METH increases the generation of ROS in rhNPC.

<p>Neurospheres derived from rhNPC were digested and cells were plated on poly-D-lysine-coated cover slips. Cells were treated with 300 µM METH for 24 hours or pre-treated with 10 mM NAC for 30 min, then treated with 300 µM METH for 24 hours. Cells were then subjected to mitochondrial ROS staining with MitoTracker® CM-H₂ROS (a reduced, nonfluorescent dye that fluoresces upon oxidation) (a2-d2, red), immunostaining with mouse nestin monoclonal antibody (a1-d1, green) and nucleus staining with DAPI (a3-d3, blue) and visualized by Zeiss Axiovert microscope. Cells were treated with 200 µM H₂O₂ for 30 min as a positive control (e1-f4).</p

METH induces mitochondrial fragmentation in rhNPC in a time-dependent manner.

<p>Neurospheres were digested and plated on poly-D-lysine-coated cover slips. After 24 hours NPC were treated with 300 µM METH for the indicated times and then subjected to mitochondrial staining with MitoTracker®Red (upper panel) and immunostaining with nestin monoclonal antibody (green) and nuclear staining with DAPI (blue) and visualized by Zeiss Axiovert microscope.</p

METH-induced apoptosis of rhNPC is accompanied by mitochondrial fragmentation.

<p>Neurospheres were digested and plated on poly-D-lysine-coated cover slips. After 24 hours rhNPC were treated with METH at indicated concentrations for another 24 hours. (A) NPC were stained with MitoTracker®Red CMXRos dye (red) and fixed with 3.7% formaldehyde. After permeabilization with 0.2% Triton X-100 in PBS at 4°C for 10 min, cells were subjected to immunostaining with monoclonal nestin antibody and nuclei were stained with DAPI (blue), then visualized by Zeiss Axiovert microscope. (B) Apoptotic nuclei, showing highly condensed and fragmented chromatin, as indicated with arrows in A-b2 and A-c2. The results are expressed as average±SD of triplicate samples. # denotes <i>p</i><0.05 in comparison to control; ## denotes <i>p</i><0.001 compared with control. (C) Cells were analyzed for mitochondrial morphology by fluorescence microscope using MitoTracker®Red. Typical mitochondrial phenotypes (A-a1) and fragmented mitochondria (A-b1 and A-c1) were quantified by counting cell number with or without fragmented mitochondria. # denotes <i>p</i><0.01 in comparison to control; ## denotes <i>p</i><0.001 compared with control.</p

Isolation and differentiation of rhNPC.

<p>rhNPC were dissociated from rat hippocampi and cultured with NPBM neural progenitor basal medium containing EGF, bFGF and NSF-1. The neurospheres were formed after culture for 7 days (Fig. 1A). Neurospheres were digested and plated on poly-D-lysine-coated cover slips and cultured with astrocyte differentiation medium or neuron differentiation medium for 7 days and then subjected to immunostaining with nestin (Fig. 1B), GFAP (Fig. 1C) and β-tubulin III (Fig. 1D) antibodies. rhNPC were cultured in six-well plates with differentiation medium for 3, 5 and 7 days, respectively, then subjected to western blotting with GFAP and β-tubulin III antibodies (Fig. 1E, 1F).</p

METH has no effect on Ca²⁺ influx or glutamate-mediated Ca²⁺ influx in rhNPC.

<p>rhNPC cultured on poly-D-lysine-coated cover slips (25 mm), were loaded with Fura-2 and monitored for calcium influx by micro-fluorescent imaging. (A) Different concentrations of METH were tested and calcium influx was measured. METH concentrations were 1: 50 µM; 2: 100 µM; 3: 500 µM; 4: 1 mM; 5: 5 mM; 6:100 µM glutamate as a positive control. (B) METH has no effect on glutamate-mediated AMPA receptor responses; a: glutamate (100 µM); b: glutamate (100 µM)+cyclothiazide (10 µM); c: glutamate (100 µM)+cyclothiazide (10 µM)+CNQX (10 µM); d: glutamate (100 µM)+500 µM METH+cyclothiazide (10 µM).</p