Laser capture microscopy

Slides were placed into the slide holder of the microscope (Leica DM6000R/CTR6500) and cells were captured using the Leica LMD7000 system. To keep contamination by surrounding cells to a minimum, cutting outlines were drawn closely around individual cells (Supplementary Figs 1c, 4b,c, 6c,g). In spinal cord sections, only cells with an area of more than 200 μm² and a visible nucleus with nucleolus in the ventral horn of the spinal cord were selected. For the analysis of small neurons, cells were collected from the dorsal motor nucleus of the vagus nerve and the hypoglossal nucleus that had an area of 130–200 μm² and 200–300 μm², respectively. mDA neurons from the SNc or ventral tegmental area (VTA) were captured based on their size (>200 μm²), the presence of melanin and a visible nucleus. The same groups of mDA neurons were dissected after a quick tyrosine hydroxylase staining on a separate membrane slide with the same tissue distribution. Cells were cut at 40 × magnification while keeping laser power to a minimum. Relative humidity for all experiments was between 17 and 65% and temperature range was between 21 and 27 °C. The Leica system uses gravity to collect cells, which fall into the cap of the collection tube (0.2 ml PCR tubes, Biozym Scientific) that is placed directly under the slide with the membrane facing down. After cells were collected in the cap a small volume of lysis buffer (0.2% Triton X-100, with 2 U μl⁻¹ recombinant RNase inhibitor, Clontech) was added, followed by pipetting up and down five to ten times. Neurons (∼150–200) isolated from human tissues were collected in 5 μl lysis buffer. For mouse MNs, amounts of lysis buffer were reduced according to sample size: for 120 and 50 cells 5 μl of lysis buffer were used, for 30 cells 4 μl and for 10, 5, 2 and 1 cell samples 2.5 μl of lysis buffer were used. Samples were spun down in a table centrifuge (VWR) for 10 s and briefly placed back into the tube holder of the microscope to check that no cells were remaining in the cap. The tubes were carefully sealed with parafilm (Pechiney Plastic Packaging), labelled, and snap frozen on dry ice. The whole process from thawing the slides until sample freezing never took longer than 3 h in any of the experiments. Samples were kept in collection tubes at −80 °C until further processing. Preparations from empty tubes that were kept in the collector while capturing cells into adjacent PCR tubes served as negative controls. For the mouse study, samples were collected randomly from three to five different animals for each experimental group. The sample size for low cell number groups was increased due to the expected higher technical and biological variation. The exact numbers of samples and animals for each group are stated in Table 1. For human post-mortem tissues, we analysed samples from at least three different individuals for each group. Exact sample sizes are listed in Table 2.