Figure 3.

The presence of SP in spines correlates with the quantity and efficacy of glutamate receptors (GluR) of the AMPA type, but not of the NMDA type. A, B, Immunostaining for GluR1 clusters demonstrates a higher mean fluorescent intensity in SP(+) spines (arrows) compared with SP(−) spines (arrowhead) (50.6 ± 4.2 and 34.2 ± 2.1 fluorescence intensity units, respectively; n = 4 cultures, 9 segments, 295 spines; p < 0.05). Scale bar, 1 μm. C, Uncaging of glutamate applied near the head of individual spines (circles) that are positive for transfected GFP-SP (yellow in colocalization with the DsRed signal) produces a larger amplitude of inward current compared with SP(−) spines. The 3D reconstruction demonstrates the subcellular localization of SP. The inset illustrates the 2D projection of the original confocal image stack. Scale bar, 5 μm. Traces of inward current, generated at the flash (arrow) demonstrate the difference between SP(+) (blue trace) and SP(−) spines. D, SP(+) spines show a 2.5-fold larger amplitude of inward current when compared with spines lacking SP (p < 0.02; n = 6 cultures; 7 cell, 47 SP(+) spines, 44 SP(−) spines). Spine size was not different in the two groups as determined by measuring the maximum cross-sectional area and spine length (p > 0.674 and 0.238, respectively). E, Current responses evoked by uncaging of glutamate near the head of a SP(+) spine, with the cell membrane held at different membrane potentials from −80 to +40 mV. The emergence of a slow component at the depolarizing potentials is evident. F, Current-voltage relations for the fast component, peaking at 2–3 ms after the flash, and for the slow component, peaking at 200 ms after the flash, and representing the NMDA component of the response to glutamate. G, An illustration of the response of a cell to flash photolysis of caged glutamate near a spine, recorded in the presence of 50 μm APV, with the cell held at −60 and +50 mV. The slow component of the response is completely blocked. H, Sample illustrations of responses of cells held at −60 and +50 mV to flash photolysis of caged glutamate near an SP(+) and an SP(−) spine, illustrating a “silent synapse.” I, J, Group data [n = 19 SP(+) spines and 17 SP(−) spines] in three different experiments (n = 5 cells, paired recording), demonstrating a significant difference when recorded at resting potential (J) and a lack of difference when recorded at +50 mV.