Four-month-old male mice were terminated, and their brains were dissected. Fixation, preparation of histological sections, staining with anti-tyrosine hydroxylase antibody (TH, mouse monoclonal antibody, clone TH-2, Sigma diluted 1:1000) and stereological counting of TH-positive neurons in the SNpc and ventral tegmental area (VTA) were performed as described [97 (link),98 (link)].
Briefly, the margins of SNpc and VTA on stained sections were outlined using distribution atlas of TH-positive cells [99 (link)]. The first section for counting was randomly chosen from the first ten sections that included the SN/VTA region. Starting from this section, on every fifth section, TH-positive cells with a clearly visible nucleus were counted through the whole region. ZEN Microscopy Software (Carl Zeiss) was employed to measure diameters of 30 nuclei of dopaminergic neurons in each of these regions of every mouse brain included in this study. The nuclei were chosen randomly, and the distance measured as the horizontal length as they appeared on the screen. A mean was calculated for each animal and used for Abercrombie’s correction [100 (link)] to obtain an actual number of TH positive cells in the structure.
Briefly, the margins of SNpc and VTA on stained sections were outlined using distribution atlas of TH-positive cells [99 (link)]. The first section for counting was randomly chosen from the first ten sections that included the SN/VTA region. Starting from this section, on every fifth section, TH-positive cells with a clearly visible nucleus were counted through the whole region. ZEN Microscopy Software (Carl Zeiss) was employed to measure diameters of 30 nuclei of dopaminergic neurons in each of these regions of every mouse brain included in this study. The nuclei were chosen randomly, and the distance measured as the horizontal length as they appeared on the screen. A mean was calculated for each animal and used for Abercrombie’s correction [100 (link)] to obtain an actual number of TH positive cells in the structure.
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