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. 2021 Mar 13;95(5):1703–1722. doi: 10.1007/s00204-021-03024-0

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© The Author(s) 2021

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — Characterization of mixed cortical cultures (MCCs). Mixed cortical cultures (MCCs) were generated from NESCs according to Reinhardt et al. (2013), by differentiating them for > 20 days on Matrigel in neuronal differentiation medium. a Schematic display of MCC differentiation (BDNF brain-derived neurotrophic factor, GDNF glial cell-derived neurotrophic factor, TGFß transforming growth factor beta, cAMP cyclic adenosine monophosphate, AA ascorbic acid). b Gene expression profile of the neuronal marker RBFOX3. Gene expression was quantified by real-time PCR. Data are given relative to reference genes (RPL13A and TBP) and the NESC starting population (= DoD0, depicted as dotted line) for DoD10, DoD15, and DoD21. c Gene expression of synaptic markers. Gene expression of d glial markers and of e NMDA receptor sub-units NR1 (GRIN1), NR2A (GRIN2A) and NR2B (GRIN2B). The symbols show the values of single biological replicates and the coloured lines show their mean. Significance was tested with a t test; *p value < 0.05. f Immunofluorescence image of MCCs on DoD24 using antibodies against the neuron-specific cytoskeletal marker beta-III-tubulin (Tuj1) and the vesicular glutamate transporter 1 (vGlut1). The composite images also include the nuclei stained with H33342 (blue) Scale bar: 50 µm. g Cells on DoD0 and DoD24 were allowed to incorperate the nucleotide analog EdU, to visualize mitotic activity. EdU-positive cells were counted and are shown as percentage of total cell number ± SD. h MCCs were differentiated on MEA plates. Spontaneous spikes of electrical activity were recorded on various days of differentiation for the same cells. From these activity measurements (30 min each day), the parameters “network burst frequency”, “number of spikes per burst”, and “synchronizity coefficient” were calculated and plotted over time. The dotted line indicates the confidence interval. i On DoD24 MCCs on MEAs were treated with the GABA_A receptor antagonist bicuculline [1 µM] (blue, addition is indicated by the black arrow, baseline in red). The generation of spikes was recorded directly before and after administration, the number of spikes was binned (bin size 0.1 s) and a representative example of the acute response is shown. A full set of data over an extended time span is presented in Fig. S2 (color figure online)