Microforge mf 830

The apical-middle part of cochleae was acutely isolated and immobilized under a cross of dental floss. Whole cell recordings were carried out with an electrophysiology amplifier (HEKA, EPC-10 USB) running the PatchMaster software. Borosilicate glass filaments (Sutter) were made with a pipette puller (Sutter, P-2000) and polished with an MF-830 microforge (Narishige) to resistances of 3–5 MOhm. The recording solution contained 144 mM NaCl, 0.7 mM NaH₂PO4, 5.8 mM KCl, 1.3 mM CaCl₂, 0.9 mM MgCl₂, 5.6 mM glucose and 10 mM H-HEPES (pH 7.4). The pipette solution contained 140 mM KCl, 1 mM MgCl₂, 0.1 mM EGTA, 2 mM MgATP, 0.3 mM Na₂GTP and 10 mM H-HEPES, pH7.2. The MET currents were evoked using a fluid jet from a pipette (tip diameter of 5–10 μm). The pipette tip delivering the fluid jet was positioned by facing the staircase side of the hair bundle at a distance of around 5 μm to elicit saturating MET currents. The 40 Hz sinusoidal force stimuli were generated by a 27-mm-diameter piezoelectric disc controlled by the HEKA amplifier and driven by a homemade piezo amplifier.

Borosilicate round
capillaries (World Precision Instruments, U.K.) with ID/OD 1.56/2.0
and 0.58/1.0 mm were used as outer and inner capillaries, respectively.
The inner capillary tube was pulled from the middle using a P-97 micropipette
puller (Sutter Instruments) and separated into two capillaries, i.e.,
injection capillary and collection capillary. The tips of both capillaries
were adjusted to the desired orifice diameter by grazing over a sandpaper.
The orifice diameters were measured using an MF-830 Microforge (Narishige).

Induced (evoked) AMPAR-mediated synaptic responses were recorded from paired hippocampal neurons (holding potential −70mV) using the whole-cell patch-clamp technique. Neurons were stimulated with 10 ms depolarization pulse from a holding potential of −70 to 60 mV. Patch pipettes were pulled from borosilicate glass (Harvard Apparatus), fire-polished (Microforge MF-830; Narishige), and had resistances of 2.5–4 MΩ when filled with the following (in mm): 130 K-gluconate, 1 MgCl₂, 10 HEPES, 5 EGTA, 4 Mg-ATP, and 0.3 Na-GTP, pH 7.2 with KOH. The bath solution contained the following (in mm): 137 NaCl, 3 KCl, 10 HEPES, 2 MgCl₂, 1.8 CaCl₂, 0.05 DL-AP5, and 10 glucose, pH 7.4 with NaOH. Currents were recorded with an EPC 10 amplifier controlled by PatchMaster software (HEKA Elektronik Dr. Schulze). To test synaptic plasticity (or paired-pulse plasticity), we applied a pair of pulses with varying interpulse intervals (10, 25, 50, 100, 250, or 500 ms) and calculated the paired-pulse amplitude ratio (P2/P1).
To test spontaneous activity of mixed cortical-MSN cultures, TTX (1 μm) and bicuculline (10 μm) or CNQX (10 μm) were added to bath solution and miniature EPSCs (mEPSCs; holding potential −70 mV) or miniature IPSCs (mIPSCs; holding potential 0 mV), respectively, were recorded.

A horizontal Flaming/Brown micropipette puller (P-97; Sutter Instruments, CA) was used to prepare injection capillaries with a tip resistance of 3 to 4 MΩ. Tips of capillars were then polished with Micro Forge MF-830 (Narishige, Japan) to reach a 10- to 30-μm tip diameter for cRNA injection. Capillars were then backfilled with sterile mineral oil and filled with 3 to 5 μl of cRNA solution. Forty nanograms of cRNA (41.4 to 50.6 nl) per oocyte was injected using the Nanoject II system (Drummond Scientific, CO) and incubated for 72 h at 19°C. Control oocytes were injected with same amount of diethylpyrocarbonate (DEPC) water.