Trkb fc

Culture media was replaced with either fresh Neurobasal media containing penicillin/streptomycin and glutamine or HUCPVCs-conditioned media (see above), for 14 h in whole neuronal cultures experiments or 24 h in local axonal application experiments. For CM and CM + TrkB Fc experiments, the media was supplemented with TrkB Fc (1 µg/ml) (R&D Systems).

Isoflurane was used at aqueous concentrations equivalent to 0.25–2 times minimum alveolar concentration (MAC) in rat (Taheri et al., 1991 (link)) as a clinically relevant dose range (1 MAC is ED₅₀). Saturated stock solutions were diluted to working concentrations for superfusion through Teflon tubing using gas-tight glass syringes. Coverslips were placed in a heated (37°C) perfusion chamber (260 μl) and Tyrode’s solution [in mM: 119 NaCl, 2.5 KCl, 2 CaCl₂, 2 MgCl₂, 25 HEPES, and 30 glucose (pH 7.4)] ± isoflurane was delivered using a custom pressurized, inline heated, gas-tight superfusion system (Hemmings et al., 2005 (link)) at 2 ml/min (equilibration time constant of ∼8 s). Cells were equilibrated with control or anesthetic solutions prior to start of experiments and anesthetic concentrations from bath and syringe samples were confirmed by gas chromatography (Herold et al., 2009 (link)). TrkB-Fc (1 μg/ml; ED₅₀ = 0.1–0.4 μg/ml; R and D Systems, Minneapolis, MN) and recombinant BDNF (75–100 ng/ml; PeproTech, Rocky Hill, NJ; Yang et al., 2016 (link)) were added 5 min prior to stimulation. Drug concentrations were based on cell type, stimulation frequency, and incubation times (Rex et al., 2007 (link)).

Silver electrode implanted mice were prepared before all mice experiments according to Christianson’s methods57 (link) (Supplementary Methods-5). All treated mice had 5 days for recovery and fasted for 24 h before CRD and EMG detection.
BDNF^+/+ and BDNF^+/− mice received an intracolonic infusion of FSN (0.3 ml, 170 μl/h) from HCs and IBS-D (with or without aprotinin preincubation) or IBS-C patients through a catheter (outside diameter 1 mm) inserted into the colon 3.5 cm from the anus. We pretreated BDNF^+/+ mice with recombinant TrkB Fc chimera, TrkB/Fc (10 ng/mouse, intracolonical infusion, 30 min before FSN infusion, R&D, USA)12 (link) and fluorocitrate (20 μmol/kg, i.p, twice a day at 9 am and 6 pm for 7 days, sigma)51 (link) to block the effect of colonic BDNF and EGCs in BDNF^+/+ mice, respectively.
CRD started at 1 hour after the intracolonic infusion of FSN under pressures of 15, 30, 45 and 60 mmHg (Supplementary Methods-6). The EMG activity was used to evaluate the visceromotor response of mice and was recorded by the Powerlab BL-420E system. Data as expressed by the area under the curve were analyzed by Labchart v5.0 software. EMG activity was corrected for the baseline activity and expressed as “% of baseline”.