epileptic seizures are characterized by excessive excitation the role of excitatory

epileptic seizures are characterized by excessive excitation the role of excitatory synaptic transmission in the induction and expression of epilepsy remains unclear. on excitatory synaptic transmission in area CA1 of the hippocampus. For this purpose we treated cultured hippocampal slices overnight with the specific GABAA receptor antagonist (-)-bicuculline methochloride (BMC 50 μm). Methods Hippocampal slice cultures Organotypic hippocampal slice cultures were prepared from 6-day-old Wistar rat pups killed by decapitation (G?hwiler 1997) following a protocol approved by the Veterinary Department of the Canton of Zurich. After Pneumocandin B0 2 weeks slices were randomly allocated into three groups that were incubated for 15 ± 3 h (overnight) either in serum-based medium alone or in medium made up of BMC (50 μm) or BMC plus the specific NMDA receptor antagonist (R E)-4-(3-phosphonoprop-2-enyl)piperazine-2-carboxylic acid (CPP 40 μm; donated by Novartis Basel Switzerland). Electrophysiology Field recordings of Pneumocandin B0 spontaneous bursting activity were performed at 35°C (incubation heat) with patch pipettes (3-5 MΩ) made up of 2 m NaCl in culture medium. Whole-cell voltage-clamp recordings of excitatory Pneumocandin B0 synaptic currents were obtained using an Axopatch 200B amplifier (Axon Devices Foster City CA USA) and pipettes made up of (mm): 140 potassium or caesium gluconate 10 KCl 5 Hepes 1.1 EGTA 4 MgCl2 10 phosphocreatine pH 7.3 285 mosmol l?1 unless otherwise stated. Slices Pneumocandin B0 were perfused with warmed (32°C) saline made up of (mm): 137 NaCl 2.7 KCl 2.8 CaCl2 2 MgCl2 11.6 NaHCO3 0.4 NaH2PO4 5.6 glucose and phenol red (10 mg l?1) pH 7.4 unless otherwise mentioned. Only cells with a series resistance between 10 and 15 MΩ were included. Miniature excitatory postsynaptic currents (mEPSCs) were recorded at -70 mV in the presence of 0.5 μm tetrodotoxin (TTX Latoxan Valence France) 50 μm picrotoxin 50 μm BMC and 40 μm CPP using potassium gluconate-based intracellular solution (see above). Miniature inhibitory postsynaptic currents (mIPSCs) were recorded at -70 Rabbit polyclonal to ALPK1. mV in the presence of 0.5 μm TTX 20 μm 2 3 2 3 4 (NBQX) and 40 μm CPP using the following intracellular solution (mm): 140 caesium gluconate 10 NaCl 1 MgCl 10 Hepes 0.4 Mg2GTP 0.1 EGTA pH 7.3 285 mosmol l?1. mEPSCs and mIPSCs were analysed offline with the ‘Mini Analysis Program’ (Synaptosoft Leonia CA USA) using a detection threshold of 5 pA. Cumulative histograms were constructed by pooling 300 consecutive events from each Pneumocandin B0 cell. Average traces were obtained for each experiment by aligning individual mEPSCs to their rising phases; fitting a single exponential around the 90-10% of the decaying signal yielded the decay time constant. Excitatory postsynaptic currents (EPSCs) were evoked using a monopolar glass stimulation electrode filled with extracellular medium and placed into stratum radiatum close to the recording electrode in the presence of 50 μm picrotoxin 50 μm BMC 4 mm Mg2+ and 4 mm Ca2+ and after a cut between CA3 and CA1. AMPA receptor-mediated EPSCs were evoked at -70 mV whereas NMDA receptor-mediated EPSCs were evoked at +40 mV during blockade of AMPA receptors by application of 20 μm NBQX. Field excitatory postsynaptic potentials (fEPSPs) were recorded in the stratum radiatum of the CA1 region with a glass electrode made up of 2 m NaCl and 3 mm BMC after cutting between areas CA3 and CA1. Stimuli (0.1 ms) were delivered to CA3 axons at 0.25 Hz by a bipolar glass electrode filled with extracellular solution. LTP was induced by theta burst stimulation composed of five trains at 5 Hz each consisting of five stimuli delivered at 100 Hz. LTD was induced by stimulating at 3 Hz for 5 min (Dudek & Bear 1992 The levels of potentiation and depressive disorder were estimated for each cell by measuring the average slope of field potentials in the middle third of its rising phase over 5 min taken 25 min after the end of LTP or LTD..