Bupivacaine hydrochloride

Exparel™ (multivesicular liposomal bupivacaine suspended in 0.9% sodium chloride; 13.3 mg bupivacaine/mL) was purchased from Pacira Pharmaceuticals, Inc. (San Diego, CA). Bupivacaine Hydrochloride was purchased from Sigma-Aldrich, Co. (St. Louis, MO).

Poloxamer 407 (P407) (BASF, New Jersey, USA), casein (C3400-Sigma-Aldrich, Minnesota, USA) (approximate casein composition of milk is (g/L): α-s1, 12–15; α-s2, 3–4; ß, 9–11; and k, 2–4), bupivacaine hydrochloride (Sigma-Aldrich, Minnesota, USA), sulforhodamine B (230162 -Sigma-Aldrich, Minnesota, USA). bupivacaine hydrochloride and sulforhodamine B were chosen as model drugs.

ATP, ADP, UDP, GDPβS and bupivacaine hydrochloride were purchased from Sigma. Pertussis toxin (PTX), forskolin, tetraethylammonium (TEA), quinine, and 4-aminopyridine (4-AP) were purchased from Tocris. Stock solutions of all drugs were made in water and diluted to the appropriate working concentrations in ACSF. Drugs were applied to the slices either through bath application or using a picopump (WPI pneumatic picopump, Sarasota, FL). The diameter of the drug application pipette tip was ∼3–4 μm. The pressure (10 psi) and duration (100 ms) of the puff was controlled and the distance between the patched cell and puff pipette was kept constant (∼15 μm). This was achieved by marking the position of the two pipettes (recording and puff) on the display screen and adjusting the distance of the puff pipette until the preferred distance was reached. The holding pressure of the puff pipette was maintained at -2 psi to prevent leakage, but there may still be minimal spontaneous leakage. For experiments involving testing of antagonists on ATP-induced current, control applications of ATP were performed and then on the same cell the effect of the antagonist was tested.