Tcs 1105

TETS (≥ 97% pure as determined by GC–MS) was synthesized in the laboratory of Dr. Bruce Hammock, UC Davis as previously described (Zhao et al., 2014 (link)). L-838417, PZ-II-029, TCS 1105 and SB-205384 were purchased from Tocris, Minneapolis, MN, USA. CL-218872 was purchased from Toronto Research Chemicals, Toronto, Ontario, Canada. SL-651498 was purchased from Axon Medchem, Reston, VA, USA. NS11394 was purchased from eNovation Chemicals, Bridgewater, NJ, USA. DS2, THIP (gabaxadol hydrochloride), and zolpidem were purchased from Sigma, St. Louis, MO, USA. Purity of all commercial chemicals based on the vendor supplied certificate of analysis was 98% or greater by HPLC. The compounds 2–261 and 2–314 were generously provided by Kelvin Gee (University of California, Irvine). TETS and PAMs were dissolved as 1000× stocks in 100% DMSO (Sigma, St. Louis, MO, USA) and kept in a −20 °C freezer until use. Further dilution to 2× sub-stocks was conducted using embryo medium immediately before exposures. Vehicle (DMSO) was kept below 0.3% for all exposure paradigms.

For volatile anesthetic treatment, coverslips in 12-well plates with a low volume of culture media (500μl to facilitate gas diffusion, were placed in airtight, humidified modular chambers (Billups-Rothenberg, Del Mar, CA, USA) as previously described (Mintz, et al. 2013 (link); Xu, et al. 2018b (link)). The chamber was connected to an agent-specific calibrated vaporizer (SuperaVet, Vaporizer Sales and Services Inc, Rockmart, GA, USA) that delivered 2.4% isoflurane mixed with 5% carbon dioxide / 95% air carrier gas at 12 L/min. Carrier gas alone was used as for controls. Gas composition was measured periodically using a 5250 RGM gas analyzer (Datex-Ohmeda, Madison, WI, USA). In some cases, co-treatment or pre-treatment with pharmacologic compounds was performed. These compounds included: TCS 1205 (10 nM, 100 nM, 1μM, Tocris), TCS 1105 (10 nM, 100 nM, 1μM, Tocris), Zolpidem (10 nM, 100 nM, 1μM, Tocris), Bumetanide (10μM, Tocris), and Chloride Ionophore I (3μM, Millipore). After a 15-min equilibration, the sealed chambers with dissociated cultures were placed in an incubator to maintain temperature at 37°C for 1 hour, followed by 20 min exposure to either vehicle control or a recombinant soluble axon guidance cue, Semaphorin 3A (R & D Systems, Bend, OR) at 100 ng/ml to induce growth cone collapse (Figure 1A, B).