Chapter 14 - Electrophysiological Methods for Caenorhabditis elegans Neurobiology
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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Advances in our understanding of nematode ion channels as potential anthelmintic targets
2022, International Journal for Parasitology: Drugs and Drug ResistanceCitation Excerpt :Jarman (1959) performed the first patch-clamp study in a nematode and analyzed the A. lumbricoides muscle cells' electrical activity. Several groups of scientists have since used the patch-clamp technique in a variety of nematode preparations, including muscle cells, pharyngeal tissue, and nerve cells (Holden-Dye et al., 1988; Martin and Pennington, 1988, 1989; Holden-Dye and Walker, 1990; Martin, 1996; Adelsberger et al., 1997; Brownlee et al., 1997; Francis et al., 2003; Qian et al., 2006; Robertson et al., 2011, 2013; Goodman et al., 2012). Two-electrode voltage clamp (TEVC) is another conventional electrophysiology approach employed by scientists to study ion channel properties.
Elegantly
2020, The Neural Control of Movement: Model Systems and Tools to Study Locomotor FunctionRole of simulation models in understanding the generation of behavior in C. elegans
2019, Current Opinion in Systems BiologyCitation Excerpt :Despite the substantial behavioral, genetic, and anatomical knowledge about C. elegans, information about the electrophysiological properties of its nervous system is less complete. Using single-cell optical recording and whole-cell patch-clamp techniques, slow but steady progress has been made [52–57]. In addition, techniques such as light sheet-microscopy and optogenetics are accelerating progress by allowing simultaneous imaging and manipulation of calcium activity of all neurons in the entire nervous system of both immobilized and freely-moving worms [58–70].
Nematode C. elegans: Genetic Dissection of Pathways Regulating Seizure and Epileptic-Like Behaviors
2017, Models of Seizures and Epilepsy: Second EditionThe whole worm: Brain-body-environment models of C. elegans
2016, Current Opinion in NeurobiologyCitation Excerpt :Of course, connectivity alone is insufficient for understanding neural activity and its role in behavior [11]; electrophysiological and neuromodulatory information is also required and the data here are much less complete. Although electrophysiological analysis of C. elegans has been difficult due to the small size of its neurons and its pressurized body, considerable progress has been made in whole-cell patch clamping techniques over the last decade [12,13,14•]. In addition, functional properties can sometimes be inferred from gene expression mapping, behavioral genetics and laser ablation [15].
C. elegans locomotion: Small circuits, complex functions
2015, Current Opinion in NeurobiologyCitation Excerpt :Because of the absence of voltage-gated sodium channels in its genome [31], it had been thought that worms lack classical action potentials. Steadily improving electrophysiological preparations, however, have revealed regenerative potentials with different molecular mechanisms [8,48••,73–78]. Calcium-dependent, regenerative plateau potentials have been observed in some neurons in the head ganglion [76].