Vocal changes in a zebra finch model of Parkinson’s disease characterized by alpha-synuclein overexpression in the song-dedicated anterior forebrain pathway
Fig 5
Asyn expression levels by molecular weight in Area X.
A) A representative Western blot loaded with low salt (LS) or urea (U) soluble fractions obtained from Area X of birds injected with either AAV5-CBA-eGFP or AAV5-CBA-ASYN following 2HRUD singing is shown on the top. A blot with non-surgical (NS) birds is a supplementary figure (S4 Fig). Western blots were labelled with an αsyn antibody for quantification of protein levels in Area X relative to GAPDH from the LS lane of the same sample. B) A representative coronal slice of zebra finch brain highlighting punched Area X, the ventral striato-pallidum (VSP) ventral and medial to Area X, and nidopallium (NP). Tissue biopsies were taken of song-dedicated nucleus Area X and neighboring non-song related motor area VSP prior to thionin staining to confirm punch accuracy. Image from Miller et al. [56] C) Quantification of blots. For quantification, αsyn expression levels for each fraction are grouped by molecular weight within each group. Western blots indicate that higher levels of monomeric (15kD) urea-soluble αsyn are expressed in the ASYN group compared to the NS group and for the GFP group. Levels of trimeric (~45-50kD) αsyn protein are lower in ASYN and GFP groups compared to NS. Interestingly, total levels of urea soluble αsyn are also lower in ASYN compared to NS. Summary statistics provided in Table 2. Subscripts under experimental condition on representative blot denote different birds (i.e., sample). The representative blot contains raw data from birds 1 and 2 of both ASYN and GFP control groups (all raw blot images can be found in Supporting Information). Horizontal dashed lines indicate where blot was cut for incubating blot with either ASYN (~15kD, 40–250+kD) or GAPDH (~35kD) antibody. Names on left side of ladder indicate primary antibody used for immuno-labeling that section of the blot. For boxplots, vertical lines are 95% confidence intervals. Bottom end of the box is first quartile (25 percentile), middle horizontal line is second quartile (50 percentile), and top end of the box is the third quartile (75 percentile). The circle points represent the mean value for each group. Statistical comparisons were made using a Welch’s test. * indicate p < 0.05. # indicates 0.05 < p < 0.1.