Automated Intracellular Pharmacological Electrophysiology for Ligand-Gated Ionotropic Receptor and Pharmacology Screening

Abstract

Communication between neuronal cells, central to brain function, is performed by several classes of ligand-gated ionotropic receptors. The gold standard technique for measuring rapid receptor response to agonist is manual patch-clamp electrophysiology, capable of the highest temporal resolution of any current electrophysiology technique. We report an automated highprecision patch-clamp system which substantially improves the throughput of these timeconsuming pharmacological experiments. The patcherBot Pharma enables recording from cells expressing receptors of interest and manipulation of them to enable millisecond solution exchange to activate ligand-gated ionotropic receptors. The solution-handling control allows for autonomous pharmacological concentration-response experimentation on adherent cells, lifted cells, or excised outside-out patches. The system can perform typical ligand-gated ionotropic receptor experimentation protocols autonomously, possessing a high success rate in completing experiments, and up to a 10-fold reduction in research effort over the duration of the experiment.Using it, we could rapidly replicate previous datasets, reducing the time it took to produce an 8point concentration response curve of the effect of propofol on GABA A R deactivation from likely weeks of recording to ~13 hours of recording. On average, the rate of data collection of the patcherBot Pharma was a data point every 2.1 minutes that the operator spent interacting with the patcherBot Pharma . The patcherBot Pharma provides the ability to conduct complex and comprehensive experimentation that yields datasets not normally within reach of conventional systems that rely on constant human control. This technical advance can contribute to accelerating the examination of the complex function of ion channels and the pharmacological agents that act on them.

De novo CLPTM1 variants with reduced GABAAR current response in patients with epilepsy

“…Meanwhile, overexpression of CLPTM1 in vivo decreased miniature inhibitory postsynaptic current amplitude in hippocampal CA1 neurons 24 . To investigate the effects of the two CLPTM1 variants on GABA A R‐mediated currents, we transfected three types of plasmids— CLPTM1 ‐WT, CLPTM1 ‐p.R454H, and CLPTM1 ‐p.R568Q—into a HEK293T cell line stably expressing GABA A R 33 . Whole‐cell voltage‐clamp recording was performed to compare the amplitude of GABA‐evoked currents among the CLPTM1 ‐WT, CLPTM1 ‐p.R454H, and CLPTM1 ‐p.R568Q groups.…”

“…HEK293T cells stably transfected with the α1, β2, and γ2 subunits of GABA A R (CRL1573, American Type Culture Collection) 33 were cultured in a 37°C incubator with 5% CO 2 . Dulbecco's modified Eagle medium/GlutaMAX medium (Life Technologies) was supplemented with 10% fetal bovine serum (Life Technologies) and 1% penicillin–streptomycin (PS, Life Technologies).…”

“…A previous study indicated that CLPTM1 altered GABA A R-mediated currents by trapping GABA A R in the endoplasmic reticulum and reducing surface expression of GABA A R, 24 current amplitude in hippocampal CA1 neurons. 24 To investigate the effects of the two CLPTM1 variants on GABA A R-mediated currents, we transfected three types of plasmids-CLPTM1-WT, CLPTM1-p.R454H, and CLPTM1-p.R568Q-into a HEK293T cell line stably expressing GABA A R. 33 Whole-cell voltage-clamp recording was performed to compare the amplitude of GABA-evoked currents among the CLPTM1-WT, CLPTM1-p.R454H, and CLPTM1-p.R568Q groups. As a result, the peak amplitude of GABA-evoked currents the variants CLPTM1-p. R454H and p.R568Q groups was significantly reduced compared to CLPTM1-WT (Figure 3A To minimize the effects of different cell sizes on the current amplitudes, the outside-out patch-clamp recording was performed to evaluate the effects of the two variants on GABA-evoked currents.…”

High-throughput combined voltage-clamp/current-clamp analysis of freshly isolated neurons

“…Another previously published method added automation to traditional manual patch-clamp rigs that use glass pipettes (as opposed to planar patch in our setup). 68 , 69 , 70 This method possesses many of the same advantages of traditional patch-clamp, with the added benefits of automation; however, it does not provide the advantages of increased throughput or the option of head-to-head comparison of multiple cell-types, as in our method.…”

Patch-walking: Coordinated multi-pipette patch clamp for efficiently finding synaptic connections

“…These advances rely on the con-current discovery that pipettes can be reused, rather than replaced after each recording attempt [23, 24, 25]. These improvements have enabled novel drug screening assays [26], deep in-vivo recordings [27], voltage indicator screening, and fluorescent cell targeted patch clamp [15].…”