Calcium and Cyclic AMP Promote Axonal Regeneration in Caenorhabditis elegans and Require DLK-1 Kinase

Ghosh-Roy, Anindya; Wu, Zilu; Goncharov, Alexandr; Jin, Yishi; Chisholm, Andrew

doi:10.1523/jneurosci.5464-09.2010

Cited by 271 publications

(365 citation statements)

References 48 publications

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“…Many of these studies have pushed these techniques a step further using image recognition and tracking techniques to enable recording from neurons in partially restrained or even freely moving animals as well as recording from multiple neurons simultaneously. Additional studies have investigated other aspects of calcium physiology such as the response to neuronal damage 13,20 as presented here as well as neuronal degeneration on longer time scales 21 .…”

Section: Discussionmentioning

confidence: 99%

In vivo Neuronal Calcium Imaging in C. elegans

Chung

Sun

Gabel

2013

JoVE

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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 geneticallyencoded 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. Video LinkThe video component of this article can be found at

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Section: Discussionmentioning

confidence: 99%

In vivo Neuronal Calcium Imaging in C. elegans

Chung

Sun

Gabel

2013

JoVE

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“…Previous studies combining laser microsurgery and C. elegans have identified how several factors, including animal age, neuronal type, synaptic branching and axon guidance signalling, influence regeneration [29][30][31] . Laser microsurgery screens, as well as screens using nematodes exhibiting spontaneous neurite breaks due to dysfunction of β-spectrin, have also revealed that axon regrowth depends on the activity of MAP kinase pathways [31][32][33] . Most recently, we have demonstrated the use of microfluidics in combination with femtosecond laser microsurgery to perform chemical screens on neural regeneration in C. elegans 19 .…”

Section: Discussionmentioning

confidence: 99%

Subcellular in vivo time-lapse imaging and optical manipulation of Caenorhabditis elegans in standard multiwell plates

Rohde

Yanik

2011

Nat Commun

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High-resolution in vivo time-lapse assays require repeated immobilization and imaging of whole animals. Here we report a high-throughput technology for screening Caenorhabditis elegans at cellular resolution over its entire lifespan inside standard multiwell plates using repeated immobilization, imaging and optical manipulation. our system does not use any fluidic or mechanical components, and can operate for tens of thousands of cycles without failure. It is also compatible with industrial high-throughput screening platforms and robotics, and it allows both chemical, and forward and reverse genetic screens. We used this technology to perform subcellular-resolution femtosecond laser microsurgery of single neurons in vivo, and to image the subsequent regeneration dynamics at subcellular resolution. our single-neuron in vivo timelapse analysis shows that neurite regrowth occurring over short time windows is significantly greater than that predicted by ensemble averaging over many animals. H igh-throughput screening (HTS) allows rapid identification of potential therapeutic targets and leads. Although high-throughput assays can be performed in vitro, thorough study of many biological phenomena, such as development, organogenesis, regeneration and aging, requires the use of animal models. The use of multicellular organisms also facilitates identification of off-target or toxic effects. The nematode Caenorhabditis elegans is one of the most commonly used multicellular organisms for HTS. As nematodes can be cultured and screened in liquid, many techniques currently used for screening cells can be adapted for C. elegans. The small size and simple physiology of C. elegans render it suitable for culture in 96-and 384-well plates in small volumes, and internal organs can be easily visualized because C. elegans is optically transparent. Investigation of C. elegans often involves high-content and time-lapse studies; however, these rapidly moving organisms must be immobilized to image cellular and subcellular processes. In addition, many physiological processes are stochastic and time-dependent (that is, development, aging), which requires multitime-point immobilization, tracking, and imaging of large numbers of individual animals.Commercially available technologies for HTS of C. elegans utilize flow cytometry [1][2][3] . Despite their speed, these systems can acquire only one-dimensional fluorescence images. Thus, assays requiring analysis of cellular and subcellular features are not feasible. In addition, optical measurements and manipulations, such as multiphoton 4 and confocal imaging, laser stimulation and laser microsurgery [5][6][7] , are not possible with such approaches. Recently, we and others developed microfluidic approaches for immobilization of C. elegans for high-throughput and highcontent screening. Mechanical immobilization (both active [8][9][10][11] and passive 12) and cooling 13 have been used to enable short-term immobilization. Three-dimensional multiphoton imaging 9,10 and femtosecond laser microsurger...

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“…The injury-induced calcium outflow via RyR is crucial for stimulating both outgrowth and guidance of regenerating neurons (18). In C. elegans PLM sensory neurons, the amplitude of injury triggered calcium waves also correlates with the extent of regeneration (19). Whether calcium release from internal stores to sustain a back propagating calcium wave represents a specific property of regenerating neurons remains to be tested.…”

Section: Figmentioning

confidence: 99%

Signaling Over Distances

Saito

Cavalli

2016

Molecular & Cellular Proteomics

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Neurons are extremely polarized cells. Axon lengths often exceed the dimension of the neuronal cell body by several orders of magnitude. These extreme axonal lengths imply that neurons have mastered efficient mechanisms for long distance signaling between soma and synaptic terminal. These elaborate mechanisms are required for neuronal development and maintenance of the nervous system. Neurons can fine-tune long distance signaling through calcium wave propagation and bidirectional transport of proteins, vesicles, and mRNAs along microtubules. The signal transmission over extreme lengths also ensures that information about axon injury is communicated to the soma and allows for repair mechanisms to be engaged. This review focuses on the different mechanisms employed by neurons to signal over long axonal distances and how signals are interpreted in the soma, with an emphasis on proteomic studies. We also discuss how proteomic approaches could help further deciphering the signaling mechanisms operating over long distance in axons. Neurons have unique and highly polarized morphologies. The axon allows intracellular communication between the cell soma and the distantly located synaptic terminal. The extreme length of axons relative to the cell soma diameter poses great challenges for neuronal development, maintenance, and repair. Anterograde and retrograde axonal transport of proteins vesicles and RNA along the microtubule tracks in the axon is crucial to sustain the required signaling events underlying development and maintenance of the nervous system. The retrograde transport system is also used following axon injury to communicate information from the site of injury back to the soma. Communication between distant axon locations and the cell soma is also ensured by a more rapid signal encoded in calcium waves. Incoming signals from distant axon locations are interpreted by the soma to regulate gene expression for survival or repair. The neuronal epigenome is also influenced by incoming signals to appropriately coordinate transcriptional responses. In this review, we discuss the state of the field regarding the different mechanisms employed by neurons to signal intracellularly over long distances in axons and how signals are interpreted in the cell soma, with an emphasis on proteomic studies. We also discuss how the use of proteomics could further help in understanding long distance signaling system in axons. The communication employed by neurons to signal along dendrites has been reviewed in detail elsewhere (1) and will not be discussed here.Calcium Influx, Back Propagation, and Long Distance Effects-Alterations in axonal calcium levels affect multiple and diverse signaling events, including local protein synthesis and degradation. These local signals can have long distance effects. Axon injury provides a powerful system to study the role of calcium in axonal signaling, because injury-induced calcium influx in the axon triggers important downstream events at the injury site and the soma (Fig.

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Calcium and Cyclic AMP Promote Axonal Regeneration in Caenorhabditis elegans and Require DLK-1 Kinase

Cited by 271 publications

References 48 publications

<em>In vivo</em> Neuronal Calcium Imaging in <em>C. elegans</em>

<em>In vivo</em> Neuronal Calcium Imaging in <em>C. elegans</em>

Subcellular in vivo time-lapse imaging and optical manipulation of Caenorhabditis elegans in standard multiwell plates

Signaling Over Distances

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