We are grateful to Dr. Tingtao Zhou for designing and writing the code for the trajectory visualizations used in our analysis. This work was supported by funding: R01 NS113119 (PWS), Chen senior postdoc fellowship, and the Tianqiao and Chrissy Chen Institute for Neuroscience.

Extraction and Quantification of Volatile Sex Pheromone Responses in C. elegans

The authors have no conflicts of interest to disclose.

This protocol provides a robust methodology for the extraction of volatile sex pheromones from C. elegans, along with establishing a robust chemoattraction assay to measure male chemoattraction responses. Additional information can be found in the WormLab user guide (see the Table of Materials); for a basic code to visualize worm movement trajectory, see protocol section 7.3.8.5. Several crucial steps in the protocol are important for the outcome. First, careful synchronization of worm populatio...

The interplay between chemical communication and reproductive success is a fundamental principle across the animal kingdom1,2,3,4,5,6,7,8,9,10. Sex pheromones trigger a wide array of sexually dimorphic behaviors essential for locating mates, coordinating the steps involved in finding and attracting a partner, and ultimately promoting the propagation of a species11,12,13,14,15,16,17. Significant progress has been made in understanding pheromone signaling, but the molecular mechanisms, neural circuits, and computational processes governing these interactions often remain incompletely defined18,19,20,21,22,23,24,25,26.
The nematode Caenorhabditis elegans provides a powerful model for dissecting these questions. Notably, C. elegans exhibits an unusual reproductive strategy-hermaphrodites can self-fertilize but also outcross with males27,28,29,30,31,32,33. This flexibility requires a robust communication system to signal reproductive status. C. elegans is known for its well-characterized water-soluble pheromones, the ascarosides, which play varied roles in development, behavior, and social interactions. Recent discoveries have unveiled a distinct class of volatile sex pheromones employed by nematodes. These pheromones are specifically produced by sexually mature C. elegans and C. remanei virgin females and sperm-depleted hermaphrodites, serving as an attractant for adult males29,34,35. This attractant exhibits remarkable sexual dimorphism in its production and perception. The female somatic gonad governs the synthesis of this volatile sex pheromone, and production dynamically reflects reproductive status, ceasing upon mating and resuming several hours later29,34.
Understanding nematode sex pheromone communication provides insights into the evolution of chemical communication systems, the interplay between reproductive state and behavior, and the mechanisms underlying sexually dimorphic neural processing24,26,36,37,38,39. Studies implicate the amphid neuron AWA in males as critical for pheromone detection, with the G-protein-coupled receptor SRD-1 playing a key role in pheromone detection in males24. C. elegans is well-suited for studying animal chemical communication, especially sex pheromone signaling, due to its reliance on the olfactory system for mate-searching. While much is known about ascaroside signaling, the volatile sex pheromone system offers unique opportunities for comparison25,26,36,40,41,42,43,44,45,46,47,48,49,50, 
51,52,53,54,55,56,57. Moreover, C. elegans is a powerful genetic model organism due to its fully sequenced genome, clearly defined cellular lineage, and well-characterized olfactory mutants.
However, the complete neural circuitry involved in processing this pheromone, the computations that translate its perception into targeted mate-searching behaviors, and its biosynthesis regulation remain to be fully elucidated. Further investigations into these processes are crucial for understanding the diverse mechanisms governing animal communication and reproductive behaviors. The identification of key genes involved in pheromone synthesis, secretion, and perception promises to unveil novel molecular players in animal communication.&#160;The assays&#160;described here provide a basis to address these&#160;questions.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 cm Petri dishes</td>
			<td>Falcon</td>
			<td>25373-100</td>
			<td>Falcon bacteriological Petri dish 100 x 15 mm</td>
		</tr>
		<tr>
