Ti e inverted live cell imaging system

Cultured midbrain neurons were treated with AAV1-GCaMP6f day 4 in vitro. After 4–6 days, neurons were imaged on a Nikon Ti-E Inverted Live Cell Imaging System. GCaMP was measured at an excitation of 470 nm. For all conditions, baseline images were acquired for 30 s followed by 4 min and 30 s of imaging after treatment. For controls, external solution or 1 μM PRE-084 was added after baseline imaging. For METH treatment, neurons were incubated with 1 μM PRE-084 or vehicle for 30 min at 37 °C prior to imaging. After baseline imaging, 10 μM METH was added. To determine the selectivity of the dose of PRE-084, neurons pretreated with 1 μM BD1063 for 15 min prior to PRE-084 treatment. The DAT-dependence of METH-mediated increase in calcium was tested by pretreating the neurons with 10 μM nomifensine for 15 min prior to METH treatment. For quantification, experimenter-defined ROIs were created around the cell body of each individual neuron. Background fluorescence was subtracted from each frame. The percent change in fluorescence over baseline was calculated as described above. Images are presented in grayscale.