Electrophysiological Methods for Caenorhabditis elegans Neurobiology

doi:10.1016/B978-0-12-394620-1.00014-X

Methods in Cell Biology

Volume 107, 2012, Pages 409-436

https://doi.org/10.1016/B978-0-12-394620-1.00014-X Get rights and content

Abstract

Patch-clamp electrophysiology is a technique of choice for the biophysical analysis of the function of nerve, muscle, and synapse in Caenorhabditis elegans nematodes. Considerable technical progress has been made in C. elegans electrophysiology in the decade since the initial publication of this technique. Today, most, if not all, electrophysiological studies that can be done in larger animal preparations can also be done in C. elegans. This chapter has two main goals. The first is to present to a broad audience the many techniques available for patch-clamp analysis of neurons, muscles, and synapses in C. elegans. The second is to provide a methodological introduction to the techniques for patch clamping C. elegans neurons and body-wall muscles in vivo, including emerging methods for optogenetic stimulation coupled with postsynaptic recording. We also present samples of the cell-intrinsic and postsynaptic ionic currents that can be measured in C. elegans nerves and muscles.

Introduction

Nonmammalian organisms continue to be effective experimental systems for the study of fundamental problems in the function of neurons and neural circuits. Nonmammalian neuroscience has coalesced around the three systems with a high level of genetic tractability: Drosophila melanogaster fruit flies, Danio rerio zebrafish, and Caenorhabditis elegans nematodes. Each animal has aspects in which it excels. The nematode, the focus of this chapter, has three main advantages: (i) a nervous system of only 302 neurons, (ii) neurons that are re-identifiable from one individual to the next, and (iii) a complete anatomical reconstruction of its nervous system.

The celebrated strengths of the C. elegans nervous system come at a price. The electrophysiologist must learn to record from neurons whose cell bodies are about 2 μm in diameter and protected by a tough, pressurized cuticle in an animal that is only 1 mm long. Our experience in training others to record from C. elegans neurons and muscles indicates that patch-clampers are able to master the aspects of the technique that are specific to C. elegans in days to weeks. The ease with which the technique can be learned is traceable to two factors. First, the approach involves simple modifications of standard patch-clamping equipment and procedures (Goodman et al., 1998), mainly to compensate for the small size of the animals and their neurons and muscles. Second, the process of establishing and maintaining whole-cell recordings from C. elegans neurons is not especially difficult relative to other electrophysiological preparations.

With the advent of genetically encoded calcium indicators and techniques for in vivo calcium imaging in C. elegans (Kerr, 2006, Kerr et al., 2000, Kerr and Schafer, 2006), it is reasonable to ask: Why bother with electrophysiology? The answer depends almost entirely on the type of information required for the problem under study. If one wants to observe neuronal activity in intact animals, then calcium imaging is indispensable, as electrophysiology requires dissection, whereas calcium imaging in C. elegans does not. For other problems, however, electrophysiology is the indispensable technique. Three main examples come to mind. First, voltage clamp is an absolute requirement for recording single-channel currents or isolating the macroscopic current flowing through a particular type of ion channel. Second, calcium imaging has neither the time resolution nor the signal-to-noise ratio to resolve voltage transients such as unitary postsynaptic potentials in neurons or single action potentials in muscle cells. Third, calcium imaging does not in general reveal synaptic inhibition, except in unusually favorable cases.

Patch-clamp electrophysiology has been available in C. elegans now for a little over a decade. During this time, patch-clamp recordings have been used to examine the muscles of the body wall and pharynx, and about a dozen types of neurons (Francis et al., 2003). Progress has been rapid along four main fronts including genetically identified, ligand-gated and voltage-gated channels, intrinsic electrical properties of C. elegans neurons, the electrical events of sensory transduction, and the physiology and molecular biology of the neuromuscular junction (NMJ).

This chapter has two main. The first is to present to a broad audience the many techniques available for patch-clamp analysis of neurons, muscles, and synapses in C. elegans. We hope this presentation will be useful in correcting the misapprehension that C. elegans remains mostly intractable to these types of studies. We would be pleased if it also helps to cultivate fruitful collaborations between C. elegans geneticists and electrophysiologists.

