Animals were anaesthetized with trifluoroethane and decapitated to prepare 300-400 μm thick coronal slices from the hippocampus of P14-P22 rats (Yang et al. 2006). All experiments conformed with the guidelines laid down by the Albert Einstein College of Medicine animal welfare committee. Whole-cell voltage clamp recordings were made from CA1 pyramidal neurons. The patch pipettes were filled with a caesium gluconate solution containing (mm): 123 caesium gluconate, 8 NaCl, 1 CaCl2, 10 EGTA, 10 Hepes, 10 glucose, 5 ATP, 0.4 GTP, 1 QX-314 (pH 7.2; 280-290 mOsmol l−1). Slices were superfused (3-4 ml min−1) at room temperature (25°C) with oxygenated physiological saline (mm: 119 NaCl, 2.5 KCl, 1.3 or 0.1 MgCl2, 2.5 CaCl2, 1 NaH2PO4, 26.2 NaHCO3, 11 glucose). All mobility experiments were performed at 34°C.
Recordings were rejected if the holding current was greater than −100 pA when pyramidal cells were voltage clamped at −60 mV. NMDA receptor currents were isolated by including NBQX (5 or 10 μm) and picrotoxin (100 μm) in the external solution. NMDA currents were recorded by relieving Mg2+ block with depolarization. Initial experiments were carried out at +30 mV (N= 4), but we subsequently determined that cells were easier to maintain when they were subjected to less depolarization. As a result, we used depolarization sufficient to elicit substantial NMDAR currents (∼−20 mV) in all subsequent experiments. We did not see any relationship between the amount of depolarization and the percent of extrasynaptic NMDAR measured (r2= 0.15).
MK-801 (50 μm in bath or 500 μm via picospritzer), d-APV (50 μm), ifenprodil (3 μm), and Ro 25-6981 (1 μm; Tocris) were used to block NMDA receptors. Synaptic stimulation was achieved by placing a monopolar or bipolar theta glass stimulating electrode adjacent to the dendrite to activate synapses (every 10-30 s; 40-200 μA, 100-200 μs). Repeated electrical stimulation (0.1 or 0.2 Hz) in the presence of MK-801 was used to block synaptic NMDARs. The different stimulating electrodes yielded similar results.
For comparisons of decay kinetics, currents were fitted with double exponentials (y=y0+A1e−τ1+A2e−τ2). The weighted tau was then calculated as τw=A1τ1/(A1+A2) +A2τ2/(A1+A2) (Stocca & Vicini, 1998). Where A1 and A2 are the amplitudes of the fast and slow decay constants and τ1 and τ2 are their associated time constants. The decays of synaptic currents (τw= 88 ± 6 ms; N= 23), photolytic EPSCs (phEPSCs; τw= 98 ± 14 ms; N= 15), single spontaneous NMDAR currents (τw= 81 ± 6 ms; mean amplitude 29 ± 5 pA; N= 7), and currents from cells in ifenprodil (τw= 90.3 ± 9.1 ms; N= 15) did not differ significantly when fitted with double exponentials (ANOVA, P > 0.6). As the mobility experiments were performed at 34°C, we saw shorter decay times for these currents (τw= 52 ± 6 ms; N= 4).
Estimation of the extrasynaptic pool was corrected for any residual synaptic current, using the calculation: E= (T′−fT)/(1 −f), where E is the extrasynaptic NMDAR current, T′ the peak photolytic current amplitude after synaptic blockade, f the fraction of residual synaptic current after synaptic blockade and T the peak photolytic current amplitude prior to synaptic blockade. We observed 100% block of synaptic NMDAR current in 7 of 12 of experiments used for estimates of extrasynaptic pool size. For the experiments with incomplete block, 6.5 ± 0.8% (N= 5) of the current remained unblocked. Estimates of extrasynaptic NMDAR population were independent of percentage block (35 ± 5 versus 32 ± 5, P > 0.6, Student’s t test).