Optical Imaging of Calcium Transients in Neurons and Pharyngeal Muscle of C. elegans

Abstract

Electrophysiology and optical indicators have been used in vertebrate systems to investigate excitable cell firing and calcium transients, but both techniques have been difficult to apply in organisms with powerful reverse genetics. To overcome this limitation, we expressed cameleon proteins, genetically encoded calcium indicators, in the pharyngeal muscle of the nematode worm Caenorhabditis elegans. In intact transgenic animals expressing cameleons, fluorescence ratio changes accompanied muscular contraction, verifying detection of calcium transients. By comparing the magnitude and duration of calcium influx in wild-type and mutant animals, we were able to determine the effects of calcium channel proteins on pharyngeal calcium transients. We also successfully used cameleons to detect electrically evoked calcium transients in individual C. elegans neurons. This technique therefore should have broad applications in analyzing the regulation of excitable cell activity in genetically tractable organisms.

Reporting neural activity with genetically encoded calcium indicators

“…Several classes of GECIs have performed well for neural imaging, encompassing single-FP and FRET sensors, and sensors based on calmodulin and troponin-C calcium-binding domains. Published GECIs are adequate for detecting high frequency bursts of neuronal activity, making them appropriate for activity localization in mouse myotubes (Nakai et al, 2001), mouse cerebellum (Diez-Garcia et al, 2005), or in invertebrate systems (Kerr et al, 2000;Higashijima et al, 2003;Wang et al, 2003;Chronis et al, 2007;Clark et al, 2007). However, reliable in vivo single-AP detection is not yet feasible with the current generation of techniques.…”

“…They are compatible with long-term, repeated, in vivo measurements (Hasan et al, 2004). GECIs have successfully recorded in vivo neural activity in brain regions with large modulation of dense spiking (Kerr et al, 2000;Fiala et al, 2002;Hasan et al, 2004), though recording of sparse spike trains remains elusive (Mank and Griesbeck, 2008). Through iterative cycles of optimization, GECIs are now able to occasionally detect single APs, at least under ideal ex vivo imaging conditions (Mao et al, 2008).…”

Generation and modulation of chemosensory behaviors in C. elegans

“…Presumably, males and hermaphrodites also produce signals to attract or repel each other, but these cues and the relevant sensory neurons are unknown [3,4]. As "...behavior is messy" (with apologies to [110]), the development of technologies for imaging neuronal activity in single neuron types in vivo in response to an applied stimulus [111][112][113][114][115] may allow for more precise functional mapping of chemicals to sensory neuron types.…”

Behavioral plasticity in C. elegans: Paradigms, circuits, genes

“…It will be extremely challenging to obtain electrophysiologic recordings from live animals engaged in a specific task. However, genetically encoded calcium sensors combined with highly sensitive imaging techniques (Kerr et al, 2000) may eventually provide cellular correlates for behavioral states that can be assayed in specific genetic backgrounds.…”