Comparing Caenorhabditis elegans gentle and harsh touch response behavior using a multiplexed hydraulic microfluidic device

AbstractThe roundworm Caenorhabditis elegans is an important model system for understanding the genetics and physiology of touch. Classical assays for C. elegans touch, which involve manually touching the animal with a probe and observing its response, are limited by their low throughput and qualitative nature. We developed a microfluidic device in which several dozen animals are subject to spatially localized mechanical stimuli with variable amplitude. The device contains 64 sinusoidal channels through which worms crawl, and hydraulic valves that deliver touch stimuli to the worms. We used this assay to characterize the behavioral responses to gentle touch stimuli and the less well studied harsh (nociceptive) touch stimuli. First, we measured the relative response thresholds of gentle and harsh touch. Next, we quantified differences in the receptive fields between wild type worms and a mutant with non-functioning posterior touch receptor neurons. We showed that under gentle touch the receptive field of the anterior touch receptor neurons extends into the posterior half of the body. Finally, we found that the behavioral response to gentle touch does not depend on the locomotion of the animal immediately prior to the stimulus, but does depend on the location of the previous touch. Responses to harsh touch, on the other hand, did not depend on either previous velocity or stimulus location. Differences in gentle and harsh touch response characteristics may reflect the different innervation of the respective mechanosensory cells. Our assay will facilitate studies of mechanosensation, sensory adaptation, and nociception.

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Identification of Nonviable Genes Affecting Touch Sensitivity in Caenorhabditis elegans Using Neuronally Enhanced Feeding RNA Interference

G3 Genes|Genome|Genetics ◽

10.1534/g3.114.015776 ◽

2015 ◽

Vol 5 (3) ◽

pp. 467-475 ◽

Cited By ~ 4

Author(s):

Xiaoyin Chen ◽

Margarete Diaz Cuadros ◽

Martin Chalfie

Keyword(s):

Rna Interference ◽

Caenorhabditis Elegans ◽

The Body ◽

Genetic Screens ◽

Tubulin Acetylation ◽

Touch Receptor Neurons ◽

Touch Sensitivity ◽

Touch Response ◽

Receptor Neurons ◽

Neuronal Functions

Abstract Caenorhabditis elegans senses gentle touch along the body via six touch receptor neurons. Although genetic screens and microarray analyses have identified several genes needed for touch sensitivity, these methods miss pleiotropic genes that are essential for the viability, movement, or fertility of the animals. We used neuronally enhanced feeding RNA interference to screen genes that cause lethality or paralysis when mutated, and we identified 61 such genes affecting touch sensitivity, including five positive controls. We confirmed 18 genes by using available alleles, and further studied one of them, tag-170, now renamed txdc-9. txdc-9 preferentially affects anterior touch response but is needed for tubulin acetylation and microtubule formation in both the anterior and posterior touch receptor neurons. Our results indicate that neuronally enhanced feeding RNA interference screens complement traditional mutageneses by identifying additional nonviable genes needed for specific neuronal functions.

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The Tactile Receptive Fields of Freely Moving Caenorhabditis elegans Nematodes

10.1101/259937 ◽

2018 ◽

Author(s):

E. A. Mazzochette ◽

A. L. Nekimken ◽

F. Loizeau ◽

J. Whitworth ◽

B. Huynh ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Receptive Fields ◽

Behavioral Responses ◽

The Body ◽

Stimulus Strength ◽

Wild Type ◽

C Elegans ◽

Speed Up ◽

Touch Sensation ◽

Freely Moving

AbstractSensory neurons embedded in skin are responsible for the sense of touch. In humans and other mammals, touch sensation depends on thousands of diverse somatosensory neurons. By contrast, Caenorhabditis elegans nematodes have six gentle touch receptor neurons linked to simple behaviors. The classical touch assay uses an eyebrow hair to stimulate freely moving C. elegans, evoking evasive behavioral responses. While this assay has led to the discovery of genes required for touch sensation, it does not provide control over stimulus strength or position. Here, we present an integrated system for performing automated, quantitative touch assays that circumvents these limitations and incorporates automated measurements of behavioral responses. Highly Automated Worm Kicker (HAWK) unites microfabricated silicon force sensors and video analysis with real-time force and position control. Using this system, we stimulated animals along the anterior-posterior axis and compared responses in wild-type and spc-1(dn) transgenic animals, which have a touch defect due to expression of a dominant-negative α spectrin protein fragment. As expected from prior studies, delivering large stimuli anterior to the mid-point of the body evoked a reversal, but such a stimulus applied posterior to the mid-point evoked a speed-up. The probability of evoking a response of either kind depended on stimulus strength and location; once initiated, the magnitude and quality of both reversal and speed-up behavioral responses were uncorrelated with stimulus location, strength, or the absence or presence of the spc-1(dn) transgene. Wild-type animals failed to respond when the stimulus was applied near the mid-point. These results establish that stimulus strength and location govern the activation of a stereotyped motor program and that the C. elegans body surface consists of two receptive fields separated by a gap.