			<td>6 cm Petri dishes</td>
			<td>Falcon</td>
			<td>25373-085</td>
			<td>Falcon bacteriological Petri dish 60 x 15 mm</td>
		</tr>
		<tr>
			<td>C. remanei (EM464)</td>
			<td>CGC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>centrifuge 5418</td>
			<td>&#160;Any brand should work.</td>
		</tr>
		<tr>
			<td>Chemoattraction assay plates</td>
			<td>Homemade solution</td>
			<td>N/A</td>
			<td>1.5% agar, 25 mM NaCl, 1.5 mM Tris-base, and 3.5 mM Tris-Cl</td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Alfa Aesar</td>
			<td>57-88-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Microscope</td>
			<td>Leica</td>
			<td>LeicaMZ75</td>
			<td>&#160;Any brand should work.</td>
		</tr>
		<tr>
			<td>E. Coli OP50</td>
			<td>CGC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Koptec</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>fog-2(q71) (JK574)</td>
			<td>CGC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>him-5(e1490)(CB4088)</td>
			<td>CGC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Household bleach</td>
			<td>Clorox Germicidal bleach concentrated</td>
			<td></td>
			<td>&#160;Bleach</td>
		</tr>
		<tr>
			<td>M9 buffer</td>
			<td>Homemade solution</td>
			<td>N/A</td>
			<td>3 g KH2PO4, 11.3 g Na2HPO4.7H2O, 5 g NaCl, H2O to 1 L. Sterilize by autoclaving. Add 1 mL 1 M MgSO4 after cool down to room temperature.</td>
		</tr>
		<tr>
			<td>Magnesium Sulfate, Anhydrous, Powder</td>
			<td>Macron</td>
			<td>M1063-500GM-EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Microwave</td>
			<td>TOSHIBA</td>
			<td>N/A</td>
			<td>&#160;Any brand should work.</td>
		</tr>
		<tr>
			<td>N2</td>
			<td>CGC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma-aldrich</td>
			<td>S318-3</td>
			<td>1 M</td>
		</tr>
		<tr>
			<td>NGM plates solution</td>
			<td>Homemade solution</td>
			<td>N/A</td>
			<td>2.5 g Peptone, 18 g agar, 3 g NaCl, H2O to 1 L.Sterilize by autoclaving. Once the autoclave is done (2 h), wait until the temperature of the medium drops to 65 &#176;C. Put on a hotplate at 65 &#176;C and stir. Then add the following, waiting 5 min between each to avoid crystallization: 1 mL CaCl2 (1 M), 1 mL MgSO4 (1 M), 25 mL K3PO4 (1 M, pH=6), 1 mL Cholesterol ( 5 mg/mL in ethanol).</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis</td>
			<td>13-374-10</td>
			<td>Bemis Parafilm M Laboratory Wrapping Film</td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>VWR</td>
			<td>97063-324</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet- aid</td>
			<td>Drummond Scientific&#160;</td>
			<td>4-000-100</td>
			<td>&#160;Any brand should work.</td>
		</tr>
		<tr>
			<td>Plastic paper&#160;</td>
			<td>Octago</td>
			<td>Waterproof Screen Printing Inkjet Transparency Film</td>
			<td>https://www.amazon.com/Octago-Waterproof-Transparency-Printing-Printers/dp/B08HJQWFGD</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma-aldrich</td>
			<td>SLBP2366V</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate</td>
			<td>Spectrum</td>
			<td>7778-77-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td>Eppendorf</td>
			<td>SKU: EPPR4331; MFG#: 2231300006</td>
			<td>20 - 200 &#181;L, 100 - 1000 &#181;L, any brand should work.</td>
		</tr>
		<tr>
			<td>Rotator</td>
			<td>Labnet</td>
			<td>SKU: LI-H5500</td>
			<td>&#160;Labnet H5500 Mini LabRoller with Dual Direction Rotator. Any brand should work.</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>VWR</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium phosphate dibasic</td>
			<td>Sigma-aldrich</td>
			<td>SLCG3888</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-base</td>
			<td>Sigma-aldrich</td>
			<td>77-86-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-Cl&#160;</td>
			<td>Roche</td>
			<td>1185-53-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>VWR</td>
			<td>97063-390</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Scientific industries</td>
			<td>Vortex-Genie 2</td>
			<td>&#160;Any brand should work.</td>
		</tr>
		<tr>
			<td>WormLab system&#160;</td>
			<td>MBF Bioscience</td>
			<td>N/A</td>
			<td>https://www.mbfbioscience.com/help/WormLab/Content/home.htm; https://www.mbfbioscience.com/products/wormlab/</td>
		</tr>
		<tr>
			<td>Wormpicker</td>
			<td>Homemade&#160;</td>
			<td>N/A</td>
			<td>made with platinum and glass pipet tips</td>
		</tr>
	</tbody>
</table>

