Carbenoxolone

Rat and mouse optic nerve head astrocytes were subcultured onto plastic 6-well plates and grown until confluent. Cells were incubated in control isotonic solution containing (in mM) 105 NaCl, 5 KCl, 4 NaHEPES, 6 HEPES acid, 1.3 CaCl₂, 5 glucose, 5 NaHCO₃ and 60 mannitol, pH 7.4 or in solution made 30% hypotonic solution by addition of dH₂0, for 4 h in the tissue culture incubator. Cells were pretreated with inhibitors Bay 11-7082 (4 μM), Brilliant blue G (BBG, 10 μM), A839977 (50 nM, Tocris Bioscience), A740003 (5 μM, Tocris Bioscience), carbenoxolone (10 μM, #C4790), probenecid (1 mM, #P8761) or ¹⁰Panx1 and scrambled peptide (100 μM, #3348 and #3708, respectively, Tocris Bioscience) for 1 h before adding test solutions. RNA was extracted immediately after the 4 h treatment.

For this study, we used chemical treatments of HMR1556 20 μM (Sigma-Aldrich), Thapsigargin 10 μM (Sigma-Aldrich), Apyrase 10U (Sigma-Aldirch), Carbenoxolone 100 μM (Tocris), GsMTx4 1 μM (Tocris), Yoda1 100 μM (Tocris), and Jedi2 40 μM (Tocris). Concentrations were based on previous work and from testing a variety of concentrations of Piezo1 agonists (S6 Fig). HMR1556 was stored at 10 mM concentration at −20°C. Thapsigargin was stored at 10 mM concentration at −20°C. Apyrase was stored at 100U in −20°C. GsMTx4 was stored at 1 mM at −20°C. Yoda1 was stored at 10 mM at 4°C in 100% DMSO. Jedi2 was stored at 10 mM at 4°C. All treatments were done in 2% DMSO. Control groups were treated with 2% DMSO throughout.

Multichannel extracellular recordings were collected at 25 kHz using a linear 16-channel probe configuration (A16x1-2 mm-100–177, NeuroNexus; electrode separation, 100 μm). This was connected to an ME16-FAI-μPA system and MC-Rack software (Multichannel Systems). The signals were filtered using an analog high-pass filter with a 300 Hz cutoff frequency. Data acquisition was conducted using a 1401–3 Analog-Digital converter (Cambridge Electronic Design) and Spike2 software (Cambridge Electronic Design). The electrode array was placed along layer V in the occipital dorsal area of neocortex, approximately corresponding to primary visual cortex (Dong, 2008 ). Channelrhodopsin was activated by a 470 nm LED, delivering light through a Nikon Plan Fluor 4× objective (numerical aperture, 0.13), using the patterned illuminator Polygon400 (Mightex). The system was controlled and the patterns were designed through the PolyScan 2 software from the same company. The light intensity was measured at ∼2 mW/mm².
AMPA currents were blocked by bathing in 20 μm NBQX (HelloBio), and NMDA currents were blocked using 50 μm d-APV (Abcam). GABA_A receptors were blocked by gabazine (20 μm). We used three different gap junction blockers, namely mefloquine (50 μm) and quinine (100 μm; both from Sigma-Aldrich); and carbenoxolone (100 μm; Tocris Bioscience).