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.

NMDA stimulates the release of VEGF from proliferating hippocampal NPCs. (A) Real-time PCR analysis of BDNF, PDGF and VEGF mRNAs 24 hours and 72 hours after a single 24-hour pulse of each substance. The expression of each gene was normalized to the amount of GAPDH to obtain the relative level of its transcript (n=5 per group) and is shown relative to the expression levels in control cells at 24 hours. (B) Western blot of VEGF. Secretion of VEGF into the culture medium 72 hours after the single 24-hour pulse of each treatment. VEGF (10 ng/ml) was loaded as a positive control. (C) Addition of VEGF or BDNF increases the fraction of BrdU⁺ (red). VEGF (50 ng/ml) or BDNF (50 ng/ml) was added to the culture medium in the absence of bFGF for 72 hours, and proliferating cells were labeled by BrdU for 1 hour before fixation. All the cells were counterstained with DAPI (blue). The percentage of BrdU⁺ cells [BrdU(+)] cells is shown. Bar, 100 μm. (D) Quantitative RT-PCR analysis showing that Bcl2 and Bax mRNA levels were increased and decreased, respectively, after exogeneous VEGF application for 24 hours. Expression levels of Bcl2 and Bax were normalized to the amount of GAPDH expression to calculate the relative amount of gene transcript (n=3 per group). Expression levels at each concentration are depicted relative to the expression of the control 3 days after the onset of stimulation for comparison. MK, MK801. Error bars indicate ± s.e.m. CTL, control. ^*P<0.05, ^**P<0.01, ^**P<0.001.

NMDA modulates the cell cycle characteristics of cultured hippocampal NPCs. (A) Flow-cytometric histograms of DNA content as indicated by fluorescence intensity (FL-2 Area) on the x-axis and number of cells after staining with propidium iodide on the y-axis. Cells were incubated for 4 days with 20 ng/ml bFGF, with or without NMDA (5 μM), thymidine (3 μM), or MK801 (5 μM) for the first 24 hours. The results shown are from a single representative experiment. (B) Cells treated with various stimuli (as described in A) were analyzed for DNA content, and the areas under the peaks were compared. The numbers of cells with DNA contents corresponding to each cell cycle phase are expressed as percentages of the total sorted events. Values are the mean ± s.e.m. of 12 independent experiments. (C) A single-pulse treatment of NMDA decreases the level of Rb and increases that of P-Rb. Quantitative RT-PCR analysis (graph) showing that Rb mRNA levels were decreased by NMDA (5 μM) treatment for 24 hours. The expression of Rb was normalized to the amount of GAPDH to calculate the relative amount of the gene transcript (n=3 per group). Expression levels at each time point are depicted relative to the expression of the control 3 days after the onset of stimulation for comparison. Representative western blots showing that the level of P-Rb^Ser780 was increased by NMDA for up to 6 days, even when NMDA stimulation was restricted to 24 hours (n=3). For comparison, blots were exposed for same duration in each antibody. AP, apoptosis; CTL, control; MK, MK801; Thy, thymidine; ^*P<0.05, ^**P<0.01.

Effect of a single systemic injection of NMDA on the number of BrdU⁺ cells in the subgranular zone of the dentate gyrus 3 days and 28 days after administration. Diagrams show the way animals were treated (see Materials and Methods); arrows indicate drug application; arrowheads indicate BrdU detection. (A,B) Stereoscopic 3D-counting revealed that treatment with NMDA (100 mg/kg) decreased the number of BrdU⁺ cells (red) when analyzed 3 days after treatment, whereas MK801 (50 mg/kg) increased it (saline, 8758±883 cells/mm³; NMDA, 5720±510 cells/mm³; MK801, 11,503±456 cells/mm³, n=11 animals in each group). (C,D) Twenty-eight days after treatment, NMDA application resulted in an increase in the number of BrdU⁺ cells, whereas MK801 produced a decrease (saline, 3048±417 cells/mm³; NMDA, 4669±455 cells/mm³, MK801, 1657±124 cells/mm³, n=12 animals in each group). All cells were counterstained with DAPI (blue). Bar, 100 μm. Data are presented as the mean ± s.e.m. ^*P<0.05, ^**P<0.01.

Excitation increases acquisition of the neuronal phenotype and neuronal maturation of NPCs. Cells were induced to differentiate by withdrawal of bFGF in the presence of NMDA or MK801 for 3-5 days, followed by either dual immunocytochemistry or western blotting. (A) Diagram illustrating the experimental procedure. (B,C) Immunolabeling for Tuj1 (red). (B) Tuj1⁺ cells were counted and numbers are expressed as the change in percentage compared with DAPI-positive cells in four experiments. (D) Dual-label immunocytochemistry for MAP2⁺ (green) and GFAP⁺ (red) cells 5 days after inducing differentiation. (E) Western blots showing that NMDA increased the expression of Tuj1 and MAP2 in differentiating NPCs and had little effect on the expression of the astroglial marker GFAP. (F) Immunocytochemical analysis showing that NMDA increased the number of neurons with axonal arborizations. Asterisks indicate soma; arrows indicate branch points. (G) The number of axons exiting the soma (1' axon) and axonal branches arborizing from 1' axons (2' axons) were counted in neurons immunostained for Tuj1 (n=100 cells per treatment). (H) Synaptic varicosities (arrows) were increased by NMDA treatment. Inset, enlargement of the field indicated by the box. (I) Representative images from Ca²⁺ imaging. Differentiated cells (8 DIV) were treated as described for Fig. 2A. The mean values of all loaded cells are shown (n=10 for KCl; n=16 for NMDA). Images show a field of cells at time points corresponding to 1 and 2. MK, MK801. ^*P<0.05, ^***P<0.001. Bars, 50 μm (B,C), 20 μm (F), 5 μm(H).

NMDA receptor subunit gene expression during NMDA-induced proliferation and differentiation. Gene expression was investigated by semi-quantitative RT-PCR (A) and quantitative RT-PCR (B). Proliferating NPCs were treated with NMDA for 24 hours in the presence of bFGF and mRNA was extracted 72 hours after the stimulation. For differentiation, NPCs were treated daily with NMDA for 3 days in NB medium, and mRNA was isolated 24 hours after the last stimulation. The primer sets used for amplification of different subunits are listed in Materials and Methods. ^*P<0.05, ^**P<0.01, ^***P<0.001.