The bathing solution was then switched back to ASW and a third application of agonist was made to study washout of the antagonist. When L-Glu and D-Asp currents were studied in the same cell, the agonist pipette was changed after washout and this protocol was repeated. Agonist order was alternated with each cell studied. For Gly and D-Ser experiments, the agonist pipette was changed from one containing D-Asp to one containing D-Asp + Gly or D-Asp + D-Ser after control currents for D-Asp were recorded. Because the noncompetitive antagonists

memantine and MK-801 require Inhibitors,research,lifescience,medical channel opening for block to occur, two applications of agonist were made during antagonist exposure before washout, and each application was compared to the control. For analysis, D-Asp and L-Glu current amplitudes in pharmacological agents Inhibitors,research,lifescience,medical were normalized to the initial control current. Unless otherwise noted, pharmacological experiments were performed at −30 mV, approximately the resting potential for cultured BSC neurons (Fieber et al. 2010). Cyclothiazide (CTZ) experiments were performed both under the conditions described above and under conditions designed to investigate

block of desensitization. For desensitization Inhibitors,research,lifescience,medical experiments, cells were exposed to repeated applications of D-Asp both in ASW and in ASW + CTZ. Three applications of D-Asp were made: an initial, control application (t0), an application at t10=t0+ 10 sec, and a final application at t20=t0+ 20 sec. Currents were normalized to the control current for each condition. Solutions Unless noted, all reagents were from Sigma-Aldrich (St. Louis, MO). Control extracellular solution consisted of ASW. Inhibitors,research,lifescience,medical Control intracellular solutions contained (mM) 458 KCl, 2.9 CaCl2 (2 H2O), 2.5 MgCl2 (6 H2O), 5 Na2ATP, 1 EGTA, and 40 HEPES-KOH, pH 7.4. For pharmacological experiments, competitive and noncompetitive Inhibitors,research,lifescience,medical antagonists of L-Glu receptors or Cl− channel blocker were

diluted in ASW from frozen stocks. The protocol entailed application of blocker to cells after control records Casein kinase 1 in response to D-Asp or L-Glu were made, then washout of any blocker effects. Blocker concentrations were selected based on prior experiments in Aplysia (Dale and Rapamycin order Kandel 1993; Armitage and Siegelbaum 1998; Klein et al. 2000; Chitwood et al. 2001; Antzoulatos et al. 2003; Jin and Hawkins 2003; Collado et al. 2007). Stocks of the following drugs were made in water, then diluted in ASW at 1:50 or greater, for their working concentrations shown in parentheses: 4-acetamido-4′-isothiocyanato-2,2′-stilbenedisulfonic acid disodium (SITS; 100 μM), DL-kynurenic acid (kynurenate; 1 mM), DL-2-amino-5-phosphonopentanoic acid (APV; 100 μM), memantine hydrochloride (Tocris, St.