Fig. 1

Locally induced protein overexpression via injection of rAAV vectors in the LC region. a rAAV1/2 vectors contain a chicken β-actin promoter hybridized with a CMV immediate early enhancer sequence (CMV/CBA) to drive expression of either A53T-aSYN or luciferase (control). ITR, inverted terminal repeat; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element; BGH-pA, bovine growth hormone polyadenylation sequence. b Experimental design and schematic illustration of the injection site. Animals were consecutively sacrificed after 3 days, 1, 3, 6 and 9 weeks for immunohistochemical evaluation. c-f Analysis of the infection or transduction rates via double immunofluorescence staining for TH (red) and viral coating proteins (VP, green) (c, d) or TH (red) and human A53T-aSYN (green) or luciferase (green) (e, f), respectively. Co-localization of TH and VP indicates successful entry of viral particles, whereas co-localization of TH and A53T-aSYN/luciferase indicates successful protein expression. Student’s t-test revealed no significant difference between the transduction rates of the two vectors (p > 0.05, n = 3 animals per protein) (d, f). Values (mean ± SEM) represent the percentage (%) of TH-positive neurons that were also positive for VP, aSYN or Luc. g Overview of the pontine brainstem (Bregma: − 5.30 mm) stained against TH (red) and human aSYN (green) depicting the transduced area 3 days post-injection. Abbreviations: L, left; R, right; PB, parabrachial nucl.; SUV, superior vestibular nucl.; MV, medial vestibular nucl.; DTN, dorsal tegmental nucl.; LDT, laterodorsal tegmental nucl. h Higher magnification overview image of the TH-positive LC region (red) transduced with human A53T-aSYN (green). Scale bars 25 μm in c, e; 500 μm in g and 100 μm in h