volatile sex pheromone extraction and chemoattraction assay in caenorhabditis elegans

Chemical communication is vital in organismal health, reproduction, and overall well-being. Understanding the molecular pathways, neural processes, and computations governing these signals remains an active area of research. The nematode Caenorhabditis elegans provides a powerful model for studying these processes as it produces a volatile sex pheromone. This pheromone is synthesized by virgin females or sperm-depleted hermaphrodites and serves as an attractant for males.
This protocol describes a detailed method for isolating the volatile sex pheromone from several C. elegans strains (WT strain N2, daf-22, and fog-2) and C. remanei. We also provide a protocol for quantifying the male chemotaxis response to the volatile sex pheromone. Our analysis utilizes measurements such as chemotaxis index (C.I.), arrival time (A.T.), and a trajectory plot to compare male responses under various conditions accurately. This method allows for robust comparisons between males of different genetic backgrounds or developmental stages. Furthermore, the experimental setup outlined here is adaptable to investigating other chemoattraction chemicals.

Trajectory analysis of volatile sex pheromone perception defective strain in chemoattraction assay 
This chemoattraction assay reliably differentiates between wild-type and mutant strains of C. elegans in their response to volatile sex pheromones. Successful experiments with him-5 males consistently demonstrate robust chemotaxis towards the pheromone source. This is reflected in a high chemotaxis index (C.I.) (Figure 2), often exceeding 0.5, indicating...

Author Spotlight: Examining Volatile Sex Pheromone Influence on Male C. elegans Behavior

This protocol establishes methods for extracting and quantifying responses to the volatile sex pheromone in C. elegans, providing tools to study chemical communication and navigation trajectory.

Volatile Sex Pheromone Extraction and Chemoattraction Assay in Caenorhabditis elegans

Chemical communication is vital in organismal health, reproduction, and overall well-being. Understanding the molecular pathways, neural processes, and ...

volatile-sex-pheromone-extraction-chemoattraction-assay

Research

JoVE Journal

Behavior

2.1K Views.  California Institute of Technology. This protocol establishes methods for extracting and quantifying responses to the volatile sex pheromone in C. elegans, providing tools to study chemical communication and navigation trajectory.

Video: Author Spotlight: Examining Volatile Sex Pheromone Influence on Male C. elegans Behavior

The Modified Hole Board - Measuring Behavior, Cognition and Social Interaction in Mice and Rats

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure multiple dimensions of unconditioned behavior in small laboratory mammals (e.g., mice, rats, tree shrews and small primates). This paradigm is a valuable alternative for the use of a behavioral test battery, since a broad behavioral spectrum of an animal&#8217;s behavioral profile can be investigated in one single test.
The apparatus consists of a box, representing the &#8216;protected&#8217; area, separated from a group compartment. A board, on which small cylinders are staggered in three lines, is placed in the center of the box, representing the &#8216;unprotected&#8217; area of the set-up. The cognitive abilities of the animals can be measured by baiting some cylinders on the board and measuring the working and reference memory. Other unconditioned behavior, such as activity-related-, anxiety-related- and social behavior, can be observed using this paradigm. Behavioral flexibility and the ability to habituate to a novel environment can additionally be observed by subjecting the animals to multiple trials in the mHB, revealing insight into the animals&#8217; adaptive capacities.
Due to testing order effects in a behavioral test battery, na&#239;ve animals should be used for each individual experiment. By testing multiple behavioral dimensions in a single paradigm and thereby circumventing this issue, the number of experimental animals used is reduced. Furthermore, by avoiding social isolation during testing and without the need to food deprive the animals, the mHB represents a behavioral test system, inducing if any, very low amount of stress.

