Real-time multimodal optical control of neurons and muscles in freely behaving Caenorhabditis elegans

Abstract

The ability to optically excite or silence specific cells using optogenetics has provided a powerful tool to interrogate the nervous system. Optogenetic experiments in small organisms have mostly been performed using whole-field illumination and genetic targeting, but these strategies do not always provide adequate cellular specificity. Targeted illumination can be a valuable alternative but to date it has only been shown in non-moving animals without the ability to observe behavior output. We present a real-time multimodal illumination technology that allows both tracking and recording the behavior of freely moving Caenorhabditis elegans while stimulating specific cells that express Channelrhodopsin-2 or MAC. We use this system to optically manipulate nodes within the C. elegans touch circuit and study the roles of sensory and command neurons and the ultimate behavioral output. This technology significantly enhances our ability to control, alter, observe, and investigate how neurons, muscles, and circuits ultimately produce behavior in animals using optogenetics.

Optogenetic analysis of GABABreceptor signaling inCaenorhabditis elegansmotor neurons

“…However, the most promising clone of GBB-2 still contained two missense mutations resulting in A484V and V572A changes of the GBB-2 amino acid sequence Behavioral assays and data analysis. Optogenetic/behavioral assays and automated video analysis for the extraction of worm body length were described previously (Liewald et al 2008;Stirman et al 2011;Weissenberger et al 2011). In brief, for body length measurements, animals were transferred onto plain NGM plates and recorded with a PowerShot G5 or G9 digital camera (Canon) while blue light from a 50-W HBO lamp [450-to 490-nm green fluorescent protein excitation filter; intensity adjusted using neutral density filters (AHF Analysetechnik)] was applied.…”

“…Velocity, bending angles, and trajectories were recorded using tracking software (Stirman et al 2011) that controls an x,y-translational stage and allows photoactivation of ChR2 via a LCD projector (450 -490 nm; ϳ4 mW/mm 2 ). Ten minutes before the start of these assays, animals were transferred to plain NGM plates.…”

“…As we previously showed, prolonged photostimulation of GABA neurons via the zxIs3 transgene causes 4 -5% body elongation that declines to 1-2% within 10 -20 s, likely due to desensitization of the UNC-49 GABA A receptor (Liewald et al 2008), but then, however, GABA effects sustain for several minutes . We thus used a recently developed tracking system, capable of selective photostimulation of freely behaving animals (Stirman et al 2011), to track locomotion trajectories, speed, and mean bending angles of wild-type and gbb-2(tm1165) animals, both containing zxIs3. When analyzing animal trajectories, we observed that gbb-2; zxIs3 animals, while being photostimulated, reached larger maximal distances from the starting point ["R max " as defined by Dittman and Kaplan (2008); note that this is not the absolute distance traveled] during a 120-s period compared with wild type (Fig.…”

An integrated platform enabling optogenetic illumination of Caenorhabditis elegans neurons and muscular force measurement in microstructured environments

“…[35][36][37] Together with precise position control of agile nematode, optogenetics has been applied to C. elegans to manipulate sensory neurons, interneurons, and motor neurons. 20,[38][39][40][41][42][43][44] Normally, certain neurons of interest in C. elegans can be selectively transferred with a gene that allows the neurons to be switched on/off when projected with light of specific wavelength (or color). In practice, as long as the color light is projected onto the worm body segment that possesses the neurons of interest, the neurons can be stimulated positively or negatively.…”

Optogenetic analyses of neuronal network function and synaptic transmission in Caenorhabditis elegans

“…4, [27,35]). Alternatively, microscopy systems have been developed which enable the specific illumination of certain body regions with light of different wavelengths, even in freely moving animals, thus making it possible to photoactivate or photoinhibit only the desired, genetically manipulated neurons [24,45,46]. This enables functional probing of different nodes of a neuronal network (.…”