Abstract
Optogenetics is a powerful tool for manipulating neuronal activity with high temporal and spatial precision. In the nematode C. elegans optogentics is especially useful and easy to apply. This is because C. elegans is translucent, so its neurons are highly accessible to optic stimulation. In addition, many of its neurons can be exclusively targeted using cell-specific promoters. We have recently taken advantage of optogenetics to deliver artificial patterns of prolonged activation to a class of mechanosensory neurons, called touch receptor neurons (TRNs) in worms that lack touch sensation due to a genetic mutation. Our aim was to examine whether we can counteract the effects of sensory loss by artificially activating the sensory neurons. Here we describe in detail the various components of the protocol that we used. This consists of exposing worms expressing the light-sensitive ion channel Channelrohdopsin 2 (ChR2) in TRNs to long-term random flashes of light.
Keywords: Optogenetics, C. elegans, Mechanosensation, Locomotion, Cross-modal plasticity
Background
Artificial optogenetic stimulation (or silencing) of neurons has become of broad use in neuroscientific research. The powerful model organism, C. elegans, is particularly amenable to optogenetic manipulation (Nagel et al., 2005), and multiple groups have developed a range of techniques for delivering artificial brief patterns of stimulation with high temporal and spatial precision (Leifer et al., 2011; Stirman et al., 2011) and in combination with behavioral (Kocabas et al., 2012) and calcium imaging (Guo et al., 2009) or electrophysiological (Lindsay et al., 2011) readouts. We were interested in establishing a long-term stimulation protocol that would substitute natural ongoing activity in mechanosensory neurons deprived of sensory input (Rabinowitch et al., 2016). Our protocol integrates previous C. elegans optogenetic protocols, but focuses on chronic rather than transient stimulation.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Each experiment is performed on at least 3 separate days, with a similar number of repetitions per day. Mean reversing frequency (number of reversals per minute) is calculated by dividing the number of reversal events by 3 min. In experiments in which a 2 x 2 design is applied (e.g., no stimulation versus stimulation and wild-type worms versus mechanosensory mutants), a 2 x 2 ANOVA is used to establish whether a significant interaction exists between the two categories. Post hoc t-tests with Bonferroni corrections are then used to compare within each category. Alternatively, reversing rate values are normalized by the average reversing rate without any stimulation for each experiment day and a t-test is used to compare between experimental conditions (e.g., wild-type versus mechanosensory mutants). Examples of raw data can be found in http://journals.plos.org/plosbiology/article?id=10.1371%2Fjournal.pbio.1002348#pbio.1002348.s001, which is a supplement to our original research paper (Rabinowitch et al., 2016).
Notes
Recipes
Acknowledgments
Support for writing the protocol was provided by an FHCRC New Development Grant, and NIH grant NINDS R01NS085214. The current protocol was adapted from previous work (Nagel et al., 2005)
References
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