In vivo Neuronal Calcium Imaging in C. elegans

Summary

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

Abstract

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon ¹ and GCaMP ² allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

Introduction

Here we present practical methods for in vivo calcium imaging in C. elegans neurons. The development of genetically encoded calcium-sensitive fluorophores with high signal-to-noise ratio makes C. elegans a comparatively straightforward and cost effective system for measurement of neurophysiology and activity. Our imaging is done with a standard compound microscope using wide-field fluorescence imaging of commonly available fluorophores. We present several techniques employing various fluorophores and different sample preparations, discussing the strengths and weakness of each. Data is then presented from two example experiments. An ....

Protocol

1. Optical Setup

1. Use a standard compound microscope with epifluorescence imaging capabilities. We use a Nikon Eclipse Ti-U inverted microscope with an Intensilight HG Illuminator.
2. For best image and signal quality use a high magnification, high numerical aperture objective. We typically use a Nikon X60 1.4 N.A. oil immersion objective. In some cases it is possible to use lower magnification (X40, X20) depending on expression level of the fluorophore and signal strength.
3. Use a high sensitivity cooled CCD camera, such as an Andor Clara Camera to maximize image sensitivity and minimize background noise.
4. For ....

Results

Here we present results from two separate experiments. The first employs GCaMP to measure the response of a specific sensory neuron to a defined external stimulus, giving a good example of how fluorescent calcium reporters can be used to optically monitor neuronal activity in intact C. elegans. The second employs cameleon to measure the intracellular calcium transient triggered within a neuron in response to specific laser damage, thus illustrating how calcium physiology can be measured within a single cell .......

Discussion

Genetically encoded calcium indicators have been widely utilized in C. elegans neurobiology. Numerous groups have employed these techniques to study response of primary sensory neurons to external stimuli as demonstrated here with the ASJ response to an electrical field. Prominent examples include sensation of mechanical touch, specific chemicals, temperature and an electric field ^{12, 16-19}. Activity of interneuron and muscle cells have also been monitored both in response to stimuli and in con.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Eclipse Ti-U inverted microscope	Nikon
Intensilight HG Illuminator	Nikon	C-HGFI	Fluorescent light source
CFI Plan Apo VC 60X Oil	Nikon
Optical table or 3'X3' optical grade breadboard	Thor Labs		If an optical table is not used an optical grade breadboard on a solid laboratory bench should suffice.
Clara Interline Camera	Andor Technology		High-sensitivity CCD camera
wtGFP Longpass Emission	Chroma Technology Corp.	41015	GFP filter set for imaging GCaMP
Filter 440 +/- 10 nm	Chroma	D440/20x EX	excitation filter for cameleon
Dichroic mirror > 455 nm longpass	Chroma	455DCLP BS	microscope dichroic for cameleon imaging
Dichroic mirror > 515 nm longpass	Chroma	515DCLP BS	dichroic mirror for cameleon imaging
Filter 535 +/- 15 nm	Chroma	D535/30m EM	YFP emission filter
Filter 480 +/- 20 nm	Chroma	D485/40m EM	CFP emission filter
Lens, 200 mm, Achromat	Thor Labs	AC508-200-A1	Relay lens for FRET optics (3)
Silver broadband mirror	Thor Labs	ME2S-P01	FRET optics (2)
NGM buffer
Levamisole	Sigma
Polybead Microspheres	Polysciences, Inc.	08691-10, 2.5% by volume, 50 nm diameter	polystyrene nanoparticles for C. elegans immobilization
Transgenic strain, Strain gpa-9::GCaMP3(in pha-1; him-5 bkg)	Sternberg Lab	Strain PS6388
Transgenic strain, mec- 4::YC3.60	Gabel Lab	Strain CG1B