Pp 830 puller

Shortly before the electrical recordings, cells (e.g., GH₃, A7r5, or H9c2 cells) were harvested and transferred to a home-made recording chamber positioned on the stage of an inverted microscope. Cells were immersed at room temperature (22–25 °C) in normal Tyrode’s solution, the composition of which was described above. Patch-clamp recordings under the whole-cell mode were achieved with either an RK-400 (Bio-Logic, Claix, France) or an Axopatch-200B amplifier (Molecular Devices, Sunnyvale, CA) (Wu et al., 2000 (link); Wu et al., 2017 (link)). Patch electrodes with tip resistances of 3–5 MΩ were made of Kimax-51 borosilicate capillaries (#34500; Kimble, Vineland, NJ) on either a PP-830 puller (Narishige, Tokyo, Japan) or a P-97 horizontal puller (Sutter, Novato, CA), and then fire-polished with an MF-83 microforge (Narishige). The signals, comprising voltage and current tracings, were stored online at 10 kHz in an ASUSPRO-BU401LG computer (ASUS, Taipei City, Taiwan) controlled by pCLAMP 10.7 software (Molecular Devices). Changes in membrane potential recorded from GH₃ cells were measured under current-clamp configuration. In a separate set of whole-cell I_K(DR) recordings with intracellular dialysis, the recording pipettes used were filled with the internal solution containing 0.3 µM CFZ.

Shortly before the experiments, the cells (i.e., GH₃ cells and mHippoE-14 neurons) were dissociated, and an aliquot of cell suspension was transferred to a home-made recording chamber that was mounted on the stage of a DM-IL inverted microscope (Leica, Wetzlar, Germany), and then left to settle. The cells were immersed at room temperature (20–25 °C) in HEPES-buffered normal Tyrode’s solution, the composition of which is described above. For the recordings, patch electrodes were fabricated from borosilicate glass capillaries (#34500; Kimble Products, Vineland, NJ, USA) on a Narishige PP-830 puller (Narishige, Tokyo, Japan), then fire-polished with an MF-83 microforge (Narishige). The resistances existing between the standard pipette and the bathing solution ranged between 3 and 5 MΩ. Recordings of different types of ionic currents were measured in the whole-cell mode using the standard patch-clamp technique with either an RK-400 (Bio-Logic, Claix, France) or an Axopatch 200B patch-clamp amplifier (Molecular Devices, Sunnyvale, CA, USA) [32 (link)], which was interfaced via a Digidata 1440A to a PC running pCLAMP suite software (Molecular Devices). The liquid junction potentials were corrected shortly before seal formation was established.

Whole-cell voltage clamp recording was performed at room temperature to measure K⁺-uptake capacity. Inward currents were measured using an EPC-8 amplifier and Patch master software (Both from HEKA Instruments, Germany). The patch pipettes (World Precision Instruments, Inc.) were constructed using a PP-830 puller (Narishige, Tokyo). When filled with pipette solution, the resistance of the pipettes was 3–6 MΩ (always less than 10 MΩ). The pipette capacitance was compensated after the formation of a giga seal. The recording chamber was continuously superfused (1–2 ml/min). Data were low-pass filtered at 2 kHz and acquired using the Patch master program. The standard bath solution contained 170 mM K-gluconate, 1 mM CaCl₂, 2.5 mM MgCl₂, and 10 mM HEPES/Tris, pH 6.8. The pipette solution contained 150 mM K-gluconate, 20 mM KCl, 1 mM CaCl₂, 10 mM HEPES, 10 mM EGTA and 4 mM Mg-ATP/Tris, pH 7.4. The protocol for recording potassium channel currents consisted of stepping the membrane potential from a holding potential of -20 mV to the test potential (ranging from -140 to +40 mV) for 1 s in 20 mV increments at 2 s intervals. To minimize changes in offset potentials during bath solution exchanges, 3 M-KCl agar salt bridges were used for the reference electrode. Data are presented as means ± standard error (SEM).

Cerebellar slices were prepared as previously described^{73 (link),74 (link)}. Briefly, P18 to P110 male mice were decapitated and the cerebellum was isolated. Sagittal slices (300 µm) were cut from the cerebellar vermis using a vibratome (Leica VT1200) in an ice-cold slicing solution (containing in mM: 81.2 NaCl, 2.4 KCl, 23.4 NaHCO₃, 1.4 NaH₂PO₄, 6.7 MgCl₂, 0.5 CaCl₂, 23.3 glucose, 69.9 sucrose, pH 7.4). Slices were then maintained in aCSF (in mM: 125 NaCl, 2.5 KCl, 26 NaHCO₃, 1.25 NaH₂PO₄, 1 MgCl₂, 2 CaCl₂, 25 glucose, pH 7.4) saturated with 95% O₂, 5% CO₂ at room temperature for at least 30 min before recording. All experiments were carried out at near physiological temperature (33–37 °C). Unless otherwise noted, all recordings were obtained in lobules V and VI of the cerebellar vermis with patch pipettes (3–6 MOhm for Purkinje cells, 5–10 MOhm for interneurons) pulled from borosilicate capillary glass (Harvard Apparatus, Holliston, MA) with a Narishige PP-830 puller. EPSCs and IPSCs were recorded in the presence of GABA_A-R blockers (100 µM PTX + 5 µM SR-95531), and a non-NMDAR inhibitor (5 µM NBQX), respectively. TTX (0.5 µM) was included during recordings of miniature events. Analog signals were filtered at 6 kHz and digitized at 20 kHz (Multiclamp 700A, Axon Instruments). Data were analyzed using Clampex 10.2.0.12 (Axon Instruments).

Viruses were front filled into a glass pipette (NanoW) filled with mineral oil (Millipore Sigma, M3516) and connected to a nanoinjector (Neurostar, Glass capillary Nanoinjector). The glass pipettes were pulled to obtain a tip diameter of ~50–80 µm (Narishige, PP-830 puller). Viruses were infused into target regions at 50 nl/min, and the pipette was kept in place for an additional 8 min, then slowly withdrawn (dorsoventral speed 0.1 mm/s, entry and exit). Mice were given postoperative buprenorphine (0.1 mg/kg, subcutaneous) and monitored for 3 consecutive days.

Immediately before each experiment, cells were transferred to a chamber positioned on the stage of an inverted microscope (Leica). The glass pipettes were fabricated from Kimax-51 capillaries (Kimble Glass, Vineland, NJ, USA) using a PP-830 puller (Narishige, Japan) and the tips were fire-polished with an MF-83 microforge (Narishige). When filled with a pipette solution, their resistance ranged between 3 and 5 MΩ. Ionic currents were recorded in different patch-clamp configurations (i.e., cell-attached, outside-out, and whole-cell variants), with the use of an RK-400 amplifier (Bio-Logic, Claix, France) controlled by the ClampEx program (Molecular Devices, Sunnyvale, CA, USA). To measure I_K(Ca), cells were bathed in a normal Tyrode solution with 1.8 mM CaCl₂ at room temperature. Each cell was held at 0 mV to inactivate other non-specific voltage-gated K⁺ current. The method elicits a family of large, noisy, and outward currents with rectification. When external Ca²⁺ was removed, the current amplitude was reduced and the residue current was specifically identified as I_K(Ca).