SIM imaging and analysis for active zone release sites in vulnerable dopamine axons

Vulnerable dopamine neurons show higher LRRK2 expression, we wondered whether active zone changes may be prominent in vulnerable Aldh1a1+ as compared to resilient Aldh1a1- dopaminergic axons in Lrrk2G2019S mice. To accomplish this, we used a intersectional/subtractive approach that allows for simultaneous labeling of Aldh1a1+ with EYFP and Aldh1a1- neuronal with mCherry, respectively. Striatal brain sections were then stained with antibodies against bassoon to analyze the number and organization of active zone sites in Aldh1a1+ (EYFP+) or Aldh1a1- (mCherry+) axons from the same mouse (Figure 6B). Due to the high signal of bassoon in the striatum, we used structured illumination microscopy (SIM) for improved resolution, followed by 3D reconstruction analysis.