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Development of a high-throughput arrayed neural circuitry platform using human induced neurons for drug screening applications.

ABSTRACT: Proper brain function relies on the precise arrangement and flow of information between diverse neural subtypes. Developing improved human cell-based models which faithfully mimic biologically relevant connectivity patterns may improve drug screening efforts given the limited success of animal models to predict safety and efficacy of therapeutics in human clinical trials. To address this need, we have developed experimental models of defined neural circuitries through the compartmentalization of neuronal cell subtypes in a 96 well plate-based platform where each microwell is divided into two compartments connected by microchannels allowing high-throughput screening (HTS) of small molecules. We demonstrate that we can generate subtype-specific excitatory and inhibitory induced neuronal cells (iNs) from human stem cell lines and that these neurons form robust functional circuits with defined connectivity. Through the use of the genetically encoded calcium indicator GCaMP6f, we monitor calcium ion transients generated during neuronal firing between and within compartments. We further demonstrate functionality of the circuit by perturbing network activity through the addition of glutamate receptor blockers using automated liquid handling. Lastly, we show that we can stimulate network activity in defined neuronal subtypes through the expression of the designer receptor exclusively activated by designer drugs (DREADD) hM3Dq and application of the ligand clozapine-N-oxide (CNO). Our results demonstrate the formation of functional neural circuits in a high-throughput platform that is compatible with compound screening, representing an important step towards developing new screening platforms for studying and ultimately treating psychiatric brain disorders that arise from disordered neural circuit function.

SUBMITTER: Fantuzzo JA

PROVIDER: S-EPMC7339603 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Development of a high-throughput arrayed neural circuitry platform using human induced neurons for drug screening applications.

Fantuzzo Joseph A JA Robles Denise A DA Mirabella Vincent R VR Hart Ronald P RP Pang Zhiping P ZP Zahn Jeffrey D JD

Lab on a chip 20200301 6

Proper brain function relies on the precise arrangement and flow of information between diverse neural subtypes. Developing improved human cell-based models which faithfully mimic biologically relevant connectivity patterns may improve drug screening efforts given the limited success of animal models to predict safety and efficacy of therapeutics in human clinical trials. To address this need, we have developed experimental models of defined neural circuitries through the compartmentalization of ...[more]

PMID: 32064487

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Project description:Particulate matter (PM) exposure is a public health burden with poorly understood health effect mechanisms and lacking an efficient model to compare the vast diversity of PM exposures. Zebrafish (Danio rerio) are amenable to high-throughput screening (HTS), but few studies have investigated PM toxicity in zebrafish, despite the multitude of advantages. To develop standardized exposure procedures, the urban PM standard reference material (SRM) 1649b was used to systematically determine sample preparation methods, design experimental controls, determine concentration ranges and evaluation procedures. Embryos (n = 32/treatment) were dechorionated and placed into 96-well plates containing SRM1649b (0-200 μg/mL) at 6 h post fertilization (hpf). Developmental toxicity was assessed at 24 and 120 hpf by evaluating morphological changes, embryonic/larval photomotor behavior, and mortality. Differences from blank medium and particle controls were observed for all biological responses measured. Differences due to SRM1649b concentration and preparation method were also observed. Exposure to SRM1649b from DMSO extraction was associated with changes in morphology and mortality and hypoactivity in photomotor responses compared to the DMSO control for the whole particle suspension (76, 68%) and soluble fraction (59, 54%) during the embryonic and larval stages, respectively. Changes in behavioral responses were not observed following exposure to the insoluble fraction of SRM1649b from DMSO extraction. The toxicity bias from PM preparation provided further impetus to select a single HTS exposure method. Based on the biological activity results, the soluble fraction of SRM1649b from DMSO extraction was selected and shown to have concentration dependent cyp1a/GFP expression. This rapid, sensitive and consistently scalable model is a potentially cost-effective vertebrate approach to study the toxicology of PM from diverse locations, and provides a path to identifying the toxic material(s) in these samples, and discover the mechanisms of toxicity.

Dataset Information

Development of a high-throughput arrayed neural circuitry platform using human induced neurons for drug screening applications.

Publications

Development of a high-throughput arrayed neural circuitry platform using human induced neurons for drug screening applications.

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