%0 Journal Article %1 Bradley2018 %A Bradley, Jenifer %A Metea, Monica %A Strock, Christopher J. %D 2018 %J Journal of Pharmacological and Toxicological Methods %K imported %R 10.1016/j.vascn.2018.01.456 %T Evaluating the pharmacology and neurotoxic predictability of neuroactive compounds using human induced pluripotent stem cell-derived glutamatergic neurons co-cultured with astrocytes using a microelectrode array platform %X Microelectrode array (MEA) technology is recognized as a robust and reliable tool for assessing the seizurogenic and neurotoxic potential of chemical entities using rodent neuronal models. Until recently, human neuronal models lacked complex burst organization and network characteristics, which made MEA electrophysiological neurotoxic prediction challenging. Advancements in hiPSC neuronal models have addressed these limitations and provided a viable option for assessing CNS drug-induced liabilities. We have developed an early screening model for neurotoxicity using hiPSC glutamatergic neurons with astrocytes. The co-cultures were plated on 48-well MEA plates and maintained for 14-18 days, allowing formation of a robust neural network displaying complex burst organization and network (synchrony) characteristics. When this maturation process was complete, the cells were treated with different target-specific neurotoxins and seizurogenic compounds, and dose-response curves were generated. These included the proconvulsants bicuculline and picrotoxin (GABA A receptor antagonists); SNC80 ($\delta$-opioid receptor agonist); strychnine (glycine receptor antagonist); 4-aminopyridine (potassium channel blocker); pilocarpine (cholinergic and muscarinic receptor agonist); and the neurotoxin domoic acid. Spike train analyses aligned with expected in vivo effects of these compounds and confirmed the predictability of the model. Compared to vehicle control 0.2\% DMSO, 10µM domoic acid caused a complete loss in spike activity; 4-aminopyridine caused an increase in burst organization and synchrony. Strychnine caused a unique dose response pattern with changes in spike/burst rates and effects on burst and network organization while SNC80 caused a significant increase in burst activity and changes in burst structure. Pilocarpine caused a complete loss in burst organization and synchrony with a decrease in firing and burst rates. In conclusion, hiPS cell-derived glutamatergic neurons plated with hiPSC derived astrocytes are a robust model suitable for the evaluation of potential neurotoxic and seizurogenic compounds when tested on a multi-well MEA platform. Cellular Dynamics International's (CDI) GlutaNeurons and iCell Astrocytes  iCell GlutaNeurons: iPS cell-derived human glutamatergic-enriched cortical neurons.  iCell Astrocytes: iPS cell-derived human astrocytes. Axion BioSystems' Maestro Microelectrode Array (MEA) Platform  All recordings were acquired on the Axion Maestro platform using 48-well configured MEA plates. The Axion ECmini was used to deliver pre-mixed CO 2 throughout the recordings. A Constant temperature of 37°C was maintained through the software controller. Figure 1A. The Maestro, Axion BioSystems. 768 recording channels with fully integrated heater and software controls. Accommodates 12, 48 and 96 well MEA plates. Figure 1B. 48 well configured MEA plate, Axion BioSystems. 16 microelectrodes per well, ANSI compliant, nano-textured gold electrodes with evaporation reducing lid. 1A 1B Methods  48-well MEA plates were pre-coated with a 0.07\% PEI solution, rinsed and allowed to dry overnight.  iCell GlutaNeurons and iCell Astrocytes were rapidly thawed and slowly diluted (to avoid osmotic shock) with BrainPhys Neuronal Medium supplemented with iCell DopaNeurons Supplement, iCell Nervous System Supplement, N2 supplement, Laminin and Penicillin/Streptomycin.  After a gentle centrifugation step, the cells were resuspended at the appropriate density with cell dotting medium (complete BrainPhys Medium supplemented with additional laminin).  A 10µL droplet of a cell suspension containing 120K iCell GlutaNeurons and 20K iCell Astrocytes was dispensed directly over the electrode grid of each well of a 48-well MEA plate.  The cells were incubated, humidified at 37°C in 5\% CO 2 for 1 hour.  500 µL of complete BrainPhys medium was slowly added to each well in a 2-step process to avoid detaching the cells.  Cells were maintained for 14-18 days by changing 50\% medium 3 times a week.  Recordings were acquired on the Axion Biosystems' Maestro periodically throughout the maintenance period to document the maturation process.  Recordings were also acquired immediately before compound treatment (baseline) and 1 hour post treatment. All experiments were performed 14-18 days after plating.  Custom MATLAB scripts were used to analyze the spike trains. Endpoints reported include: firing rate, burst rate, number of spikes in bursts, percent isolated spikes, ISI CV, normalized IQR burst duration, burst duration, interburst interval, mean of ISI-distance, IQR/median ISI, skewness ISI, normalized MAD burst spike number, median/mean ISI and median ISI.  Raster plots were generated with Axion BioSystems' Neural Metric Tool.