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Caenorhabditis elegans exhibits positive gravitaxis

BMC Biology ◽

10.1186/s12915-021-01119-9 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Wei-Long Chen ◽

Hungtang Ko ◽

Han-Sheng Chuang ◽

David M. Raizen ◽

Haim H. Bau

Keyword(s):

Caenorhabditis Elegans ◽

Molecular Mechanisms ◽

Life Forms ◽

The Body ◽

C Elegans ◽

Neural Processes ◽

Sensory Cilia ◽

Touch Response ◽

Vector Direction ◽

Hydrodynamic Effects

Abstract Background Gravity plays an important role in most life forms on Earth. Yet, a complete molecular understanding of sensing and responding to gravity is lacking. While there are anatomical differences among animals, there is a remarkable conservation across phylogeny at the molecular level. Caenorhabditis elegans is suitable for gene discovery approaches that may help identify molecular mechanisms of gravity sensing. It is unknown whether C. elegans can sense the direction of gravity. Results In aqueous solutions, motile C. elegans nematodes align their swimming direction with the gravity vector direction while immobile worms do not. The worms orient downward regardless of whether they are suspended in a solution less dense (downward sedimentation) or denser (upward sedimentation) than themselves. Gravitaxis is minimally affected by the animals’ gait but requires sensory cilia and dopamine neurotransmission, as well as motility; it does not require genes that function in the body touch response. Conclusions Gravitaxis is not mediated by passive forces such as non-uniform mass distribution or hydrodynamic effects. Rather, it is mediated by active neural processes that involve sensory cilia and dopamine. C. elegans provides a genetically tractable system to study molecular and neural mechanisms of gravity sensing.

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Structural and functional diversity in the neuronal microtubules of Caenorhabditis elegans.

The Journal of Cell Biology ◽

10.1083/jcb.93.1.15 ◽

1982 ◽

Vol 93 (1) ◽

pp. 15-23 ◽

Cited By ~ 147

Author(s):

M Chalfie ◽

J N Thomson

Keyword(s):

Caenorhabditis Elegans ◽

Colchicine Treatment ◽

Variable Number ◽

Sensory Transduction ◽

Partial Replacement ◽

C Elegans ◽

Touch Receptor Neurons ◽

Touch Sensitivity ◽

Receptor Neurons

Tannic acid fixation reveals differences in the number of protofilaments between microtubules (MTs) in the nematode Caenorhabditis elegans. Most cells have MTs with 11 protofilaments but the six touch receptor neurons (the microtubule cells) have MTs with 15 protofilaments. No 13-protofilament (13-p) MT has been seen. The modified cilia of sensory neurons also possess unusual structures. The cilia contain nine outer doublets with A subfibers of 13 protofilaments and B subfibers of 11 protofilaments and a variable number of inner singlet MTs containing 11 protofilaments. The 15-p MTs but not the 11-p MTs are eliminated by colchicine-treatment or by mutation of the gene mec-7. Concomitantly, touch sensitivity is also lost. However, whereas colchicine treatment leads to the loss of all MTs from the microtubule cells, mutations in mec-7 result in the partial replacement of the 15-p MTs with 11-p MTs. Benzimidazoles (benomyl and nocodazole) have more general effects on C. elegans (slow growth, severe uncoordination, and loss of processes from the ventral cord) but do not affect the 15-p MTs. Benomyl will, however, disrupt the replacement 11-p MTs found in the microtubule cells of mec-7 mutants. The 11-p and 15-p MTs also respond differently to temperature and fixation conditions. It is likely that either type of MT will suffice for the proper outgrowth of the microtubule cell process, but only the 15-p MT can function in the specialized role of sensory transduction of the microtubule cells.

