Ca2+ and current responses of the total extrasynaptic NMDA receptor pool in single hippocampal neurons. (A) The protocol used to isolate and measure extrasynaptic NMDA receptor responses is shown schematically. Application of 50 μM bicuculline in the presence of 10 μM glycine initiates recurrent AP bursting as recorded in current clamp (IC). 10 μM MK-801 is then applied for 4 to 20 bursts (48 to 163 action potentials, 2 to 10 min) to block synaptic NMDA receptors (synaptic blockade) before halting synaptic activity with TTX in the absence of glycine. In voltage clamp (VC, Vhold = -71 mV) the extrasynaptic pool of receptors is then activated with a 30 s application of 100 μM NMDA in the absence of Mg2+ and the presence of 10 μM glycine. Spontaneous activity of all cells was measured in cell attached mode before commencing experiments. (B-D) Example recordings from the same cell are shown for the (B) IC and (C, D) VC components of the recording. Ca2+ recordings in C show the isolated extrasynaptic NMDA receptor response measured in the nucleus (light grey), soma (grey) and a dendritic region 50 μm from the soma (black) measured with cell impermeant bis-FURA2.