2.5. Quantification of Dopaminergic Cell Loss in SNpc

Dopaminergic neurons in the SNpc were quantified by stereology of TH+ cells according to the optical fractionator principle as described previously [23]. Every fourth section (section sampling fraction, ssf = 4) covering the full extent of the SNpc (−4.04 to −7.56 from bregma) was sampled for analysis, yielding 10–12 sections per animal. The average mounted section thickness (h) was 24.1 μm (±2.3) and no guard zones were used (thickness sampling fraction (tsf) = 1). Section thickness was measured at every fourth site while counting, and the area-sampling fraction (asf) was on average 0.112. Dissector volume (h*A_frame) was 86,400 mm³ on average, and the average number of dopaminergic neurons counted in each individual was 210 (±41). A maximal Gundersen coefficient of error (CE) [24] of 0.08 was accepted. For cells to be counted, we relied on the following criteria: the cell body had to be clearly defined by the TH+ marker with a visible lighter-stained nucleus; if cells were too dark and the nucleus was not clearly visible, they were still counted if the projections were distinctly visible as being part of their respective cell bodies. Of 24 animals, 6 were excluded from the analysis due to complications during surgery or with tissue processing, leaving 10 DA, and 8 DA.VRA1 for quantification. When correlating stereological cell counts with fiber O.D., 10 DA and 7 DA.VRA1 animals were included.