1. Application of High-Throughput Automated Patch-Clamp Electrophysiology to Study Voltage-Gated Ion Channel Function in Primary Cortical Cultures
- Author
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Kosalvisal Ouk, Bruce L Roth, Rodolfo Haedo, Julie M Brooks, Sokhom S. Pin, Tim Strassmaier, Corey B. Puryear, and May Fern Toh
- Subjects
0301 basic medicine ,Patch-Clamp Techniques ,Primary Cell Culture ,Endogeny ,Biochemistry ,Ion Channels ,Analytical Chemistry ,Cerebellar Cortex ,03 medical and health sciences ,0302 clinical medicine ,Automated patch clamp ,Drug Discovery ,Animals ,Humans ,Ion channel ,Voltage-gated ion channel ,Chemistry ,Drug discovery ,Multielectrode array ,High-Throughput Screening Assays ,Rats ,Electrophysiology ,030104 developmental biology ,Gene Expression Regulation ,Molecular Medicine ,Neuroscience ,030217 neurology & neurosurgery ,Function (biology) ,Biotechnology - Abstract
Conventionally, manual patch-clamp electrophysiological approaches are the gold standard for studying ion channel function in neurons. However, these approaches are labor-intensive, yielding low-throughput results, and are therefore not amenable for compound profiling efforts during the early stages of drug discovery. The SyncroPatch 384PE has been successfully implemented for pharmacological experiments in heterologous overexpression systems that may not reproduce the function of voltage-gated ion channels in a native, heterogeneous environment. Here, we describe a protocol allowing the characterization of endogenous voltage-gated potassium (Kv) and sodium (Nav) channel function in developing primary rat cortical cultures, allowing investigations at a significantly improved throughput compared with manual approaches. Key neuronal marker expression and microelectrode array recordings of electrophysiological activity over time correlated well with neuronal maturation. Gene expression data revealed high molecular diversity in Kv and Nav subunit composition throughout development. Voltage-clamp experiments elicited three major current components composed of inward and outward conductances. Further pharmacological experiments confirmed the endogenous expression of functional Kv and Nav channels in primary cortical neurons. The major advantages of this approach compared with conventional manual patch-clamp systems include unprecedented improvements in experimental ease and throughput for ion channel research in primary neurons. These efforts demonstrated feasibility for primary neuronal ion channel investigation with the SyncroPatch, providing the foundation for future studies characterizing biophysical changes in endogenous ion channels in primary systems associated with disease or development.
- Published
- 2020