Pp 830

Mice 6–8 week old were anesthetized using Ketamine-Xylazine (83 and 3.5 mg/kg, respectively) and placed on a heating blanket to maintain the body temperature at 37 °C. The animal was head fixed on a stereotaxic apparatus (Kopf model 940, Tujunga, CA) to perform the surgery.
The bone was exposed at the desired position through a small skin incision and small craniotomies (<0.5-mm diameter) were performed above the site of injection at (anteroposterior (AP), mediolateral (ML), depth from cortical surface (DV), in stereotaxic coordinates from Bregma): 0.7; ±0.3; −1.2 to target the ACC. Each solution of miRNA were injected with a thin glass pipette (5-000-1001-X, Drummond Scientific, Broomall, PA) pulled on a vertical puller (Narishige PP-830, Tokyo, Japan).
At the end of the surgery, mice received a dose of analgesic (Buprenorphine, s.c. 0.1 mg/kg body weight). After 4 days, mice were injected i.p. with a lethal dose of penthobarbital and intracardial injection of about 50 ml of paraformaldehyde 4% was done to collect the organs. The brains were collected in PBS 0.1 M 30% sucrose overnight at 4 °C and sliced with a microtome (Microm HM440E, section thickness: 60 μm). The slices were disposed in 12-well plates filled with 0.1 M PB for immunohistochemistry.

Whole-cell voltage clamp recordings were made from hemisectioned retinas continuously superfused in oxygenated ACSF (32–34°C) at a rate of 2–4 ml/min. Retinas were visualized under infrared illumination (870 nm). The ILM was removed using a glass recording pipette. Borosilicate glass recording pipettes (Sutter Instruments) were pulled (PP-830; Narishige) with tip resistance of 3–6 MΩ and filled with cesium gluconate internal solution containing the following: 110 mM CsMeSO₄, 2.8 mM NaCl, 20 mM HEPES, 4 mM EGTA, 5 mM TEA-Cl, 4 mM ATP-Mg, 0.3 mM GTP-Na₃, 10 mM phosphocreatine-Na₂, and 5 mM QX-314, pH adjusted to 7.2 and with an osmolarity of 290 mOsm/kg H₂O. The liquid junction potential correction for this solution was −13 mV. Voltage-clamp recordings were obtained from somas of RGCs (holding potential of −60 mV) as 10-min gap free recording using pCLAMP10 recording software and a Multiclamp 700A amplifier (Molecular Devices), sampled at 20 kHz and low-pass filtered at 2 kHz. For current clamp experiments, internal solution contained the following: 116 mM K⁺ D-gluconate, 6 mM KCl, 2 mM NaCl, 20 mM HEPES, 0.5 mM EGTA, 4 mM ATP-Mg²⁺, 0.3 mM GTP-Na₃, and 10 mM phosphocreatine-Na₂.

Transmembrane action potentials and ion currents were recorded from isolated myocytes using a patch clamp amplifier (EPC-10, Heka Electronic, Lambrecht, Pfalz, Germany) with a current or voltage clamp technique, respectively. All experiments were performed at 37±0.5°C and bath solutions were bubbled with 95% O₂ and 5% CO₂. Before patch clamp recording, myocytes were transferred into a recording chamber mounted on an inverted microscope stage (Nikon TE2000-S) and allowed to adhere to the glass bottom of the chamber for 5 min. A motorized micromanipulator (MP285, Sutter, USA) propelled the recording electrode to the chosen cells. Membranes were then sealed and ruptured with negative pressure. Sealing resistance was maintained >1GΩ. A 80% compensation of series resistance was achieved without ringing. Capacitance and series resistances were adjusted to minimize the contribution of the capacitive transients. Patch electrodes were pulled with a two-stage patch pipette puller (PP-830, Narishige Group, Tokyo, Japan) and then fire-polished. Patch electrodes had a resistance of 1.8–2.5 MΩ when filled with pipette solution. Current signals were conducted by the Ag/AgCl electrode and amplified by an EPC-10 amplifier, then filtered at 1.5 kHz, digitized at 10 kHz, and stored on a computer hard disk for analysis.

Ciliary membrane currents were recorded from single ORCs with a WC recording configuration (Hamill et al., 1981 (link)) under the voltage-clamp mode (V_h = −54 mV) as previously described (Takeuchi and Kurahashi, 2002 (link); 2019 (link)). The culture dish was mounted on the stage of laser scanning microscopy (LSM, Axiovert 510 system; Carl Zeiss Microimaging GmbH). Patch pipettes (resistance, 10–15 MΩ) were made from borosilicate tubing with filaments (outer diameter, 1.2 mm; World Precision Instruments) using a two-stage vertical patch electrode puller (PP-830; Narishige). The recording pipette was filled with Cs⁺ solution containing (in mM) 119 CsCl, 1 CaCl₂, 5 EGTA, 10 HEPES, and 0.001% phenol red (pH 7.4 adjusted using CsOH), as well as 1 mM caged cAMP (catalog number 116810; Calbiochem; Merck Millipore) and 50 µM Fluo-4 (F14200; Invitrogen; Thermo Fisher Scientific). Current signals were I-V converted using a 200B amplifier (Molecular Devices LLC), and data were sampled using pCLAMP ver.10 (Molecular Devices LLC) at 10 kHz, after being low-pass filtered at 2 kHz. For curve drawings of the membrane current, some data were low-pass FFT-filtered at 0.02 kHz. Care was taken to avoid saturation of response, particularly when evaluating adaptation. All experiments were performed at room temperature (23–25°C).