This protocol describes the modified hole board, which is a behavioral test set-up that comprises the characteristics of an open field and a traditional hole board. This set-up enables the differential analysis of unconditioned behavior of small laboratory mammals as well as the analysis of cognitive abilities.

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure ...

Profiling Anti-Neu5Gc IgG in Human Sera with a Sialoglycan Microarray Assay

Cells are covered with a cloak of carbohydrate chains (glycans) that is commonly altered in cancer and that includes variations in sialic acid (Sia) expression. These are acidic sugars that have a 9-carbon backbone and that cap vertebrate glycans on cell surfaces. Two of the major Sia forms in mammals are N-acetylneuraminic acid (Neu5Ac) and its hydroxylated form, N-glycolylneuraminic acid (Neu5Gc). Humans cannot produce endogenous Neu5Gc due to the inactivation of the gene encoding cytidine 5&#39;monophosphate-Neu5Ac (CMP-Neu5Ac) hydroxylase (CMAH). Foreign Neu5Gc is acquired by human cells through the dietary consumption of red meat and dairy and subsequently appears on diverse glycans on the cell surface, accumulating mostly on carcinomas. Consequently, humans have circulating anti-Neu5Gc antibodies that play diverse roles in cancer and other chronic inflammation-mediated diseases and that are becoming potential diagnostic and therapeutic targets. Here, we describe a high-throughput sialoglycan microarray assay to assess such anti-Neu5Gc antibodies in the human sera. Neu5Gc-containing glycans and their matched pairs of controls (Neu5Ac-containing glycans), each with a core primary amine, are covalently linked to epoxy-coated glass slides. We exemplify the printing of 56 slides in a 16-well format using a specific nano-printer capable of generating up to 896 arrays per print. Each slide can be used to screen 16 different human sera samples for the evaluation of anti-Neu5Gc antibody specificity, intensity, and diversity. The protocol describes the complexity of this robust tool and provides a basic guideline for those aiming to investigate the response to Neu5Gc dietary carbohydrate antigen in diverse clinical samples in an array format.

A sialoglycan microarray assay can be used to evaluate anti-Neu5Gc antibodies in human sera, making it a potential high-throughput diagnostic assay for cancer and other chronic inflammation-mediated human diseases.

Cells are covered with a cloak of carbohydrate chains (glycans) that is commonly altered in cancer and that includes variations in sialic acid (Sia) ...

A Simple and Low-cost Assay for Measuring Ambulation in Mouse Models of Muscular Dystrophy

Measuring functional outcomes in the treatment of muscular dystrophy is an essential aspect of preclinical testing. The assessment of voluntary ambulation in mouse models is a non-invasive and reproducible activity assay that is directly analogous to measures of patient ambulation such as the 6-minute walk test and related mobility scores. Many common methods for testing mouse ambulation speed and distance are based on the open field test, where an animal&#39;s free movement within an arena is measured over time. One major downside to this approach is that commercial software and equipment for high-resolution motion tracking is expensive and may require transferring mice to specialized facilities for testing. Here, we describe a low-cost, video-based system for measuring mouse ambulation that utilizes free and open-source software. Using this protocol, we demonstrate that voluntary ambulation in the dystrophin-null mdx mouse model for Duchenne muscular dystrophy (DMD) is decreased relative to wild-type mouse activity. In mdx mice expressing the utrophin transgene, these activity deficits are not observed and the total distance traveled is indistinguishable from wild-type mice. This method is effective for measuring changes in voluntary ambulation associated with dystrophic pathology, and provides a versatile platform that can be readily adapted to diverse research settings.

This protocol describes a flexible, low-cost system for measuring mouse ambulation in an open field activity assay. We show that a 6-minute ambulation assay based on this system detects a decrease in voluntary movement in mdx mice, and accurately distinguishes improvement in a muscle-specific rescue of these animals.

