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

Abstract

Communication between neuronal cells, which is 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 high-precision patch-clamp system that substantially improves the throughput of these time-consuming 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 data sets, reducing the time it took to produce an eight-point concentration-response curve of the effect of propofol on GABA type A receptor 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 data sets 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. SIGNIFICANCE STATEMENT: This work presents an automated intracellular pharmacological electrophysiology robot, patcherBot_Pharma, that substantially improves throughput and reduces human time requirement in pharmacological patch-clamp experiments. The robotic system includes millisecond fluid exchange handling and can perform highly efficient ligand-gated ionotropic receptor experiments. The patcherBot_Pharma is built using a conventional patch-clamp rig, and the technical advances shown in this work greatly accelerate the ability to conduct high-fidelity pharmacological electrophysiology.

Graphical abstract

Fig. 1.

Comparison of patcherBot vs. patcherBot_Pharma. (A) Cartoon of the previously published patcherBot (Kolb et al., 2019), assembled from an upright microscope, high sensitivity camera, custom pressure control box, quasi-four-axis electrode manipulator, and a motorized stage. (B) Cartoon of the patcherBot_Pharma, assembled from an inverted microscope, high sensitivity camera, custom pressure control box, quasi-four-axis electrode manipulator, a motorized microscope manipulator, two solution valves, and a solution exchange manifold. A manual detailing the components and the operation of the patcherBot_Pharma is provided on GitHub (https://github.com/riley-perszyk/patcherBot_pharma).

Fig. 2.

Repeatability of the physical manipulations required for fast solution exchange electrophysiological experiments. (A) Image of the recording chamber. (B) Cartoon illustrating the large distances (e.g., X-Y mm scale) the electrode must translate during experimentation. (C) Open-tip solution exchange times, using piezoelectric translator, across many repeated experimental cycles (cell locations, solution manifold interface, cleaning/wash bath). (D–F) Cell lifting procedure. (D) Image of an isolated cell in the whole-cell conformation before lifting (isolated cells are more reliably lifted than those with cellular processes to adjacent cells). (E) Spiral path (100 discrete segments) employed to lift isolated cells. (F) Resulting resistance plot showing a high-resistance seal is robustly maintained during the lifting process. (G–I) Patch-pulling procedure. (G) Image of a cell in the whole-cell conformation before pulling an outside-out patch. (H) Arc path (100 discrete segments) employed to pull outside-out patches. (I) Resulting capacitance and resistance plots showing successful high-resistance, low-capacitance outside-out patches. We speculate the low resistance prior to pulling the outside-out patches is due to electrical connections due to gap-junctions between multiple cultured cells in physical contact with one another.

Fig. 3.

Exemplary fast solution exchange electrophysiological experimental results. (A) NMDAR responses from transiently transfected HEK cells stimulated by 100 μM glutamate and 30 μM glycine. Recordings are from a lifted whole cell (left) and an outside-out patch using a 4-MΩ electrode (right) at −60 mV in 0 mM Mg²⁺. (B) GABA_AR responses from stably transfected HEK cells (α1β2γ2L) stimulated by 1 mM GABA. Recordings are from a lifted whole cell (left, 1-second application) and an outside-out patch (right, 5-millisecond application).

Fig. 4.

Representative experimental timeline of patcherBot_Pharma operation. (A) Timeline of experimental progress. The time periods of operator interaction with the patcherBot_Pharma and recording duration are highlighted, along with recording outcome. (B) GABA_AR responses (1 mM GABA, 1-second application) from all successful outside-out patches pulled. Scale bars indicate 20 pA and 0.5 seconds. (C) Post-experiment open-tip position validation utilizing a 50% H₂O/50% wash solution. Scale bars indicate 200 pA and 20 milliseconds. The average [± S.D. (range)] 20–80 rise and fall times for piezoelectric jumps were 3.06 ± 0.78 (1.30 4.11) and 3.56 ± 0.32 (2.27 6.55).

Fig. 5.

GABA_AR propofol deactivation time-constant concentration-response case study; the patcherBot_Pharma has the capability to collect pharmacological data at an accelerated rate. (A) A flowchart illustrating the patcherBot_Pharma operation, timing, and success rate of individual steps. The manual (white boxes) and automated (gray boxes) steps are indicated. After the one-time calibration and cell selection step, the patcherBot_Pharma loops through and serially records from the selected cells. Quality control measures are in place to terminate the current experiment and continue to the next iteration. (B) A more detailed depiction of the manual steps is shown. The calibration and cell selection step (left) includes: 1) aligning the electrode and microscope coordinate systems, 2) ensuring the saved locations of the solution manifold are correct, and 3) selecting a set of cells for experimentation (typically 7–12 cells). The cell approach and patching step (right) at the beginning of each loop (coinciding with an auditory signal so that the operator need not always be present) starts when the patcherBot_Pharma translates the stage to the next cell selected, and then the electrode is brought to a position just above (100 μm) the cell. The operator then only needs to lower the electrode to the optimal position on the cell and has the option of manually sealing and breaking in or can elect to have the patcherBot_Pharma conduct those processes. (C) A more detailed look at the experimental protocol step of the patcherBot_Pharma process. In this case, there were six sets of solutions that would be used during each experiment (two control and four propofol solution sets, detailed on the left). Each phase of each experiment would start with the valves changing to the next set to be tested, with a wait step to allow for the solutions to be primed, followed by the collection of 10 replicates of the intended jump protocol (right). (D) The results from one experiment (all phases), showing all replicates (top) and the average (± S.D., shown by shaded gray area) response. The desensitization and deactivation of all recordings were fitted simultaneously and are depicted on the averaged responses (white line). (E) The relationship between the average (± S.E.M.) deactivation τ and propofol concentration is shown and fitted with the Hill equation. The 100 μM propofol response was omitted from fitting because of the reduced response amplitude as a result of the enhanced desensitized state in the presence of such a high concentration of propofol. att., Attempt; conc., concentration.