Abstracts

Rationale: Human stem cell-derived neurons represent a novel substrate for the development of in vitro models of epilepsy. Human induced pluripotent stem (iPS) cells generated from patients can be differentiated into diverse cell types, which enables investigation of disease biology in the context of a human genetic background. CRISPR/Cas9 can be used to introduce mutations in control iPS cells or correct mutations in disease cells, creating isogenic cell line pairs for probing the physiological effects of gene mutations. Human iPS cell systems will serve to complement existing rodent models or, in certain cases, supplant them if disease biology is not faithfully captured in the animal model. Novel assays with high information content are needed for the discovery of robust epilepsy-relevant cellular phenotypes as well as their response to pharmacological interventions. Methods: We have created an optogenetic platform called Optopatch that rapidly and robustly characterizes electrophysiological activity of neurons. Stimulation of action potentials is achieved with a blue light-activated channelrhodopsin (CheRiff). Fluorescence readout of changes in transmembrane potential is attained with an Archaerhodopsin variant (QuasAr). Optopatch components can be expressed in all cells or expressed in disjoint populations using Cre-recombinase to enable stimulation of presynaptic neurons and readout of post-synaptic neurons. A hybrid spatial-temporal PCA/ICA algorithm segments high-speed Optopatch recordings to identify active neurons. Optopatch assays yield more than 80 different functional parameters. Parameters are combined with information from other modalities such as morphological measures and average spike waveforms. Statistical significance testing is performed to identify the set of phenotype-specific properties. We use dimensionality reduction and regression models to maximize the sensitivity of the phenotype. Results: We have demonstrated robust Optopatch measurements of intrinsic excitability recordings in both NGN2 excitatory and inhibitory human iPS cell-derived neurons. Using combinations of synaptic blockers, we pharmacologically isolate GABAR/NMDAR/AMPAR signals, and extract excitatory and inhibitory post-synaptic potential (PSP) properties including amplitude, rise time decay time, width and area from tens of thousands of cells in a single day. In addition to high-throughput assays of PSPs, we have also developed assays to interrogate network connectivity and microcircuits by using a digital mirror device to deliver spatially targeted stimulation to individual cells. We have extended the Optopatch technology to microtiter plates, enabling high-throughput screening applications. Assessment of excitability changes in the presence of the Kv7 agonist ML213 demonstrates that a Z’>0.5 can be achieved using our approach. Conclusions: Optopatch enables measurements of intrinsic neuronal excitability and synaptic activity from thousands of neurons, both rodent and human iPS cell-derived, with single-cell spatial resolution, millisecond temporal resolution and vastly higher throughput than manual patch clamp. Application of these capabilities to cellular models of genetically defined epilepsies promises to deliver novel, in vitro phenotypic disease signatures that can serve as the basis for screening campaigns in drug discovery. Funding: NIH NIMH Phase I SBIR: 1R43MH112273-01NIH NINDS Phase I and Phase II SBIR: 5R43NS087714-02