 95% O2/5% CO2 and recovered for 30 min before recording.
Whole-cell patch-clamp recordings
Whole-cell patch-clamp recordings were performed in pyramidal neurons (PNs) from the CA1 regions by using 3–5 MΩ borosilicate glass pipettes (World Precision Instruments, 1B150F-4) pulled with a Brown-Flaming micropipette puller P-97 (Sutter Instruments Company). The recording pipettes were filled with the internal solution consisting of 145 mM KCl, 5 mM NaCl, 10 mM HEPES, 5 mM EGTA, 4 mM Mg-ATP, and 0.3 mM Na2-GTP (pH 7.25 with KOH, 300 to 310 mOsm) for miniature excitatory postsynaptic/inhibitory currents (mEPSCs/mIPSCs) recording, and with additional 5 QX-314 for action potential recording. The cesium (Cs) based internal solution consisting of 120 Cs-methanesulfonate, 2.8 NaCl, 5 TEA-Cl, 0.4 EGTA, 20 HEPES, 2.5 Mg-ATP, 0.25 Na-GTP (pH 7.25 with CsOH, 300 to 310 mOsm) was used for evoked EPSCs (eEPSCs) and AMPAR/NMDAR-mediated EPSCs recordings.
Under voltage-clamp mode, mEPSCs were recorded at a holding potential of − 60 mV in the presence of tetrodotoxin (TTX, 1 μM) and picrotoxin (PTX, 100 μM). Evoked EPSCs (eEPSCs) were triggered with a concentric stimulation electrode placed in the Schaffer collateral projection to CA1 with a stimulus intensity of 30 μA in the presence of PTX (100 μM). The AMPAR-mediated EPSCs were recorded in voltage-clamp mode holding at − 70 mV in the presence of PTX (100 μM) and D-2-amino-5-phosphonovalerate (D-AP5, 50 μM). Then the cell was switched to + 40 mV to evoke NMDAR-mediated EPSCs in the presence of PTX (100 μM) and cyanquixaline (CNQX, 10 μM). The NMDA/AMPA ratio was calculated by dividing the peak amplitude of the NMDA EPSC by the AMPA EPSC. Under voltage-clamp mode, mIPSCs were recorded at a holding potential of − 60 mV in the presence of TTX (1 μM), CNQX (10 μM) and D-AP5 (50 μM). The amplitude and frequency of mEPSC/mIPSC were determined for subsequent analysis. The amplitude, rise slope and decay slope of eEPSCs were calculated.
The data were digitized at 10 kHz with a 2.9 kHz low-pass filter. Recorded cells with series resistances of > 25 MΩ were rejected. All data were recorded using MultiClamp 700B amplifier and pCLAMP 10.6 software (Molecular Devices).
Determination of passive and active electrophysiological properties
The membrane capacitance (Cm) and membrane time constant (τm) were measured after the initial break-in from giga-seal to the whole-cell configuration in voltage clamp mode. Once the Cm and τm were determined, the amplifier configuration was switched to current-clamp mode for action potential recording. For action potential recording, cells were held at − 70 mV to prevent spontaneous spike activity. Spikes were evoked by a series of current injection from -50 to +400 pA for 400 ms duration in 50-pA increments. The action potential kinetic properties were determined as previously reported [27]. The rheobase current required to elicit an action potential was measured. The amplitude of action potential was measured from the threshold to the peak. The after hyperpolarization (AHP) size was measured from AP threshold to the negative peak of the AHP. The AP widths were measured at halfwidth. The voltage threshold was measured in the first derivative of AP (dV/dt) considering the point where the velocity was close to 20 V/s. The interspike interval (ISI) was measured the duration between the first and the second action potential. A hyperpolarizing current pulse (400 ms) at − 50 pA was delivered to measure membrane input resistance (Rin).
Chemicals and drugs
All chemicals and drugs used in this study including sucrose (V900116), KCl (P3911), NaHCO3 (S5761), NaH2PO4 (S9638), MgCl2 (M9272), CaCl2 (C1016), glucose (G8270), NaCl (S7653), HEPES (H3375), EGTA (03777), Mg-ATP (A9187), Na2-GTP (51120), CNQX (C127), D-AP5 (A8054) and aripiprazole (PHR1784) were purchased from Sigma Aldrich except for PTX (C0375) was purchased from TGI and TTX (abs44200985a) was purchased from Absin.
Drug administration
In experiments to test aripiprazole effect on synaptic transmission, aripiprazole was diluted into 0.625 mg/mL using 5% (vol/vol) Tween 80/physiological saline. We weighed the mice and then gave aripiprazole administration (i.p., 2.5 mg/kg) according to the body weight while with the same volume of 5% (vol/vol) Tween 80/physiological saline as the vehicle group. Then we decapitated the mice and cut coronary hippocampus slices for later whole-cell recording after aripiprazole acute administration 30 min. The sequence of aripiprazole or vehicle administration, the brain slices preparation and patch clamp recording in wildtype and Opcml-deficient mice are random and blind, to avoid the random error of the brain slice quality and drug metabolism due to sequential order.
Data analysis
Data were analyzed by Clampfit 10.6 (Molecular Devices) and Igor Pro 6.22 (WaveMetrics) software. All values are presented as mean ± SEM. Statistical comparisons were performed by GraphPad Prism 8.0.2 (GraphPad Software). Differences of P < 0.05 were considered significant (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
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