Tc 344b

The external recording solution (145 mM NaCl, 2.5 mM KCl, 2 mM BaCl₂, 1 mM MgCl₂, 10 mM HEPES, and 11 mM d-glucose) was adjusted to pH 7.4 with NaOH. Patch electrodes were fabricated from borosilicate glass capillaries using an electrode puller (P97; Sutter Instrument Co., Novato, CA). The tip resistances of patch electrodes were 2.4 to 4.6 MΩ when filled with an internal solution (135 mM K-gluconate, 5 mM KCl, 10 mM HEPES, 1.1 mM EGTA, 2 mM MgCl₂, 3 mM ATP-Mg, and 0.3 mM GTP-Tris adjusted to pH 7.2 with KOH). In the whole-cell configuration, membrane potentials were recorded with an EPC-10 amplifier and PatchMaster software (HEKA, Freiburg, Germany) and digitized at 10 kHz with PowerLab and LabChart software (AD Instruments, Colorado Springs, CO). Dorsal root ganglion neurons sufficiently separated from other culture cells were selected under a microscope. All patch-clamp experiments were performed at room temperature (25°C) or 37°C. The chamber temperature was controlled with a heater controller (TC-344B; Warner Instruments, Hamden, CT) and monitored using a temperature sensor probe. GSK2193874 (1 μM) was bath applied for 1 to 5 minutes.

The method of measuring [Ca²⁺]_i was present previously [22] (link). Briefly, [Ca²⁺]_i was determined with Fura-2 AM (Invitrogen) fluorescence emitted at 510 nm after excitation at 340 and 380 nm every 12 sec intervals. PASMCs loaded with 7.5 µM Fura-2 AM were incubated for 60 min at 37°C, then mounted in a closed polycarbonate chamber and perfused with Krebs-Ringer bicarbonate solution for 10 min (KRBS, 1 ml/min), which consisted of (in mM) 118 NaCl, 4.7 KCl, 2.5 CaCl₂, 0.57 MgSO₂, 1.18 KH₂PO₄, 25 NaHCO₃, and 10 glucose. Changes in [Ca²⁺]_i were assessed with a Nikon TSE 100 Ellipse inverted microscope (Nikon, Melville, NY). Chamber temperature was maintained at 37°C with an in-line heat exchanger and dual-channel heater controller (models SF-28 and TC-344B, Warner Instruments). Data on changes in F340/F380 (Fura-2) were analyzed with InCyte software (Intracellular Imaging, Cincinnati, OH). [Ca²⁺]_i is presented as an average from 20–30 cells.

Mice were euthanized by cervical dislocation, and whole retinas were removed under long-wavelength illumination (630 nm, 800 μW/cm², FND/FG, Ushio, Cypress, CA). To aid in the removal of vitreous membrane from the retinal surface, we incubated retinas in a solution containing collagenase (LS005273, Worthington Biochemical, Lakewood, NJ), hyaluronidase (LS002592, Worthington Biochemical, Lakewood, NJ), and carbogen-saturated Ames’ medium (US Biologic, Memphis, TN) for 10 mins on a rocker as described by [44 ]. Afterwards, retinas were rinsed within fresh Ames’ medium and placed into physiological chamber mounted on an upright microscope (BX50, Olympus, Center Valley, PA). Retinas were maintained in the dark at 32 °C (TC-344B; Warner Instruments), and constantly perfused (2 mL/min) with carbogen-saturated NaHCO₃-buffered (22.6 mM) Ames’ medium plus 20 mM glucose (Osm 290, pH 7.4). For RGC intracellular filling and patch-clamp recordings, we used fire-polished borosilicate glass pipettes containing (in mM) 125 K-gluconate,10 KCl, 10 HEPES, 10 EGTA, 4 Mg-ATP, 1 Na-GTP, and 0.1 ALEXA 555 (Invitrogen, Carlsbad CA; Osm 285, pH 7.35). RGC light responses were evoked using full-field light flashes generated by a light-emitting diode (365 nm, 300 μW/cm2, 3-s duration; Roithner Lasertechnik) delivered through a shutter in the microscope condenser [29 (link), 34 (link)].