Figure 3.

Localization of HA-DAT in somatic regions of DA neurons. A, B) Specificity of HA11-Alexa Fluor 488 binding to neurons. Live postnatal DA neurons derived from WT pups were incubated with HA11-Alexa Fluor 488 for 4 h at 37°C (green), fixed, and stained with TH antibody (red) to identify DA neurons. Images depict examples of soma (A) and axons (B). Individual optical section of z stack of images is shown. C) In the soma, a large pool of HA-DAT is in intracellular compartments. Living postnatal DA neurons from HA/HA (HA-DAT) pups were incubated with HA11-Alexa Fluor 488 for 4 h at 37°C to label HA-DAT (green); then they were fixed and stained with the TH antibody (red) under conditions identical to those in A and B. D) Living postnatal HA-DAT DA neurons were incubated with HA11-Alexa Fluor 488 (green; HA-DAT), washed, and stained with Hoechst 33342 (blue; labels nucleus). Time-lapse imaging was performed through FITC and DAPI filter channels at 33°C. Somatodendritic area of the neuron is shown. E, F) Magnified images of the boxed regions in D, corresponding to several time points of the time-lapse sequence. Image acquisition times (s) are indicated. Arrows indicate examples of mobile vesicular-tubular endosomes. G) Distal dendrites. Several time points of the time-lapse sequence are shown. Intracellular HA-DAT vesicles displayed rapid bidirectional movement. Red arrows indicate vesicles moving in the retrograde direction; green arrows indicate vesicles moving anterogradely. Time (s) is indicated. Scale bars = 5 μm (A–F); 10 μm (G).