S, IN, or Charles River, Wilmington, MA) and cut transversely into 400 thick slices employing a custommade wireslicer fitted with 20 thick goldcoated platinum wire (PozzoMiller et al. 1995). Hippocampal slices were individually plated on MillicellCM filter inserts (Millipore; Billerica, MA) and cultured in 36 , five CO2, 98 relative humidity incubators (ThermoForma; Tetraethylammonium MedChemExpress Waltham, MA). Slices have been maintained in culture media (NeurobasalA plus B27, InVitrogen; Carlsbad, CA) containing 20 equine serum for the very first 4 days in vitro (div). To prevent the confounding effects of hormones and development things in the serum, its concentration was progressively lowered more than a period of 48 h starting at four div (24 h each in 10 and five serum), as Tartrazine Epigenetics described (Tyler and PozzoMiller 2001). Right after a period of 24 h in serumfree media (NeurobasalA plus B27), 70 div slices have been employed for simultaneous electrophysiology and Ca2 imaging. Simultaneous electrophysiology and Ca2 imaging Person 70 div slices were transferred to a recording chamber mounted on a fixedstage upright microscope (Zeiss Axioskop FS; Oberkochen, Germany) and constantly perfused (two ml/min) with artificial cerebrospinal fluid (ACSF) at space temperature (24 ), containing (in mM) 124 NaC1, two KC1, 1.24 KH2PO4, 1.three MgSO4, 17.6 NaHCO3, two.5 CaCl2, 10 glucose, and 29.2 sucrose (31020 mosM); ACSF was bubbled with 95 O25 CO2 (pH 7.four). Superficial CA1 pyramidal neurons had been visualized using a waterimmersion 40objective (0.9 NA) utilizing IRDIC microscopy. Simultaneous whole cell recording and microfluorometric Ca2 imaging was performed as described (McCutchen et al. 2002; Petrozzino et al. 1995; PozzoMiller 2006; PozzoMiller et al. 1996, 1999). Briefly, unpolished patch pipettesJ Neurophysiol. Author manuscript; obtainable in PMC 2010 January 14.Amaral and PozzoMillerPagecontained (in mM) 120 Csgluconate, 17.five CsCl, ten NaHEPES, 4 MgATP, 0.4 NaGTP, ten Na2 creatine phosphate, and 0.2 mM fura2 (or 0.five mM bisfura2); 28090 mosM; pH 7.2 (resistance 3 M). Nominally calciumfree extracellular ACSF was ready by replacing CaCl2 with an equimolar concentration of MgCl2. Some drugs were dissolved in DMSO (0.01 ) and others directly into the ACSF or intracellular option; automobile controls working with 0.01 DMSO had been routinely performed yielding no effects on membrane currents or BDNFinduced responses. Membrane currents had been recorded within the voltageclamp mode at a holding prospective of 65 mV using an Axoclamp 200B amplifier (Molecular Devices; Sunnyvale, CA), filtered at 2 kHz, and digitized at ten kHz. Recordings had been accepted only if access (series) resistance was 30 M. CA1 neurons had complete cell capacitances of one hundred pF. Input resistance (Ri) was measured with hyperpolarizing voltage pulses (50 ms, 20 mV), and cells were discarded if any of these cell parameters (Cm, Ri, Rs) changed by 20 through the course of an experiment. All experiments were performed inside the presence of TTX (0.five ) to block voltagegated Na channels. As noted, some experiments included Cd2 (200 ) and D,L2amino5phosphonovaleric acid (D,LAPV, 50 ) to block voltagegated Ca2 channels and NMDA receptors, respectively. Fura2 or bisfura2 (Molecular Probes; Carlsbad, CA) had been alternatively excited at 360 and 380 nm applying a monochromator (PolychromeII, TILL Photonics; Munich, Germany), and its emission (510 nm) filtered and detected with a frametransfer cooled CCD camera (PXL37, Roper Scientific; Duluth, GA); digital image pairs have been acquired each and every 4 s (50ms exposures for.