Chapter 2 74 compounds. Compounds were mixed in and electrical activity was recorded for 5 minutes at 37°C and 5% CO2 flow after a 5 minute incubation period under the same conditions. Plain Neurobasal medium was used to wash out the compounds. After washing, 50% of old and 50% of new Neurobasal/B27 medium with fresh supplements (BDNF, GDNF, IGF, cAMP) were added. Cultures were let to recover for 24h before addition of the next compound. ImageJ analysis For synaptic analysis the NeuronJ plugin was used to trace MAP2+ dendrites. In SynaptoCount, the dendritic tracing was loaded and used to determine the total number of synapses by counting the number of Synaptophysin1+ puncta present on the dendrites. The amount of VGAT+ synapses was determined by the number of VGAT+/Synaptophysin1+ puncta present on dendrites. The synaptic density was calculated by dividing the total number of synapses by the pixel length of the dendrites. The percentage of VGAT+ synapses was determined by dividing the number of VGAT+ synapses by the total number of synapses. For myelin analysis the NeuronJ plugin was used to trace NF-200+ axons, and the pixel length of axons was measured. Neurite tracings were then loaded on MBP staining, and the pixel length of neurites that showed co-localization of MBP and NF-200 staining was determined. The percentage of myelinated axons was determined by dividing the length of MBP+/NF200+ neurites by the total length of NF200 neurites. References Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9(4):357-359. Liao Y, Smyth GK, Shi W. The Subread aligner: fast, accurate and scalable read mapping by seed-and-vote. Nucleic Acids Res. 2013;41(10):e108. Robinson MD, McCarthy DJ, Smyth GK. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2010;26(1):139-140. Consortium EP. An integrated encyclopedia of DNA elements in the human genome. Nature. 2012;489(7414):57-74. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA- seq data with DESeq2. Genome Biol. 2014;15(12):550. Nadadhur AG, Emperador Melero J, Meijer M, et al. Multi-level characterization of balanced inhibitory-excitatory cortical neuron network derived from human pluripotent stem cells. PLoS One. 2017;12(6):e0178533. Dooves S, Nadadhur AG, Gasparotto L, Heine VM. Co-culture of Human Stem Cell Derived Neurons and Oligodendrocyte Progenitor Cells. Bio Protoc. 2019;9(17):e3350.
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