To examine the generation of action potentials, spontaneous synaptic activity, and AMPA and NMDA responses, an internal recording solution was used containing 4mM KCl, 125mM K-Gluconate, 10mM HEPES, 10mM phosphocreatine, 1mM EGTA, 0.2mM CaCl2, 4mM Na2-ATP, and 0.3mM Na-GTP (pH 7.3). To examine GABA responses, the pipette solution contained 130mM KCl, 10mM HEPES, 10mM phosphocreatine, 1mM EGTA, 0.1mM CaCl2, 1.5mM MgCl2, 4mM Na2-ATP, and 0.3mM Na-GTP (pH 7.3), which allowed GABA-induced synaptic currents to be recorded at normal resting membrane potential. Whole cell current clamp recordings were used to examine the ability of cells to generate action potentials and to observe AMPA responses to 50µM glutamate (in the presence of Mg) and NMDA responses to a combination of 50µM glutamate and 10µM glycine (in the presence of DNQX and the absence of Mg2+). Whole cell voltage clamp recordings were used to examine spontaneous synaptic activity in the neural culture as well as to observe responses to 50µM GABA. To elicit AMPA, NMDA, and GABA responses, a micropipette containing the appropriate solution was placed adjacent to the recorded cell. The solution was puffed onto the cell using 5psi of positive pressure.