Measuring functional outcomes in the treatment of muscular dystrophy is an essential aspect of preclinical testing. The assessment of voluntary ambulation ...

A Behavioral Assay for Investigating the Role of Spatial Memory During Instinctive Defense in Mice

Evolution has selected a repertoire of defensive behaviors that are essential for survival across all animal species. These behaviors are often stereotyped actions elicited in response to innately aversive sensory stimuli, but their success requires enough flexibility for adapting to different spatial environments, which can change rapidly. Here, we describe a behavioral assay to evaluate the influence of learned spatial knowledge on defensive behaviors in mice. We have adapted the widely used Barnes maze spatial memory assay to investigate how mice navigate to a shelter during escape responses to innately aversive sensory stimuli in a novel environment, and how they adapt to acute changes in the environment. This new assay is an ethological paradigm that does not require training and exploits the natural exploration patterns and navigation strategies in mice. We propose that the set of protocols described here are a powerful means of studying goal-directed behaviors and stimulus-triggered navigation, which should be of interest to both the fields of instinctive behaviors and spatial memory.

This protocol describes a behavioral assay, based on the Barnes maze test, to study how instinctive defensive actions are modified by the knowledge of spatial environment.

Evolution has selected a repertoire of defensive behaviors that are essential for survival across all animal species. These behaviors are often ...

Continuous Noninvasive Measuring of Crayfish Cardiac and Behavioral Activities

A crayfish is a pivotal aquatic organism that serves both as a practical biological model for behavioral and physiological studies of invertebrates and as a useful biological indicator of water quality. Even though crayfish cannot directly specify the substances that cause water quality deterioration, they can immediately (within a few seconds) warn humans of water quality deterioration via acute changes in their cardiac and behavioral activities.
In this study, we present a noninvasive method that is simple enough to be implemented under various conditions due to a combination of simplicity and reliability in one model.
This approach, in which the biological organisms are implemented into environmental evaluation processes, provides a reliable and timely alarm for warning of and preventing acute water deterioration in an ambient environment. Therefore, this noninvasive system based on crayfish physiological and ethological parameter recordings was investigated for the detection of changes in an aquatic environment. This system is now applied at a local brewery for controlling&#160;quality of the water used for beverage production, but it can be used at any water treatment and supply facility for continuous, real-time water quality evaluation and for regular laboratory investigations of crayfish cardiac physiology and behavior.

This article presents a noninvasive biomonitoring system for the continuous recording and analyses of crayfish cardiac and locomotor activities. This system consists of a near-infrared optical sensor, a video-tracking module, and software for evaluating crayfish heartbeats that reflects its physiological condition and characterizes crayfish behavior during heartbeat fluctuations.

A crayfish is a pivotal aquatic organism that serves both as a practical biological model for behavioral and physiological studies of invertebrates and as ...

Swimming Induced Paralysis to Assess Dopamine Signaling in Caenorhabditis elegans

The swimming assay described in this protocol is a valid tool to identify proteins regulating the dopaminergic synapses. Similar to mammals, dopamine (DA) controls several functions in C. elegans including learning and motor activity. Conditions that stimulate DA release (e.g., amphetamine (AMPH) treatments) or that prevent DA clearance (e.g., animals lacking the DA transporter (dat-1) which are incapable of reaccumulating DA into the neurons) generate an excess of extracellular DA ultimately resulting in inhibited locomotion. This behavior is particularly evident when animals swim in water. In fact, while wild-type animals continue to swim for an extended period, dat-1 null mutants and wild-type treated with AMPH or inhibitors of the DA transporter sink to the bottom of the well and do not move. This behavior is termed "Swimming Induced Paralysis" (SWIP). Although the SWIP assay is well established, a detailed description of the method is lacking. Here, we describe a step-by-step guide to perform SWIP. To perform the assay, late larval stage-4 animals are placed in a glass spot plate containing control sucrose solution with or without AMPH. Animals are scored for their swimming behavior either manually by visualization under a stereoscope or automatically by recording with a camera mounted on the stereoscope. Videos are then analyzed using a tracking software, which yields a visual representation of thrashing frequency and paralysis in the form of heat maps. Both the manual and automated systems guarantee an easily quantifiable readout of the animals' swimming ability and thus facilitate screening for animals bearing mutations within the dopaminergic system or for auxiliary genes. In addition, SWIP can be used to elucidate the mechanism of action of drugs of abuse such as AMPH.