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Quantitative fluorescence imaging of mitochondria in body wall muscles of Caenorhabditis elegans under hyperglycemic conditions using a microfluidic chip

Integrative Biology ◽

10.1093/intbio/zyaa011 ◽

2020 ◽

Vol 12 (6) ◽

pp. 150-160 ◽

Cited By ~ 1

Author(s):

Samuel Sofela ◽

Sarah Sahloul ◽

Sukanta Bhattacharjee ◽

Ambar Bose ◽

Ushna Usman ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Fluorescence Imaging ◽

Microfluidic Device ◽

Microfluidic Chip ◽

Body Wall ◽

The Body ◽

C Elegans ◽

Technological Advantage ◽

Body Wall Muscles ◽

Quantitative Fluorescence

Abstract Type 2 diabetes is the most common metabolic disease, and insulin resistance plays a role in the pathogenesis of the disease. Because completely functional mitochondria are necessary to obtain glucose-stimulated insulin from pancreatic beta cells, dysfunction of mitochondrial oxidative pathway could be involved in the development of diabetes. As a simple animal model, Caenorhabditis elegans renders itself to investigate such metabolic mechanisms because it possesses insulin/insulin-like growth factor-1 signaling pathway similar to that in humans. Currently, the widely spread agarose pad-based immobilization technique for fluorescence imaging of the mitochondria in C. elegans is laborious, batchwise, and does not allow for facile handling of the worm. To overcome these technical challenges, we have developed a single-channel microfluidic device that can trap a C. elegans and allow to image the mitochondria in body wall muscles accurately and in higher throughput than the traditional approach. In specific, our microfluidic device took advantage of the proprioception of the worm to rotate its body in a microfluidic channel with an aspect ratio above one to gain more space for its undulation motion that was favorable for quantitative fluorescence imaging of mitochondria in the body wall muscles. Exploiting this unique feature of the microfluidic chip-based immobilization and fluorescence imaging, we observed a significant decrease in the mitochondrial fluorescence intensity under hyperglycemic conditions, whereas the agarose pad-based approach did not show any significant change under the same conditions. A machine learning model trained with these fluorescence images from the microfluidic device could classify healthy and hyperglycemic worms at high accuracy. Given this significant technological advantage, its easiness of use and low cost, our microfluidic imaging chip could become a useful immobilization tool for quantitative fluorescence imaging of the body wall muscles in C. elegans.

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Combinatorial control of touch receptor neuron expression in Caenorhabditis elegans

Development ◽

10.1242/dev.119.3.773 ◽

1993 ◽

Vol 119 (3) ◽

pp. 773-783 ◽

Cited By ~ 7

Author(s):

S. Mitani ◽

H. Du ◽

D.H. Hall ◽

M. Driscoll ◽

M. Chalfie

Keyword(s):

Caenorhabditis Elegans ◽

Cell Types ◽

Wild Type ◽

Negative Regulators ◽

C Elegans ◽

Combinatorial Control ◽

Touch Receptor Neurons ◽

In The Wild ◽

Receptor Neurons ◽

Touch Receptor Neuron

Six touch receptor neurons with distinctive morphological features sense gentle touch in Caenorhabditis elegans. Previous studies have identified three genes (lin-32, unc-86 and mec-3) that regulate touch cell development. However, since other cell types also require these genes, we suspected that other genes help restrict the expression of touch cell characteristics to the six neurons seen in the wild type. To identify such genes, we have examined mutants defective in genes required for the development of other C. elegans cells for changes in the pattern of touch cell-specific features. Mutations in seven genes either reduce (lin-14) or increase (lin-4, egl-44, egl-46, sem-4, ced-3 and ced-4) the number of touch receptor-like cells. The combinatorial action of these genes, all of which are required for the production of many cell types, restrict the number of cells expressing touch receptor characteristics in wild-type animals by acting as positive and negative regulators and by removing cells by programmed cell death.

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Regulated distribution of mitochondria in touch receptor neurons of C. elegans influences touch response

10.1101/2020.07.26.221523 ◽

2020 ◽

Author(s):

Anjali Awasthi ◽

Souvik Modi ◽

Sneha Hegde ◽

Anusheela Chatterjee ◽

Sudip Mondal ◽

...

