Axioplan 2 inverted fluorescence microscope

Dopamine neuron neurodegeneration and dendritic blebbing was determined using worms that express GFP only in dopamine neurons under the dat-1 dopamine transporter promoter. Treatments that cause a loss of GFP-positive neurons cause a corresponding loss of dopamine neuron cell bodies (Nass et al., 2002 (link)). Pdat-1:GFP worms were aged on agar plates containing 25 μM FUdR to be observed at designated ages. At every time point, worms were mounted onto a 1% agar pad on a glass slide, paralyzed using 2 mM levamisole, and enclosed with a coverslip. Imaging of these animals was carried out with an Axioplan 2 inverted fluorescence microscope (Zeiss, Oberkochen, Germany). At each time point for the neurodegeneration assay remaining CEP, ADE, and PDE neurons were quantified. At each time point for the dendritic blebbing assay the total number of fluorescent blebs observed in the four rostral dopaminergic dendrites were quantified. Blebs, bead-shape protrusions from the neuronal processes, were defined as distortions of the dendrite structure with an area greater than approximately 5 μm². Blebbing increases with age in several classes of neurons and correlates with the dysfunction of behaviors associated with those neurons (Pan et al., 2011 (link)). Three replicates of 10 animals per time point were completed.