Most Beneficial YES1 Hints One Could Obtain
The success rate of these experiments was low: we documented evoked AMPAR currents only in five out of otherwise successful 112 whole-cell excisions. In all cases, however, AMPAR kinetics were clearly voltage independent (marked ��excised�� in Figures 1D and 1E). However, the current decay was notably slower than Cell Cycle inhibitor that of EPSCs in?situ (2.63?�� 0.43?ms and 1.61?�� 0.07?ms, n?= 5 and n?= 23, respectively; p?Icotinib mouse 1996) and therefore have no diffusion barrier for applied glutamate. In these?experiments, the AMPAR current decay (1.63?�� 0.05?ms at ?60?mV, n?= 6) was (1) indeed similar to the EPSC decay in?situ, and (2) voltage independent (Figures 1D and 1E, marked ��outside-out�� or ��O-O��). Furthermore, decreasing the glutamate pulse concentration 5-fold (from 1.0 to 0.2?mM) in the same membrane patch reduced the AMPAR response amplitude with no effect on its kinetics (Figures 1E, S1H, and S1I), thus arguing against concomitants pertinent to partial receptor saturation. To test whether the electric field effect on EPSCs was biophysically plausible, we integrated the environment of MF-GC synapses (Nielsen et?al., 2004) into the tested Monte Carlo model that incorporates glutamate electrodiffusion in the cleft (Savtchenko and Rusakov, 2007; Sylantyev et?al., 2008) (Experimental Procedures). Simulations readily YES1 reproduced the voltage asymmetry of the EPSC decay (Figures 1F and 1G), which remained robust over a physiological range of synaptic sizes and AMPAR?numbers (Figure?S2A). If the decay asymmetry indeed relies on intracleft electric fields, then decreasing the current at the same voltage should reduce this asymmetry. To test this, we recorded AMPAR EPSCs while halving the extracellular free sodium by partly replacing extracellular NaCl with N-methyl-D-glucamine (NMDG, 65?mM). This manipulation did indeed decrease both the amplitude and?the voltage asymmetry of EPSCs (the decay constant ratio?at?+40 and ?70?mV, ��+40 /��-70, was reduced in NMDG from 1.59?�� 0.11 to 1.15?�� 0.05, n?= 6, p?