QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

Help viewing high resolution images Return to article

Fig. 2. Excitation-induced Ca²⁺ influx and expression of Ca²⁺ channels in proliferating NPCs. (A) Representative images from Ca²⁺ imaging. 1, Before drug treatment; 2, after drug treatment. Cells cultured for 3 DIV in the presence of bFGF were incubated with 5 µM fura-2/AM, then exposed to either 50 mM KCl (left graph) or 100 µM NMDA (right graph) for 10 seconds at the times indicated by arrows. The acetoxymethylester form of fura-2 (fura-2/AM, Molecular probes, Eugene, OR) was used as the fluorescent Ca²⁺ indicator. Cells were incubated for 40-60 minutes at room temperature with 5 µM fura-2/AM and 0.001% Pluronic F-127 in a HEPES-buffered solution composed of 153 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 2.5 mM CaCl₂, 10 mM HEPES, 10 mM glucose, pH adjusted to 7.4 with NaOH. The cells were then washed with HEPES-buffered solution and placed on an inverted microscope (Olympus, Japan). The 5 mM KCl-HEPES buffer contained: 150 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 2.5 mM CaCl₂, 10 mM HEPES, 10 mM glucose, pH adjusted to 7.4 with NaOH. For experiments without extracellular Ca²⁺, Ca²⁺ was not added to the external solution containing 2 mM EGTA. The cells were illuminated using a xenon arc lamp, and the required excitation wavelengths (340 and 380 nm) were selected by means of a computer-controlled filter wheel (Sutter Instruments, CA). Emitter fluorescence light was reflected through a 515 nm long-pass filter to a frame transfer cooled CCD camera, and the ratios of emitted fluorescence were calculated using a digital fluorescence analyzer. All imaging data were collected and analyzed using Universal Imaging software (West Chester, PA). Fura-2/AM fluorescence is expressed as F/F₀; increased fluorescence indicates elevated [Ca²⁺]_i. Values of the mean ± s.e.m. of all the loaded cells are shown (n=52 for KCl; n=19 for NMDA). The images are of a field of cells at the time points corresponding to 1 and 2. (B) Continuous fluorometric [Ca²⁺]_i recording in proliferating NPCs loaded with fura-2/AM. Bath application of 5 mM KCl (left graph) or 5 µM NMDA (right graph) causes a time-dependent elevation in [Ca²⁺]_i when Ca²⁺-containing buffer was used (KCl, n=50; NMDA, n=50). However, the KCl- or NMDA-induced Ca²⁺ influx was abolished when Ca²⁺-free extracellular buffer was used (KCl, n=50; NMDA, n=50). Values of the ± s.e.m. of all observed cells are shown. (C) Ba²⁺ currents were recorded using a whole-cell patch clamp technique (see Materials and Methods). Cells cultured for 3 DIV in the presence of bFGF were incubated with NMDA (5 µM) before recordings. (a-d) Currents recorded in the (a) absence or (b) presence of the L-type Ca²⁺ channel blocker nifedipine (10 µM). Current traces induced by test pulses of –20, 0, 10, 20 and 30 mV are superimposed in the middle and right panels. The NPCs, of which an image is shown in the left panel, were subjected to depolarizing test pulses every 10 seconds (pulse duration of 100 mseconds) from a holding potential of -40 mV. The I–V relationships were obtained by plotting peak current amplitudes as a function of test potentials ranging from –30 to +30 mV (n=4, , control; , 10 µM nifedipine) (c). (d) Summary of the effect of nifedipine on the current density measured at 0 mV. Bath application of 10 µM nifedipine attenuated the I_Ba densities (control, 26.7±3.0 pA/pF; nifedipine, 13.9±2.0 pA/pF; n=4, ^**P<0.01). Error bars indicate ± s.e.m.

Return to article