Swimming Induced Paralysis to Assess Dopamine Signaling in Caenorhabditis elegans

Swimming induced paralysis (SWIP) is a well-established behavioral assay used to study the underlying mechanisms of dopamine signaling in Caenorhabditis elegans (C. elegans). However, a detailed method to perform the assay is lacking. Here, we describe a step-by-step protocol for SWIP.

The swimming assay described in this protocol is a valid tool to identify proteins regulating the dopaminergic synapses. Similar to mammals, dopamine (DA) ...

Assessing the Coherence of Parents' Short Narratives Regarding their Child Using the Five-Minute Speech Sample Procedure

Valid and efficient measures for assessing the quality of parent-child relationships are needed to facilitate research and evidence-based practice with parents. This paper focuses on such a method, namely Five-Minute Speech Sample-Coherence (FMSS-Coherence). In this method, a parent is asked to speak for five uninterrupted minutes about her/his child and their relationship. The resulted narrative is coded for coherence, namely the extent to which the parent provides&#160;in the narrative&#160;a clear, consistent, multidimensional and well-supported portrayal of the child. FMSS-Coherence is based on attachment research that shows that the coherence of parents&#8217; narratives is indicative of the quality of the parent-child relationship and child adjustment. It overcomes the limitations of attachment narrative measures which are typically labor intensive. FMSS-Coherence is less nuanced than extant attachment narrative measures. Yet, studies of families from different cultural backgrounds, across different child ages and in the context of typically developing children as well as children with special needs suggest that coherence can be reliably evaluated using the FMSS procedure. Furthermore, parents&#8217; FMSS-Coherence is associated with parenting quality and children&#8217;s socio-emotional adjustment. Thus, it holds promise for researchers and practitioners who seek a relatively time- and cost-effective method for assessing the coherence of parents&#8217; narratives regarding their child.

This paper introduces a method for assessing the coherence of parents' short narratives regarding their child and their relationship using the five-minute speech sample procedure.

Valid and efficient measures for assessing the quality of parent-child relationships are needed to facilitate research and evidence-based practice with ...

Calcium Imaging in Freely Behaving Caenorhabditis elegans with Well-Controlled, Nonlocalized Vibration

Nonlocalized mechanical forces, such as vibrations and acoustic waves, influence a wide variety of biological processes from development to homeostasis. Animals cope with these stimuli by modifying their behavior. Understanding the mechanisms underlying such behavioral modification requires quantification of neural activity during the behavior of interest. Here, we report a method for calcium imaging in freely behaving Caenorhabditis elegans with nonlocalized vibration of specific frequency, displacement, and duration. This method allows the production of well-controlled, nonlocalized vibration using an acoustic transducer and quantification of evoked calcium responses at single-cell resolution. As a proof of principle, the calcium response of a single interneuron, AVA, during the escape response of C. elegans to vibration is demonstrated. This system will facilitate understanding of neural mechanisms underlying behavioral responses to mechanical stimuli.

Calcium Imaging in Freely Behaving Caenorhabditis elegans with Well-Controlled, Nonlocalized Vibration

Reported here is a system for calcium imaging in freely behaving Caenorhabditis elegans with well-controlled, nonlocalized vibration. This system allows researchers to evoke nonlocalized vibrations with well-controlled properties at nano-scale displacement and to quantify calcium currents during responses of C. elegans to the vibrations.

Nonlocalized mechanical forces, such as vibrations and acoustic waves, influence a wide variety of biological processes from development to homeostasis. ...

