Figure 3.

METH induces intracellular calcium signaling in GCaMP6s-transfected astrocytes. Primary human astrocytes were transfected with GCaMP6s, a plasmid expressing an ultrasensitive protein calcium sensor, and allowed to recover overnight. MOCK-transfected astrocytes were maintained as controls in parallel. Transfected cells were treated with METH (500 μM, hatched bars) and ionomycin (10 μM) as a positive control [images not shown, (A–C)]. Fluorescence was visualized by confocal microscopy, and images were captured every 500 msec. (A–C) depict images taken from one specific cell prior to METH addition (A), and post-METH addition at 12 and 100 s, respectively (B,C). The histogram shows cumulative data of the pictured astrocyte captured over the entire imaging period (D). [Ca⁺²]_i were quantified with a Fura-2 standard curve, representing changes in emission at 340 and 380 nm at an excitation of 510 nm (image not shown). Absolute [Ca⁺²]_i are represented in (E,F). ((A–E)) is a single astrocyte traced over time representing average changes (E). Several astrocytes (n = 15) are represented as a bar graph (F). (A–E) are a single cell representing average change of multiple astrocyte donors that were tested; each was analyzed in a minimum of triplicate determinations. ***p < 0.001.