Tetrodotoxin ttx

MB was purchased from American Reagent, Inc (Shirley, NY) and directly diluted in saline to the final concentrations. Tetrodotoxin (TTX) was purchased from Tocris (Bristol, UK) and prepared in ddH₂O. 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX) and 2-amino-5-phosphonopentanoic acid (AP-5) were provided from NIMH Chemical Synthesis and Drug Supply Program, and prepared in ddH₂O. Picrtoxin (PTX) was purchased from Sigma and prepared in DMSO. The stock solutions were diluted in saline so that the final DMSO concentration (v/v) was < 0.1%.

Drugs used in this study were HIV-1gp120 IIIB (Immunodiagnostics, Inc. Woburn, MA), T140 was kindly provided by Professor Nobutaka Fujii (Kyoto, Japan). 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 2-amino-5-phosphonovalerate (APV), (R)-3-(2-Carboxypiperazin-4-yl) propyl-1-phosphonic acid (R-CPP), ifenprodil, tetrodotoxin (TTX) and picrotoxin were purchased from TOCRIS Bioscience (Ellisville, MO). picrotoxin was dissolved in dimethyl sulfoxide (DMSO) and the final DMSO concentration in artificial cerebrospinal fluid (ACSF) was less than or equal to 0.1%. TTX, CNQX, APV, R-CPP, and ifenprodil were pre-prepared separately in 1000× stock solutions and stored at -20°C refrigerator, thawed on experimental day just before use and diluted to the test concentrations. Drugs were applied onto brain slices via bath perfusion or added to the culture media. For bath perfusion, the time needed for a drug to reach the chamber was about 1 min. Unless otherwise indicated, all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).

Cortical neurons were prepared as previously described^{15 (link)}. Briefly, E15.5 mouse embryo cortices were dissected and then dissociated in 1× Hank’s Balanced Salt Solution (HBSS) in the presence of 0.1% trypsin (Invitrogen). trypsin treatment was terminated with trypsin inhibitor and triturated in presence of DNase I (Sigma). Neurons were pooled after genotyping and seeded on poly-D-lysine coated dishes. Neurons were maintained in Neurobasal medium containing B27 supplement, antibiotics and glutamine. Neurons were cultured in vitro for 10 days. One third of the medium was replaced with fresh warm medium every two days.
For KCl depolarization, neurons were quieted overnight in 1 μM tetrodotoxin (TTX, Tocris), and then were incubated for 0, 1, 3, 4 or 6 hours in 55 mM KCl as described previously^{15 (link)}. ChIP-seq experiments were performed after 0 or 1 h KCl-mediated depolarization. RNA-seq experiments were performed after 0 or 6 hours KCl-mediated depolarization. GRO-seq experiments were performed after 0, 1 or 3 hours KCl-mediated depolarization. 3X depolarization buffer (170mM KCl, 1mM MgCl₂, 2 mM CaCl₂, 10mM HEPES pH=7.9) was added as 1:3 ratio into culture medium (final 55mM KCl) to induce depolarization of cortical neurons.

Slices were kept in oxygenated artificial cerebral spinal fluid (aCSF), as described previously.^{51 (link)} aCSF was comprised of 126 mM NaCl, 2.5 mMKCl, 1.2 mM NaH₂PO₄ monohydrate, 2.4 mM CaCl₂ dihydrate, 1.2 mM MgCl₂ hexahydrate, 25 mM NaHCO₃, 11 mM glucose, and 15 mM tris(hydroxymethyl)aminomethane and was adjusted to pH 7.4 immediately prior to experimentation. A 10 mM stock solution of adenosine was prepared in 0.1 mM HClO₄ and this was diluted daily in aCSF to 1 μM for post-calibration of the CFMEs. One mM stock solutions of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, Sigma-Aldrich) were prepared in dimethyl sulfoxide (DMSO) and kept frozen until used. Stock DPCPX was added to perfusion aCSF to make a 100 nM solution and slices were perfused for 15 min before the electrode was implanted and adenosine measurements made while DPCPX was perfused. Tetrodotoxin (TTX, Tocris) was reconstituted to 3 mM in 0.2 M citrate buffer, diluted to 50 μM aliquots, and kept frozen until used. 50 μM aliquots were added to perfusion aCSF to make a 200 nM working solution and perfused over slices in the same manner as DPCPX. After each experiment all solutions and surfaces that came into contact with TTX were treated with 10% bleach to deactivate residual TTX.