Agarose hydrogel microcompartments for imaging sleep- and wake-like behavior and nervous system development in Caenorhabditis elegans larvae

Bringmann, Henrik

doi:10.1016/j.jneumeth.2011.07.013

Cited by 63 publications

(79 citation statements)

References 28 publications

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“…Two commonly observed examples of motion that may be difficult to detect are head oscillations, which can persist without directed progression of the body, or minor fluctuations (5–15% of the body length) in positions of body-bends, often observed in a state of dwelling. The dynamics of head motion and quiescence is of particular interest in the context of C. elegans lethargus, since it was found to approximate those of the entire animal [15, 16]. Therefore, a method such as centroid tracking, which is not sensitive to head motion, is expected to incur measurement errors and over-estimate quiescence.…”

Section: Resultsmentioning

confidence: 99%

Measurements of behavioral quiescence in Caenorhabditis elegans

Nagy

Raizen

Biron

2014

Methods

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The nematode Caenorhabditis (C.) elegans, a long time work horse for behavioral genetic studies of locomotion, has recently been studied for quiescent behavior. Methods previously established for the study of C. elegans locomotion are not well-suited for the study of quiescent behavior. We describe in detail two computer vision approaches to distinguish quiescent from movement bouts focusing on the behavioral quiescence that occurs during fourth larval stage lethargus, a transition stage between the larva and the adult. The first is the frame subtraction method, which consists of subtraction of temporally adjacent images as a sensitive way to detect motion. The second, which is more computationally intensive, is the posture analysis method, which consists of analysis of the rate of local angle change of the animal’s body. Quiescence measurements should be done continuously while minimizing sensory perturbation of the animal.

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Section: Resultsmentioning

confidence: 99%

Measurements of behavioral quiescence in Caenorhabditis elegans

Nagy

Raizen

Biron

2014

Methods

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“…Inspired by similar methods using agar gels, 17 we applied these techniques to our C. elegans culture system. By using a FEP mold, we were to create an array of pre-formed indentations in the PEG gel (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…One approach traps individual larval-stage animals under a coverslip in microchambers molded into agarose, allowing tens to hundreds of individuals to be imaged through the initial stages of development. 17–19 To study late larval development and early adulthood, the animals must be manually transferred to larger microchambers. 19 Another system consists of an array of agar pads with a geometry that prevents escape, 20 with each pad surrounded by a moat of chemo-repellant solution.…”

Section: Introductionmentioning

confidence: 99%

A simple culture system for long-term imaging of individual C. elegans

et al. 2017

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We have miniaturized standard culture techniques to rear arrays of isolated, individual C. elegans throughout their lives on solid gel media. The resulting apparatus is compatible with brightfield and fluorescent microscopy, enabling longitudinal studies of morphology and fluorescent transgene expression. Our culture system exploits a novel crosslinking reaction between a polyethylene glycol hydrogel and a silicone elastomer to constrain animals to individual “corrals” on the gel surface. These devices are simple to construct on the benchtop with commercially available reagents, and, unlike microfluidic isolation methods, does not rely on micropatterned materials. We demonstrate that this new culture method has negligible effects on the physiology of C. elegans compared to standard culture on agar plates. In addition, RNAi techniques are effective in this system. Finally, the hydrogel–silicone binding chemistry that we developed also allows traditional microfluidic devices to be covalently attached to gel substrates instead of glass.

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“…This system can image many worms in one run because animals are restricted to their defined chambers. Thus, this method can be easily scaled up 9,27,[34][35][36] .…”

Section: Limitations Of the Techniquementioning

confidence: 99%

“…Two principal approaches can be used to observe motile animals over long time scales. The first approach is following the animal during movement using an automated stage or camera [2][3][4][5][6][7][8] and the second is to restrict the movement to a range that is at least as small as the field of view of the camera [9][10][11][12][13][14] . Both methods have their advantages.…”

Section: Introductionmentioning

confidence: 99%

Agarose Microchambers for Long-term Calcium Imaging of <em>Caenorhabditis elegans</em>

Turek

Besseling

Bringmann

2015

JoVE

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Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Longterm calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

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Agarose hydrogel microcompartments for imaging sleep- and wake-like behavior and nervous system development in Caenorhabditis elegans larvae

Cited by 63 publications

References 28 publications

Measurements of behavioral quiescence in Caenorhabditis elegans

Measurements of behavioral quiescence in Caenorhabditis elegans

A simple culture system for long-term imaging of individual C. elegans

Agarose Microchambers for Long-term Calcium Imaging of <em>Caenorhabditis elegans</em>

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