A vertical flow chamber for Xenopus oocyte electrophysiology and automated drug screening

doi:10.1016/j.jneumeth.2003.09.002

Journal of Neuroscience Methods

Volume 132, Issue 1, 15 January 2004, Pages 69-79

https://doi.org/10.1016/j.jneumeth.2003.09.002 Get rights and content

Abstract

Xenopus laevis oocytes are used extensively in the study of ion channel coupled receptors. Efficient use of oocytes for ion channel characterization requires a system that is inherently stable, reproducible, minimizes drug volumes, and maximizes oocyte longevity. We have constructed a vertical flow oocyte recording chamber to address the aforesaid issues, where the oocyte is placed in a funnel-shaped chamber and perfused from the bottom of the funnel. The vertical rather than horizontal flow of perfusate results in an unusually stable environment for oocyte recording. Two-electrode voltage clamp recordings from a single oocyte are acquired easily and routinely over several hours while maintaining stable baseline currents and reproducible response profiles. Chamber characteristics were tested using a serotonin ligand-gated ion channel receptor (5-HT₃R). Data obtained from this system corresponds well with published data. To further test the stability and reliability of this perfusion chamber, we constructed an automated oocyte perfusion system utilizing a commonly available HPLC autosampler. We were able to obtain dose–response curves for various 5-HT_3AR ligands using the automated perfusion system with minimal user intervention. Such a system can easily satisfy need for automated oocyte electrophysiology in academic settings, especially small to medium sized laboratories.

Introduction

The Xenopus laevis oocyte, a heterologous expression system, has been successfully used to study various biological systems and ion channels including ligand gated ion channels such as the nicotinic acetylcholine and serotonin type three receptors (5-HT₃R) (Bertrand et al., 1997, Maricq et al., 1991). The functional aspects of these receptors can be studied electrophysiologically using two-electrode voltage clamp. In this technique, oocytes are injected with cRNA (cytoplasmic) or cDNA (nuclear) coding for the ion channel receptor under study. After incubation at 19–21 °C for an appropriate length of time (usually 24–48 h), an oocyte expressing ion channel receptors is placed in a perfusion chamber and voltage clamped. Rapid exposure of such an oocyte to an agonist results in inward currents with characteristic shapes and distinct desensitization characteristics.

In the conventional perfusion method, an oocyte is placed in a circular or elongated depression, with perfusion inlet from one end and drainage from the other end. Depending on specific requirements, the chamber volume can vary from small (∼50–250 μl) to large (∼1 ml). Most of the conventional perfusion chambers involve a solution flow across the oocyte and the electrodes (Fig. 1D). Such a flow results in a constant application of force to the oocyte at a direction that is perpendicular to its points of anchorage (base and the recording electrodes). Minute vibrations of the electrodes impaled in the oocyte may damage the oocyte membrane and increase the leak of electrode filling solution (usually 3 M KCl). These effects may not be obvious in short term studies although they may contribute towards oocyte mortality on a long-term basis. Additionally, in many of the standard oocyte chambers, the oocyte may not be exposed to the drug rapidly from all sides, thus limiting rise times.

Oocyte electrophysiology is commonly employed in high-throughput drug screening assays. These assay systems must meet certain requirements in order to produce reliable and reproducible data. Simplicity and rapidity of operation are important considerations, in addition to low drug volumes, rapid drug exchange rates, and stability. Among the various factors, stability appears to be of paramount importance to the successful execution of automation. An increase in the longevity of the oocyte will result in a highly productive system; where as reduced longevity will necessitate constant monitoring and intervention.

We describe a novel oocyte perfusion chamber where the oocyte is placed in a small volume funnel-shaped chamber on top of a wire mesh and perfused from an inlet at the bottom of the funnel. We have utilized ligand gated ion channel coupled receptors, the 5-HT_3A and 5-HT_3AB receptors (Davies et al., 1999, Maricq et al., 1991), to demonstrate various features of the funnel shaped chamber. A vertical flow as opposed to a sideway flow is expected to provide a stable environment to the oocyte. We show that an oocyte is stable for an extended period in the vertical flow chamber. The stability and reliability of the perfusion chamber have allowed us to construct an automated oocyte perfusion system utilizing a commonly available HPLC autosampler. We were able to obtain dose–response curves for various 5-HT_3AR ligands using the automated perfusion system with minimal user intervention.

Section snippets

Materials

Sigma type II collagenase was purchased from Sigma–Aldrich (MO, US). mMESSAGE mMACHINE™ High Yield Capped RNA Transcription Kit was purchased from Ambion (TX, US). The OC-725C oocyte clamp and 7.5–150GT glass electrodes were purchased from Warner Instruments (CT, US). Datapac 2000 data acquisition software was purchased from RUN Technologies (CA, US). X. laevis frogs and frog food were purchased from Xenopus Express (FL, US). All other chemicals were obtained from Fisher Scientific (TX, US).

Chamber construction

Results

We utilized oocytes expressing 5-HT_3A receptors to assess the functionality of the newly designed chamber. The characteristics of 5-HT_3A-mediated inward currents recorded from microinjected oocytes are well established (Davies et al., 1999, Dubin et al., 1999, Jackson and Yakel, 1995, Yakel et al., 1991). Application of 5-HT to oocytes expressing 5-HT_3AB receptors resulted in characteristic inward currents. Fig. 4A shows representative responses obtained from application of 0.3, 3, 10 and 30 μM

Discussion

A reproduction of the established pharmacological profile for the 5-HT₃ receptor using the vertical flow apparatus validates our novel drug exposure method. Ligand affinities as well as other physiological characteristics (desensitization, voltage dependence) are in close agreement with previously reported values. The oocyte in a vertical flow chamber is suspended in the path of drug flow, achieving exposure to the complete oocyte surface area. This is akin to certain whole-cell patch-clamp

Acknowledgements

This work is supported by the National Science Foundation (NSF CAREER 9985077) and the American Heart Association (AHA 0151065B).

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A vertical flow chamber for Xenopus oocyte electrophysiology and automated drug screening

Abstract

Introduction

Section snippets

Materials

Chamber construction

Results

Discussion

Acknowledgements

J. Biol. Chem.

J. Neurosci. Methods

Electrophysiology: a method to investigate the functional properties of ligand-gated channels

J. Recept. Signal Transduct. Res.

The 5-HT3B subunit is a major determinant of serotonin-receptor function

Nature

The 5-HT_3B subunit is a major determinant of serotonin-receptor function