In both cases, 10 M of FM1-43 was added to the incubation medium. The R12 culture medium contains a pH indicator, and has a slight purple color at healthy pH. presynaptic. = 26) stored in 24-well plates or inside a circular chamber glued on top of the MEA (= 2). The tradition chambers were filled with R12 medium (Romijn et al., 1984) and all cultures were stored in an incubator, under standard conditions of 36C, 100% moisture, and 5% CO2 in air flow. We kept cells in tradition for at least 3 weeks, allowing for the development of adult networks. Medium was Rabbit Polyclonal to BRF1 refreshed twice a week (300 L of aged medium was replaced by 400 L new medium). For the induction of hypoxia, we placed the ethnicities under a Plexiglas hood, referred to as hypoxic chamber, where under a continuous circulation of SCR7 a computer controlled mixture of air flow and nitrogen was kept. Five % CO2 was added to the gas combination and moisture was managed. Ethnicities on coverslips were put in the hypoxic chamber in 24-well plates, the MEA tradition chambers were sealed with watertight but O2 and CO2 permeable foil (MCS; ALA medical). For experiments, cultures were transferred to a confocal microscope (Zeiss LSM 510). Experiments began after an accommodation period of at least 20 min. Immediately before the start of an experiment covers were removed from the tradition chambers to optimize visual access and to facilitate quick medium changes. This allowed oxygen to re-enter the medium, which occurred at a relatively slow rate (observe below). From that point, maintenance of sterility was not necessary any longer, because experiments typically lasted less than 10 min. Hypoxia Prior to the measurements, cultures were exposed to SCR7 hypoxia during 6 h. This was accomplished in the hypoxic chamber by replacing 90% of air flow by nitrogen, which yielded a decreasing of partial oxygen pressure (pO2) from pO2 160 mmHg to pO2 20 mmHg. Partial oxygen pressures were measured using an optical oxygen sensor (PHOSPOR, Ocean Optics). Earlier electrophysiological measurements clearly showed synaptic failure during 6 h of hypoxia at this depth (le Feber et SCR7 al., 2016). All solutions utilized for imaging were also kept in the hypoxic chamber to obtain equivalent pO2, prior to administration. Besides controlled hypoxia, the hypoxic chamber allowed keeping the neurons under standard conditions (36C, 100% moisture, and 5% CO2). Control coverslips (without hypoxia) were incubated inside a CO2 incubator under standard conditions. After this incubation period (exposure to hypoxia) the medium of coverslips was changed to medium comprising ionotropic receptor inhibitors 50 M DL-2 amino-5-phosphonovaleric acid (APV; a selective blocker of the NMDA glutamate receptor; Sigma-Aldrich) and 10 M 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; a selective AMPA receptor blocker; Sigma-Aldrich), which had been exposed to the same hypoxic conditions. Figure ?Number1A1A illustrates the experimental protocol. In the two ethnicities plated SCR7 on MEAs, we recorded spontaneous activity and reactions to electrical activation as explained in le Feber et al. (2016). These ethnicities were used to verify the effectiveness of excitatory blockade at this concentration by evaluation of their reactions to electrical activation. Two types of imaging solutions were used in different experiments: R12 cell tradition medium and the colorless altered Tyrode solutions (136 mM NaCl, 2.5 mM KCl, 10 mM HEPES, 10 mM glucose, 1.3 mM MgCl2, pH 7.4), to verify that the color of R12 did not interfere with detection of the FM dye. Open in a separate windows Number 1 Timeline of the experiments and verification of experimental conditions. (A) Cultures were exposed to 6 h of hypoxia at pO2 20 mmHg. Then, excitatory synaptic transmission was clogged by.
In both cases, 10 M of FM1-43 was added to the incubation medium
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