An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae

Published assays for mechanical nociception in Drosophila have led to variable assessments of behavior. Here, we fabricated, for use with Drosophila larvae, customized metal nickel-titanium alloy (nitinol) filaments. These mechanical probes are similar to the von Frey filaments used in vertebrates to measure mechanical nociception. Here, we demonstrate how to make and calibrate these mechanical probes and how to generate a full behavioral dose-response from subthreshold (innocuous or non-noxious range) to suprathreshold (low to high noxious range) stimuli. To demonstrate the utility of the probes, we investigated tissue damage-induced hypersensitivity in Drosophila larvae. Mechanical allodynia (hypersensitivity to a normally innocuous mechanical stimulus) and hyperalgesia (exaggerated responsiveness to a noxious mechanical stimulus) have not yet been established in Drosophila larvae. Using mechanical probes that are normally innocuous or probes that typically elicit an aversive behavior, we found that Drosophila larvae develop mechanical hypersensitization (both allodynia and hyperalgesia) after tissue damage. Thus, the mechanical probes and assay that we illustrate here will likely be important tools to dissect the fundamental molecular/genetic mechanisms of mechanical hypersensitivity.

An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae

The goal of this protocol is to show how to perform an improved assay for mechanical nociception in Drosophila larvae. We use the assay here to demonstrate that mechanical hypersensitivity (allodynia and hyperalgesia) exists in Drosophila larvae.

Published assays for mechanical nociception in Drosophila have led to variable assessments of behavior. Here, we fabricated, for use with Drosophila ...

Comparative Analysis of Experimental Methods to Quantify Animal Activity in Caenorhabditis elegans Models of Mitochondrial Disease

Caenorhabditis elegans is widely recognized for its central utility as a translational animal model to efficiently interrogate mechanisms and therapies of diverse human diseases. Worms are particularly well-suited for high-throughput genetic and drug screens to gain deeper insight into therapeutic targets and therapies by exploiting their fast development cycle, large brood size, short lifespan, microscopic transparency, low maintenance costs, robust suite of genomic tools, mutant repositories, and experimental methodologies to interrogate both in vivo and ex vivo physiology. Worm locomotor activity represents a particularly relevant phenotype that is frequently impaired in mitochondrial disease, which is highly heterogeneous in causes and manifestations but collectively shares an impaired capacity to produce cellular energy. While a suite of different methodologies may be used to interrogate worm behavior, these vary greatly in experimental costs, complexity, and utility for genomic or drug high-throughput screens. Here, the relative throughput, advantages, and limitations of 16 different activity analysis methodologies were compared that quantify nematode locomotion, thrashing, pharyngeal pumping, and/or chemotaxis in single worms or worm populations of C. elegans at different stages, ages, and experimental durations. Detailed protocols were demonstrated for two semi-automated methods to quantify nematode locomotor activity that represent novel applications of available software tools, namely, ZebraLab (a medium-throughput approach) and WormScan (a high-throughput approach). Data from applying these methods demonstrated similar degrees of reduced animal activity occurred at the L4 larval stage, and progressed in day 1 adults, in mitochondrial complex I disease (gas-1(fc21)) mutant worms relative to wild-type (N2 Bristol) C. elegans. This data validates the utility for these novel applications of using the ZebraLab or WormScan software tools to quantify worm locomotor activity efficiently and objectively, with variable capacity to support high-throughput drug screening on worm behavior in preclinical animal models of mitochondrial disease.

Comparative Analysis of Experimental Methods to Quantify Animal Activity in Caenorhabditis elegans Models of Mitochondrial Disease

This study presents protocols for two semi-automated locomotor activity analysis approaches in C. elegans complex I disease gas-1(fc21) worms, namely, ZebraLab (a medium-throughput assay) and WormScan (a high-throughput assay) and provide comparative analysis among a wide array of research methods to quantify nematode behavior and integrated neuromuscular function.

Caenorhabditis elegans is widely recognized for its central utility as a translational animal model to efficiently interrogate mechanisms and therapies of ...