1 CaCl2, 15 HEPES (pH7.2), osmolarity 300 ± 2 mOsm/l. Dissected hippocampal CA1-CA3 regions were placed into a holding chamber containing protease type XIV (1 mg/ml, Sigma-Aldrich) dissolved in oxygenated HEPES-buffered Hank’s balanced salt solution (HBSS 6136: Sigma-Aldrich) and maintained at 37°C, pH 7.4, osmolarity 300 ± 5 mOsm/l. After 30 min incubation in the enzyme solution, LY294002 manufacturer the tissue was rinsed three times with the Low-Ca2+ HBS and triturated using fire-polished Pasteur pipettes. The cell suspension was placed into a 50 mm plastic petri dish for electrophysiological recordings. Hippocampal pyramidal neurons were selected on the basis of their characteristic morphology. Agonist-evoked currents were recorded

from

transfected HEK293T cells, acutely isolated neurons, and primary hippocampal cultures as described (Kato et al., 2008). Recordings were made using thick-walled borosilicate glass electrodes pulled and fire-polished to a resistance of 2–5 MΩ. All cells were voltage-clamped at −80 mV and data were collected and digitized using Axoclamp 200 and Axopatch software and hardware (Molecular Devices, Sunnyvale, CA). For whole cell recordings, the transfected www.selleckchem.com/products/epz-6438.html HEK293T cells were bathed in external solution containing the following (in mM): 117 TEA, 13 NaCl, 5 BaCl2, 1 MgCl2, 20 CsCl, 5 glucose, and 10 Na-HEPES pH 7.4 ± 0.03. For acutely isolated and cultured primary neurons, 10 μM CPP, 10 μM bicuculline, 1 μM TTX, and 300 nM 7-chlorokynurenic acid were added in the external solution and the extracellular concentration of NaCl was increased to 130 mM and TEA was omitted. 7-Chlorokynurenic acid (7-CK) was omitted for acutely isolated neurons. The intracellular electrode solution contained the following (in mM): 160 N-methyl-D-glucamine, 4 MgCl2, 40.0 Na-HEPES pH 7.4, 12 phosphocreatine, 2.0 Na2-ATP pH7.2 ± 0.02 adjusted by H2SO4. For neuronal

recordings, 1 mM QX314 were added to the internal solution. For outside-out patches and whole cell recordings using fast perfusion, the internal solution contained (in mM): 130 CsCl, 10 CsF, 10 Cs-HEPES pH 7.3, 10 ethylene glycol tetraacetic acid (EGTA), 1 MgCl2, and 0.5 MycoClean Mycoplasma Removal Kit CaCl2 and was adjusted to ∼290 mOsm. The transfected HEK293T cell or the acutely isolated neuron was lifted and perfused with ligand-containing solutions from a sixteen-barrel glass capillary pipette array positioned 100–200 μm from the cells (VitroCom). Each gravity-driven perfusion barrel is connected to a syringe ∼30 cm above the recording chamber. The solutions were switched by sliding the pipette array with an exchange rate of less than 20 ms. For fast application experiments with a junction potential rise time of less than 300 μs, rapid solution exchange (1 and 200 ms application for deactivation and desensitization, respectively) from a θ tube containing external solution (in mM: 140 NaCl, 3 KCl, 10 glucose, 10 HEPES pH 7.