Keyword(s):

Molecular Motors ◽

Neuronal Function ◽

Mitochondrial Density ◽

Neuronal Process ◽

C Elegans ◽

Touch Receptor Neurons ◽

Touch Response ◽

Receptor Neurons ◽

Touch Receptor Neuron

AbstractDensity of mitochondria and their localization at specific sub-cellular regions of the neurons is regulated by molecular motors, their adaptors and the cytoskeleton. However, the regulation of the mitochondrial density, the positioning of mitochondria along the neuronal process and the role of axonal mitochondria in neuronal function remain poorly understood. This study shows that the density of mitochondria in C. elegans touch receptor neuron processes remains constant through development. Simulations show that mitochondrial positioning along parts of the neuronal process that are devoid of synapses is regulated. Additionally, we also demonstrate that axonal mitochondria are necessary for maintaining touch responsiveness.

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Tracking mitochondrial density and positioning along a growing neuronal process in individual C. elegans neuron using a long-term growth and imaging microfluidic device

10.1101/2020.07.27.222372 ◽

2020 ◽

Author(s):

Sudip Mondal ◽

Jyoti Dubey ◽

Anjali Awasthi ◽

Guruprasad Reddy Sure ◽

Sandhya P. Koushika

Keyword(s):

Microfluidic Device ◽

Neuronal Process ◽

C Elegans ◽

Cellular Events ◽

Touch Receptor Neurons ◽

Intracellular Organelles ◽

Resolution Imaging ◽

Receptor Neurons

AbstractThe long cellular architecture of neurons requires regulation in part through transport and anchoring events to distribute intracellular organelles. During development, cellular and sub-cellular events such as organelle additions and their recruitment at specific sites on the growing axons occur over different time scales and often show inter-animal variability thus making it difficult to identify specific phenomena in population averages. To measure the variability in sub-cellular events such as organelle positions, we developed a microfluidic device to feed and immobilize C. elegans for high-resolution imaging over several days. The microfluidic device enabled long-term imaging of individual animals and allowed us to investigate organelle density using mitochondria as a testbed in a growing neuronal process in vivo. Sub-cellular imaging of an individual neuron in multiple animals, over 36 hours in our microfluidic device, shows the addition of new mitochondria along the neuronal process and an increase in the accumulation of synaptic vesicles at synapses, both organelles with important roles in neurons. Long-term imaging of individual C. elegans touch receptor neurons identifies addition of new mitochondria and interacts with other moving mitochondria only through fission and fusion events. The addition of new mitochondria takes place along the entire neuronal process length and the threshold for the addition of a new mitochondrion is when the average separation between the two pre-existing mitochondria exceeds 24 micrometers.

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Mutations Affecting Nerve Attachment of Caenorhabditis elegans

Genetics ◽

10.1093/genetics/157.4.1611 ◽

2001 ◽

Vol 157 (4) ◽

pp. 1611-1622 ◽

Cited By ~ 1

Author(s):

Go Shioi ◽

Michinari Shoji ◽

Masashi Nakamura ◽

Takeshi Ishihara ◽

Isao Katsura ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Nerve Cord ◽

Ventral Nerve Cord ◽

Body Wall ◽

The Body ◽

Genetic Loci ◽

Muscle Attachment ◽

C Elegans ◽

Ventral Cord ◽

Excretory Canal

Abstract Using a pan-neuronal GFP marker, a morphological screen was performed to detect Caenorhabditis elegans larval lethal mutants with severely disorganized major nerve cords. We recovered and characterized 21 mutants that displayed displacement or detachment of the ventral nerve cord from the body wall (Ven: ventral cord abnormal). Six mutations defined three novel genetic loci: ven-1, ven-2, and ven-3. Fifteen mutations proved to be alleles of previously identified muscle attachment/positioning genes, mup-4, mua-1, mua-5, and mua-6. All the mutants also displayed muscle attachment/positioning defects characteristic of mua/mup mutants. The pan-neuronal GFP marker also revealed that mutants of other mua/mup loci, such as mup-1, mup-2, and mua-2, exhibited the Ven defect. The hypodermis, the excretory canal, and the gonad were morphologically abnormal in some of the mutants. The pleiotropic nature of the defects indicates that ven and mua/mup genes are required generally for the maintenance of attachment of tissues to the body wall in C. elegans.

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