Brain derived neurotrophic factor (bdnf)

Hypoxia modeling was performed on day 14 of culture development in vitro (DIV) by replacing the normoxic cultural medium with a medium containing low oxygen for 10 min. The oxygen was displaced from the medium in sealed chamber in which the air was replaced with an inert gas (argon). The oxygen concentration decreased from 3.26 mL/L (normoxia) to 0.37 mL/L (hypoxia) [18 , 19 ].
All cultures were divided into the following experimental groups: (1) BDNF (1 ng/mL) (Millipore, GF029, USA) (N = 9); (2) k252a (150 nM) (Sigma, K2015, Germany), an inhibitor of the Trk receptors (N = 9); and (3) BDNF (1 ng/mL) plus k252a (150 nM) (N = 9) added to the medium 20 min before hypoxia. In the control group (N = 9), hypoxia was induced without additional treatment.

Prior to differentiation, iPSC lines were adapted to feeder-free conditions using Matrigel (BD) and EBs were formed. After 4 days, neural induction was initiated as previously described (32 (link),40 ). Briefly, EBs were plated onto Geltrex-coated plates in Neural Induction medium 1 (DMEM/F12 supplemented with L-Glutamine (2 mM), N2 supplement, bovine serum albumin (BSA) (1 mg/ml), Y27632 (10 μM; Tocris), SB431542 (10 μM, Tocris), Noggin (200 ng/ml) and antibiotic/antimycotic (1% v/v)). After 4 days, medium was changed to Neural Induction medium 2 (as NI1, without SB431542 and Noggin and with addition of SHH C24II (200 ng/ml; SHH C24II; R&D Systems). After 6 days, medium was supplemented with FGF8a (100 ng/ml; R&D Systems), Heparin (5 μg/ml; Sigma), BDNF (20 ng/ml) and Ascorbic Acid (200 μM; Sigma)) and incubated for 7 days, until the appearance of dense neural rosette structures. Neural progenitor cells were manually selected and re-plated onto Poly-D-Lysine/Laminin-coated plates in final differentiation medium (DMEM/F12 supplemented with L-Glutamine (2 mM), N2 supplement, BDNF (20 μg/ml), GDNF (20 μg/ml), N6, 2′ -O-dibutyryladenosine 3′,5′ -cyclic monophosphate sodium salt (dcAMP, 0.5 mM; Sigma), Laminin (1 μg/ml) and antibiotic/antimycotic (1% (v/v)). Neurons were matured for 2 weeks in this medium before experimental procedures were carried out.

Rats were implanted with a bilateral intra-VTA cannula, as described above. Seven days after surgery, the effect of intra-VTA BDNF on cocaine CPP was assessed. In the BDNF group, BDNF (Sigma Aldrich; 25 ug in 0.5 ul saline) was injected into the VTA, over 3 min. A separate vehicle group received saline injections of the same volume. One day after BDNF or saline injection, cocaine (10 mg/kg, i.p.) place preference was run, as described above.
Opioid-naïve rats received systemic 7,8-dihydroxyflavone injections (Tocris Bioscience, Bristol, UK, 10 mg/kg, i.p.), a potent trkB agonist, 1 h prior to cocaine conditioning sessions. Control animals received vehicle (saline, i.p.) injections prior to cocaine conditioning sessions.

Recombinant BDNF (1 ng/mL, Merck, GF301, Germany), ANA-12 (a selective TrkB receptor blocker, 1 μM, Sigma-Aldrich, SML0209, Germany) or BDNF (1 ng/mL) and ANA-12 (1 μM) in combination were added to the culture medium daily beginning on the third day of culture development in vitro (DIV) (Figure 1).
Analyses of spontaneous bioelectrical and calcium activities, which included defining the internal neural network structure, morphological studies, and evaluation of the dynamics of functional characteristics of the mitochondrial respiratory chain in neural cells, were performed on DIV 7, 10, and 14.