Excitatory Pharmacology Induces Seizurogenic Phenotypes in Ipsc-Derived Neuronal Cultures in Vitro

P-232 Human iPSC-derived neurons provide relevant mechanistic insight into neurotoxicity

○ Blake D ANSON1, Akihiko AZUMA2, Susan DELAURA1, Elisabeth ENGHOFER1, Lisa HARMS1, Lori NORKOSKY1, Rachel LEWIS1, Kwi Hye KIM1, Kile MANGAN1, Michael HANCOCK1, Christian KANNEMEIER1, Coby CARLSON1, Brad SWANSON1 1&HOOXODU'\QDPLFV,QWHUQDWLRQDO$)XML)LOP&RPSDQ\2&HOOXODU'\QDPLFV-DSDQ,QF$)XML)LOP&RPSDQ\ Human cell types differentiated from induced pluripotent stem cells (iPSC) offer a unique access to human cellular material for toxicity screening. Such access is valuable for tissues such as those of neuronal origin that have been traditionally impossible to obtain. An additional key advantage to iPSC systems is contextual relevance for mechanistic studies that overcome many limitations of rodent primary cells and immortalized cell lines. Here we provide examples of using iPSC-derived cortical GABAergic, glutamatergic, and midbrain dopaminergic neurons in structural and electrical toxicity investigations. Interrogations focus on unique neuronal activity including Ca2+ oscillations, excitotoxicity, and seizurogenic activity. The utility of the various models is demonstrated by; 1) sub-type specific behavior 2) differential responses to toxicants, and 3) multiplexing experiments to move from phenotypic to mechanistic interpretations. The highly-excitatory iPSC-glutamatergic neurons display synchronized network-level bursting behaviors on the MEA, while cortical GABAergic neurons also display electrical activity｠but lack｠network synchronization. iPSC-neurons are susceptible to neurotoxicants with both the broad- spectrum kinase inhibitor staurosporine as well as glutamate inducing cell death. The effects of glutamate are inhibited by the antagonists DNQX and D-AP5 indicating excitotoxicity as the underlying mechanism for glutamate. Further, multiplexed experiments enable easy movement from phenotypic responses such as LDH release and ATP production to specific aspects of cell death such as apoptosis via caspase activation. Overall, iPSC-derived neurons exhibit functional glutamate pathways that respond appropriately to known agonists and antagonists, thus providing biologically relevant models for identifying emerging targets for neuro-based toxicity research and beyond.

P-233 Excitatory pharmacology induces seizurogenic phenotypes in iPSC-derived neuronal cultures in vitro

○ Kile MANGAN1, Ko ZUSHIDA2, Kwi Hye KIM1, Coby CARLSON1, Christian KANNEMEIER1, Tromondae FEASTER1, Blake ANSON1, Brad SWANSON1 1&HOOXODU'\QDPLFV,QWHUQDWLRQDO$)XML)LOP&RPSDQ\2&HOOXODU'\QDPLFV-DSDQ,QF$)XML)LOP&RPSDQ\ Some‘regular’ prescription medications are known to display seizurogenic potential. Such adverse events can be mis- diagnosed as epilepsy. Therefore it is crucial to test for seizurogenesis during drug development. Central to this vision is induced pluripotent stem cell (iPSC) technology, which provides a human cell-based platform to expand our understanding of how pharmacology affects the human condition. We have produced human neuronal populations from iPSC that, when combined with micro-electrode array (MEA) technology, advances and provides unparalleled investigation into how pharmacology affects human neuron-networked activity. Excitatory populations of iPSC-derived cortical neurons (e.g. iCell GlutaNeurons) develop and display network-level coordinated neuronal activity in vitro, evident by synchronized bursts captured and measured via MEA. Assay optimization dictates best practice timelines for‘seizurogenic potential’ screening occurs between DIV20-23, post-thaw. Excitatory pharmacology that displays dose-dependent seizurogenic-effects include picrotoxin [0.3-100 µM], GABAzine [1-100 µM], bicuculline [4-400 µM], pentylenetetrazol [7 µM-2 mM], 4-aminopyridine [1.6-50 µM], and kainic acid [0.4-300 µM]. Activity metrics displaying dose-dependent changes with pharmacology include: mean firing rate,‘single-channel’ burst rate, intensity and duration,‘network-level’ burst rate, intensity and duration, and synchrony measures. The presented data illustrate and marry the“seizure-in-a-dish” technology, previously limited to rodent-only investigation, with human iPSC-technology to create an unprecedented investigatory space for drug screening.