Figure 1.

Model system using cultured hippocampal neurons from human-α-synuclein:GFP transgenic mice. A, To develop a model system for α-synuclein overexpression, heterozygous h-α-syn:GFP+ pups (P0–P2) were optically screened (see Materials and Methods) and dissociated hippocampal neurons were plated from these mice (left); neurons from WT littermates were used as controls for all experiments (right). For quantitative studies, GFP+ (α-synuclein-overexpressing) boutons were isolated and colocalization of presynaptic proteins/dyes within the GFP+ve boutons was analyzed (see Materials and Methods for specific details of quantization algorithm). B, Top left, Native GFP fluorescence in a 3-month-old h-α-syn:GFP mouse brain section. Robust GFP expression was seen in the forebrain and hippocampus (boxed), with lower fluorescence levels throughout the neocortex. Low-power scanning representative image of native GFP fluorescence from live DIV-21 hippocampal cultures are shown. Note that numerous h-α-syn-positive punctate, bouton-like varicosities can be readily visualized in these cultures, with diffuse fluorescence in cell-bodies (magnified in inset). These methods allowed us to obtain robust α-synuclein expression in large neuronal populations and examine the evolving pathology over time.