@article{Bradley2018, abstract = {Microelectrode array (MEA) technology is recognized as a robust and reliable tool for assessing the seizurogenic and neurotoxic potential of chemical entities using rodent neuronal models. Until recently, human neuronal models lacked complex burst organization and network characteristics, which made MEA electrophysiological neurotoxic prediction challenging. Advancements in hiPSC neuronal models have addressed these limitations and provided a viable option for assessing CNS drug-induced liabilities. We have developed an early screening model for neurotoxicity using hiPSC glutamatergic neurons with astrocytes. The co-cultures were plated on 48-well MEA plates and maintained for 14-18 days, allowing formation of a robust neural network displaying complex burst organization and network (synchrony) characteristics. When this maturation process was complete, the cells were treated with different target-specific neurotoxins and seizurogenic compounds, and dose-response curves were generated. These included the proconvulsants bicuculline and picrotoxin (GABA A receptor antagonists); SNC80 ($\delta$-opioid receptor agonist); strychnine (glycine receptor antagonist); 4-aminopyridine (potassium channel blocker); pilocarpine (cholinergic and muscarinic receptor agonist); and the neurotoxin domoic acid. Spike train analyses aligned with expected in vivo effects of these compounds and confirmed the predictability of the model. Compared to vehicle control 0.2{\%} DMSO, 10µM domoic acid caused a complete loss in spike activity; 4-aminopyridine caused an increase in burst organization and synchrony. Strychnine caused a unique dose response pattern with changes in spike/burst rates and effects on burst and network organization while SNC80 caused a significant increase in burst activity and changes in burst structure. Pilocarpine caused a complete loss in burst organization and synchrony with a decrease in firing and burst rates. In conclusion, hiPS cell-derived glutamatergic neurons plated with hiPSC derived astrocytes are a robust model suitable for the evaluation of potential neurotoxic and seizurogenic compounds when tested on a multi-well MEA platform. Cellular Dynamics International's (CDI) GlutaNeurons and iCell Astrocytes  iCell GlutaNeurons: iPS cell-derived human glutamatergic-enriched cortical neurons.  iCell Astrocytes: iPS cell-derived human astrocytes. Axion BioSystems' Maestro Microelectrode Array (MEA) Platform  All recordings were acquired on the Axion Maestro platform using 48-well configured MEA plates. The Axion ECmini was used to deliver pre-mixed CO 2 throughout the recordings. A Constant temperature of 37°C was maintained through the software controller. Figure 1A. The Maestro, Axion BioSystems. 768 recording channels with fully integrated heater and software controls. Accommodates 12, 48 and 96 well MEA plates. Figure 1B. 48 well configured MEA plate, Axion BioSystems. 16 microelectrodes per well, ANSI compliant, nano-textured gold electrodes with evaporation reducing lid. 1A 1B Methods  48-well MEA plates were pre-coated with a 0.07{\%} PEI solution, rinsed and allowed to dry overnight.  iCell GlutaNeurons and iCell Astrocytes were rapidly thawed and slowly diluted (to avoid osmotic shock) with BrainPhys Neuronal Medium supplemented with iCell DopaNeurons Supplement, iCell Nervous System Supplement, N2 supplement, Laminin and Penicillin/Streptomycin.  After a gentle centrifugation step, the cells were resuspended at the appropriate density with cell dotting medium (complete BrainPhys Medium supplemented with additional laminin).  A 10µL droplet of a cell suspension containing 120K iCell GlutaNeurons and 20K iCell Astrocytes was dispensed directly over the electrode grid of each well of a 48-well MEA plate.  The cells were incubated, humidified at 37°C in 5{\%} CO 2 for 1 hour.  500 µL of complete BrainPhys medium was slowly added to each well in a 2-step process to avoid detaching the cells.  Cells were maintained for 14-18 days by changing 50{\%} medium 3 times a week.  Recordings were acquired on the Axion Biosystems' Maestro periodically throughout the maintenance period to document the maturation process.  Recordings were also acquired immediately before compound treatment (baseline) and 1 hour post treatment. All experiments were performed 14-18 days after plating.  Custom MATLAB scripts were used to analyze the spike trains. Endpoints reported include: firing rate, burst rate, number of spikes in bursts, percent isolated spikes, ISI CV, normalized IQR burst duration, burst duration, interburst interval, mean of ISI-distance, IQR/median ISI, skewness ISI, normalized MAD burst spike number, median/mean ISI and median ISI.  Raster plots were generated with Axion BioSystems' Neural Metric Tool.}, added-at = {2019-06-17T23:34:05.000+0200}, author = {Bradley, Jenifer and Metea, Monica and Strock, Christopher J.}, biburl = {https://www.bibsonomy.org/bibtex/2d2a82ffaca901d42b9a06319ed807018/fcdi}, doi = {10.1016/j.vascn.2018.01.456}, interhash = {9c247d8ecc809e61abe66372be8c3306}, intrahash = {d2a82ffaca901d42b9a06319ed807018}, issn = {10568719}, journal = {Journal of Pharmacological and Toxicological Methods}, keywords = {imported}, timestamp = {2019-06-18T22:58:44.000+0200}, title = {{Evaluating the pharmacology and neurotoxic predictability of neuroactive compounds using human induced pluripotent stem cell-derived glutamatergic neurons co-cultured with astrocytes using a microelectrode array platform}}, year = 2018 }