Brain derived neurotrophic factor (bdnf)

Drugs were prepared as stock solutions for frozen aliquots at − 20 °C. They were diluted from the stock solution to the final desired concentration in the ACSF before being applied to the perfusion solution. BDNF (1, 20, 50 ng/ml), nimodipine (10 μM), d (−)-2-amino-5-phosphonopentanoic acid (AP-5, 100 μM), (+)-α-methyl-4-carboxylphenlyglycine (MCPG, 500 μM), K252a (200 nM), NASPM (50 μM), ZIP (5 μM) and NB001 (20 μM) were used in the current study. Drugs were infused during the period of the horizontal bar on the graphs. NB001 was provided by NeoBrain Pharmac Inc (Canada). BDNF, nimodipine, MCPG, K252a, ZIP was purchased from Tocris Bioscience. AP-5, NASPM were purchased from Hello Bio.

Cultured cells were incubated for 24 h with 40 μM NTS (Calbiochem/Merck Millipore, Fontenay sous Bois, France) or 100 ng/ml human recombinant BDNF (Alomone labs, Jerusalem, Israel). Optimal concentrations of exogenous BDNF and NTS were determined previously.^{14 (link), 21 (link)} The non-peptide NTSR antagonist SR142948A (Tocris Biosciences, Bristol, UK) was used at 67 nM,^{56 (link)} PTX (Sigma-Aldrich, St Louis, MO, USA) at 200 ng/ml^{35 (link), 57 (link)}; TrkB inhibitors were K252a (Alomone Labs) at 100 nM^{58 (link), 59 (link)} or ANA12 (Tocris Biosciences) at 100 μM.^{60 (link)}

For derivation of motor neurons, an established differentiation protocol was used (Guo et al. 2017 (link); Nijssen et al. 2019 (link)). After dissociation of embryoid bodies at day 10, motor neuron progenitors were plated in clear bottom, black 96-well plates (CLS3603, Corning), coated with laminin (Sigma-Aldrich), Fibronectin (Sigma-Aldrich), and poly-L-ornithine (Thermo Fisher Scientific), at 17,000 cells/well in Neurobasal supplemented with B27, 10 ng/mL of both brain-derived neurotrophic factor (BDNF, Peprotech) and glial cell-derived neurotrophic factor (GDNF, Peprotech), 200 nM retinoic acid (Sigma-Aldrich) for 1 d, and 10 μM DAPT (Tocris) for 4 d. After day 14, the media consequently contained only BDNF, GDNF, and ascorbic acid; media was changed every other day. For the growth factor deprivation, BDNF and GDNF were removed from the media at day 21. From this time point and onward, the motor neurons were treated with GDF15 (Peprotech) at the indicated concentrations with media changes every other day.