Figure 6.
NMDA-R subunit-specific [Ca2+] signals in spines. A, Analysis of uEPSC decay time constants. Far left, All spines in control conditions (black; 84.1 ± 0.9 ms; 98 spines). Middle left, Spines with small (<0.1) and large (>0.4) Δ[Ca2+]/uEPSC ratios (<0.1, 77.8 ± 1.1 ms, 25 spines; >0.4, 91.7 ± 2.3 ms, 23 spines). Middle right, Spines with large (>0.11 fl) and small (<0.07 fl) volumes (>0.11 fl, 71.7 ± 1.3 ms, 14 spines; <0.07 fl, 95.7 ± 1.4 ms, 44 spines). Far right, All spines in 6 μm ifenprodil (gray; 71.0 ± 1.3 ms; 55 spines). B, uEPSC size as a function of spine volume (control, r = -0.11; 6 μm ifenprodil, r = -0.09). C, Left, Histograms of Δ[Ca2+]/uEPSC in control conditions and in 6 μm ifenprodil. Inset, Average uEPSC in control conditions (black) and in ifenprodil (gray). Calibration: 2 pA, 100 ms. Right, Reduction of CV of Δ[Ca2+]/uEPSC distribution in 6 μm ifenprodil compared with control (control, CV = 0.85; ifenprodil, CV = 0.44). Error bars were computed using the bootstrap method. D, Δ[Ca2+]/uEPSC as a function of spine volume in control conditions (black) and in 6 μm ifenprodil (gray) (control, r = -0.24, *p < 0.05; 6 μm ifenprodil, r = -0.37, *p < 0.05). For A-D, control, 98 spines, 12 cells (same dataset as in Figs. 2, 3 B, C, 4); 6 μm ifenprodil, 55 spines, six cells. E, Application of MK-801 (10 μm; 6 spines; 4 cells) blocks Δ[Ca2+] and uEPSC equally in a use-dependent manner. Δ[Ca2+] and peak uEPSC were binned (bin size, 2 acquisitions) and normalized to the first bin. Inset, Binned Δ[Ca2+]/uEPSC normalized to the first bin. F, The NR2B subunit-specific antagonists ifenprodil (6 μm; 14 spines; 7 cells) or Ro 25-6981 (0.6 μm; 24 spines; 5 cells) significantly reduced Δ[Ca2+]/uEPSC (*p < 0.05) compared with control (40 spines; 14 cells), independent of uEPSC size (measured at 2 different uncaging laser powers).G, Δ[Ca2+]/uEPSC is independent of uncaging power and uEPSC size. Left, Δ[Ca2+] and uEPSCs recorded in control condition as a function of uncaging power. Right, Δ[Ca2+]/uEPSC (control, black; 6 μm ifenprodil, gray; same dataset as in F). The divergent point (90 mW) in control conditions is attributable to partial saturation of the Ca2+ indicator. Note that in the experiments in E-G, the Ca2+ indicator was Fluo-4FF (2 mm). Error bars represent SEM.