Bicuculline methiodide

Action potentials were generated in the presynaptic dGC using 5 ms square current pulses (1 nA). For connectivity analysis, 5 action potentials were induced at 20 Hz (inter-sweep-interval: 10 s; at least 30 repetitions) while recording unitary excitatory postsynaptic currents (uEPSCs) from CA3-PCs. Neurons were considered to be connected if >5% of action potentials evoked time-locked inward uEPSCs [26 (link)]. Recordings were performed in the presence of (-)-bicuculline-methiodide (10 μM, Abcam, Cambridge, UK, #ab120108) to prevent inhibitory synapse recruitment. Moreover, glutamine (200 μM, Gibco, New York, NY, USA, #11539876) was added to the recording medium to avoid synaptic depletion [27 (link)].

AP5, Bicuculline methiodide (BIM), CGP 55845, picrotoxin, NBQX, and TTX citrate were purchased from Abcam, isoflurane from Abbott, and 5α-THDOC and all other substances/salts from Sigma-Aldrich. Drugs were applied by bath application.

Cultured hippocampal neurons (DIV 13–14) were transferred to a submerged chamber and continuously perfused (2–3 ml/min) with (in mM): 140 NaCl, 5.4 KCl, 1.25, 1 MgCl₂, 1.3 CaCl₂, 10 HEPES and 3.6 D-glucose with pH adjusted to 7.35 using NaOH. All chemicals and drugs were purchased from Sigma or BioShop, Canada unless otherwise stated. YFP transfected neurons were identified with a microscope equipped with fluorescent and phase contrast optics. All recordings were performed at room temperature with a MultiClamp 700B amplifier. Cells were voltage clamped at −60 mV. Recording of mEPSCs was conducted using pipettes filled with an intracellular solution containing (in mM): 122.5 Cs-methanesulfonate, 17.5 CsCl, 2 MgCl₂, 10 EGTA, 10 HEPES, 4 ATP (K), and 5 QX-314, with pH adjusted to 7.2 by CsOH. Tetrodotoxin (500 nM; Abcam) and bicuculline methiodide (10 μM; Abcam) were added to perfused saline to block action potentials and GABA receptor-mediated inhibitory synaptic currents, respectively. mEPSCs were recorded using WinLTP software in continuous acquisition mode. Analyses for frequency and amplitude were conducted using MiniAnalysis software.