The second goal of this chapter is to provide a methodological introduction to the techniques for patch-clamping C. elegans neurons and body-wall muscles in vivo. Patch clamping in any organism requires a level of practical and analytical know-how that is not easily reduced to recipes and protocols. Thus, the methodological components of this chapter are written mainly for experimentalists who are already familiar with the basic practice and principles of patch-clamp electrophysiology. Accordingly, we emphasize the ways in which patch clamping in C. elegans is similar to and different from patch clamping neurons in other animals. Important techniques not covered in this chapter are sharp-electrode and patch-clamp recording in pharyngeal muscle (Shtonda and Avery, 2005) and patch-clamp recording from cultured neurons (Bianchi and Driscoll, 2006, Christensen et al., 2002).

Section snippets

Recording Currents from Identified Neurons

Here, we provide an overview of a typical workflow followed for obtaining in vivo patch-clamp recordings from C. elegans neurons as well as detailed descriptions of each of the steps in the workflow (Fig. 1). This work incorporates improvements devised in the 10 years that have passed since reporting the first such experiments (Goodman et al., 1998).

Recording Synaptic Events from Identified Neurons

Whereas the above methodology allows for routine recordings from single, identified C. elegans neurons, simultaneous patch-clamp recording of identified pairs of neurons remains impractical. An alternative approach for investigating synaptic transmission is to record from postsynaptic cells while stimulating presynaptic neurons. Conventionally, two approaches have been considered: electrical stimulation and sensory stimulation. The former approach offers temporal precision and has been used to

Recording Currents and Synaptic Events at the Neuromuscular Junction

The time required to master recording from body wall muscles can be several months, mostly due to the challenging dissection, particularly the initial steps of gluing worms and cutting the cuticle. To facilitate and possibly accelerate the learning process, we provide the following description and refer readers to this visualized experiment (Richmond, 2009). Figure 6 outlines a typical workflow for obtaining in vivo patch-clamp recordings from C. elegans body wall muscles.

Transgenic C. elegans Strains that Label Target Cells

In order to identify the desired neuron or muscle for patch clamp recording, we use transgenic C. elegans strains in which target cells are labeled with a soluble fluorescent protein such as GFP (Chalfie et al., 1994). A soluble, rather than a membrane-bound construct is preferred, as dilution of soluble GFP is a useful indicator of successful break-in for whole-cell recording. In building a transgenic strain suitable for in situ electrophysiology (or choosing a published strain from the

Discussion

Considerable technical progress has been made in C. elegans electrophysiology in the decade since the initial publication of the technique for whole-cell patch-clamp recordings from C. elegans neurons (Goodman et al., 1998) and muscle (Richmond and Jorgensen, 1999). Notable advances include technical adaptations for quantitative analysis of sensory mechano-, thermo-, and phototransduction (O’Hagan et al., 2005, Ramot et al., 2008, Ward et al., 2008) and for studying the physiology of chemical (

Summary

Here, we provide the methods and materials needed to obtain patch-clamp recordings from C. elegans neurons and muscles in situ, including emerging methods for optogenetic stimulation coupled with postsynaptic recording. We emphasize procedures and equipment that are essential for successful patch-clamp recordings with this small animal, including critical factors in choosing transgenic lines for study.

Acknowledgments

We thank the members of our laboratories for comments and many colleagues in the worm community for contributing unpublished data: Shangbang Gao, Alex Gottschalk, Jana Liewald, Ping Liu, Richard Martin, Daniel Ramot, Alan Robertson, Zhao-Wen Wang, Mei Zhen. This work was supported by John Simon Guggenheim Foundation (S.R.L), McKnight, Alfred P. Sloan and Klingenstein Foundations (M.B.G) and by research grants from NIH (M.B.G., S.R.L., J.E.R.) and NSF (M.B.G.).

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Chapter 14 - Electrophysiological Methods for Caenorhabditis elegans Neurobiology

Abstract

Introduction

Section snippets

Recording Currents from Identified Neurons

Recording Synaptic Events from Identified Neurons

Recording Currents and Synaptic Events at the Neuromuscular Junction

Transgenic C. elegans Strains that Label Target Cells

Discussion

Summary

Acknowledgments

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Curr. Biol.

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Neuron

Curr. Biol.

J. Biol. Chem.

Cell

The Caenorhabditis elegans unc-49 locus encodes multiple subunits of a heteromultimeric GABA receptor

J. Neurosci.

The composition of the GABA receptor at the Caenorhabditis elegans neuromuscular junction

Br. J. Pharmacol.

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Green fluorescent protein as a marker for gene expression

Science

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