Mcpbg

Acetylcholine chloride, 5-CT maleate, 5-HT creatinine sulfate, forskolin, and norepinephrine hydrochloride were obtained from Sigma Chemical Company (St. Louis, MO). 8-OH-DPAT, AS-19, BW723C86, CP93129, LP-44, LY215840, LY272015, mCPBG, PNU109291, SB269970, and TCB-2 were purchased from Tocris (R& D systems, Minneapolis, MN). ET-1 (1-21) was purchased from Bachem (Torrance, CA).

Microdissection of the CGE was performed at E14.5, trypsinized dissociated cells were plated on 96-well plates and cultured in NMB supplemented with mCPBG (10 μM; Tocris) and the mitotic inhibitor arabinofuranosyl cytidine (1 μM). Quantification of protein levels was performed using ELISA kit (Thermo scientific) and 5-HT_3AR antibody (1:1,000, LSBio) on E14.5 day in vitro 1 (+DIV1) or E14.5 (+DIV4) cells. Absorbance levels were measured on a SpectraMax Paradigm Multimode Microplate detection reader (Molecular Devices). Absorbance levels for the 5HT_3AR were normalized to absorbance levels calculated for the total amount of cells via Janus Green whole-cell staining.

Cells were superfused with a standard external medium containing (mM): NaCl, 140; KCl, 2.8; CaCl₂, 2; MgCl₂, 2; Hepes–NaOH, 10; glucose, 10; pH 7.3. The Ca²⁺-free solution contained EGTA, 2 mM. The standard intracellular solution for whole-cell recording contained (mM): CsCl, 140; Hepes–CsOH, 10; Mg₂ATP₃, 2; and BAPTA, 5; pH 7.3. F/Q calibrations were performed in a medium (calibration medium) containing Ca²⁺ as the only permeant ion (mM): N-methyl-D-glucamine, 142; CaCl₂, 2; and Hepes–Ca(OH)₂, 10, pH 7.3; or N-methyl-D-glucamine, 130; CaCl₂, 10; and Hepes–Ca(OH)₂, 10, pH 7.3. For all the measurements of Pf, patch-clamp electrodes were filled with an internal solution containing (mM): N-methyl-D-glucamine, 140; Hepes–HCl, 10; fura-2, 0.25; thapsigargin, 0.001, pH 7.3. All chemicals were purchased from Sigma-Aldrich (Saint Louis, MO, USA), except for fura-2 and fura-2 AM, from Molecular Probes (Eugene, OR, USA), and for bicuculline and mCPBG, from Tocris (Bristol, UK).