We are grateful to Anthony Rosner, PhD., with his organizational support for the early preparation of this project, and to Erin Haus for contributing to protocol analysis. This work was funded by the Juliet&#39;s Cure FBXL4 Mitochondrial Disease Research Fund, the Jaxson Flynt C12ORF65 Research Fund, and the National Institutes of Health (R01-GM120762, R01-GM120762-08S1, R35-GM134863, and T32-NS007413). The content is solely the responsibility of the authors and does not necessarily represent the official views of the funders or the National Institutes of Health.

1. Worm locomotor activity analysis in liquid media on glass slides using ZebraLab software
<ol>
	<li>Nematode growth and handling
	<ol>
		<li>Grow C. elegans on Petri plates containing nematode growth media (NGM) and spread with Escherichia coli OP50 as food source. Maintain worm culture at 20 &#176;C, as previously described8.</li>
		<li>Synchronize worms performing a timed egg lay52 and study worms at the desired stage. In this protocol, L4 stage worms were analyzed.</li>
		<li>Grow control and mutant worm strains on NGM plates with and without drug treatments to be tested or buffer control. To evaluate the drug treatment effects, prepare the desired drug stock concentration in S. basal solution; spread the calculated specific volume onto the NGM plates and allow it to dry. Transfer the worms at a specific larval or adult stage and maintain on the drug treatment plate for the desired duration before analysis.</li>
	</ol>
	</li>
	<li>Experimental set-up of worms for locomotor activity video recording and ZebraLab analysis
	<ol>
		<li>Pick 5 synchronized L4 worms per strain and condition using a worm pick. Pipette a single 20 &#181;L drop of S. basal solution onto a glass slide located under a stereomicroscope connected to a camera and transfer 5 worms into it (Figure 1A,B). Transfer the 5 worms from the Petri dish containing NGM and E. coli OP50 to the liquid drop only at the moment preceding the recording. 
		NOTE: Continue to maintain the other worms on the Petri dish until the prior video is recorded. This will avoid the damage caused on the other worms due to the drying up of the 20 &#181;l drops during the procedure (dry time ~15-20 min).</li>
		<li>Pipette multiple drops on one slide to obtain multiple technical replicates (Figure 1A). Select worms from different NGM plates (biological replicate). Do not use cover slip.</li>
		<li>Adjust the microscope&#39;s working distance to visualize the complete area of a single drop. Set and maintain a low video resolution (&#60;1024 x 768) to upload the files in the software.</li>
		<li>Allow worms to acclimate on the slide at room temperature for 1 min before recording.</li>
		<li>Record the worm swim activity in 1 drop for 1 min at 15 frames per second (fps). Repeat the imaging for each additional drop on the plate.</li>
	</ol>
	</li>
	<li>C. elegans locomotor activity recording analysis in ZebraLab software
	<ol>
		<li>Use the option ZEBRALAB AVI to upload videos to the software. Click on the option Quantization with AVI Files&#160;(Figure 1C).</li>
		<li>To create a new protocol, select File &#62; Generate Protocol, and then add the number of areas selected for the analysis. Choose Location Count: 1.</li>
		<li>Open Protocol Parameters and select 1 min in the Experiment Duration window. Select a different experimental duration for different experimental durations. Select or deselect the window No Time Bin and choose Integration Period, depending on the data output desired. In this study No Time Bin was selected (Figure 1D). 
		&#8203;NOTE: Time bin is the time over which the activity will be averaged.</li>
		<li>If a protocol was already created, select Open Protocol and select the saved protocol (in .vte format).</li>
		<li>Select File and Open a Movie to upload each individual video file that was previously recorded.</li>
		<li>Select the Area icon indicated in Figure 1E (black arrow) to build a single area of detection and create an area around the whole liquid drop where worms are located. Click on Select, then the green circle icon (gray arrow) under Areas &#62; Build &#62; Clear marks. 
		NOTE: The activity of all worms in the defined drop will be detected in the selected area (Figure 1E,F).</li>
		<li>Go to Calibration &#62; Draw scale (Figure 1E) and draw a horizontal line from the left to the right of the video area. Indicate the real distance to calibrate. Then select Apply to Group.</li>
		<li>Unselect the icon selected to build the area (arrow in Figure 1E) and select or deselect Transparent. 
		NOTE: In this study, Transparent was selected and gave better results.</li>
		<li>Adjust Detection Sensitivity and Activity Threshold to allow the detection of all the different C. elegans worm strains analyzed. 
		NOTE: In this experiment, the Detection Sensitivity was set at 8 with Burst and Freezing values of 15 and 2, respectively (Figure 1F).</li>
		<li>Set Display Scale at 70 to visualize the track made by the animal while activity analysis is underway. Then select Apply to Group (Figure 1F).</li>
		<li>Click on Experiment &#62; Execute &#62; Save as, and then on Start. A window opens. Choose Do you want to process the video media at maximum computer speed? to analyze the video quickly (e.g., a 1 min video recording is analyzed by the in ZebraLab software in 5 s).</li>
		<li>Another window opens: Running Experiment; click on Start to proceed with the experiment.</li>
		<li>After the video recording is complete, the analysis stops. Click on Experiment &#62; Stop. This saves the activity analyzed from a single drop in a spreadsheet.</li>
		<li>Repeat the analysis for each video of individual drop. Each drop is one technical replicate.</li>
	</ol>
	</li>
	<li>Output and analysis of ZebraLab data 
	NOTE: After the experiment, data from each video is individually saved as separate spreadsheets in the chosen folder. In the data output file, the integrated activity level of all worms moving in an individual drop is recorded as pixel changes under actinteg.
	<ol>
		<li>Open each spreadsheet obtained from the analysis of each video. Compile them manually into a single file. 
		&#8203;Normalize the mutant and wild-type data to a percent of control. Here, statistical analyses were performed to compare mean activity levels between groups.</li>
	</ol>
	</li>
</ol>
2. Worm locomotor activity analysis in liquid media in 96-well plate format by WormScan software analysis
<ol>
	<li>Nematode growth and handling
	<ol>
		<li>Grow C. elegans as described in section 1.1.1.</li>
		<li>Synchronize worms as described in section 1.1.2.</li>
		<li>Grow worms on specific media as described in section 1.1.3 until L4 stage or day 1 adult.</li>
	</ol>
	</li>
	<li>Experimental set-up of worms in 96-well plate for WormScan activity analysis
	<ol>
		<li>Add 50 &#181;L of 2% weight per volume of E. coli OP50 in liquid suspension in S. basal medium to each well of a 96-well, clear, flat-bottom, microplate, as previously described49,53.</li>
		<li>Under a stereomicroscope, manually pick 15 synchronized worms at L4 stage or day 1 adult from their NGM plates into liquid media within each experimental well of the 96-well microplate. Allow the worms to acclimate to the liquid media for 20 min before scanning. 
		NOTE: Other animal stages and ages can be readily substituted for study.</li>
	</ol>
	</li>
	<li>WormScan activity analysis in 96-well plate and data export to spreadsheet.
	<ol>
		<li>Scan each 96-well, clear, flat-bottom, microplate twice sequentially using a standard flatbed scanner, with less than 10 s between scans. 
		NOTE: Here, the photo scanner with resolution of 1,200 dots/in and 16-bit grayscale was used to produce jpeg images. Time required to scan four 96-well plates using the photo scanner is less than 10 min.</li>
		<li>Align the two sequential scans (Figure 2A) using open source software49. 
		NOTE: The software generates a difference image to evaluate pixel changes between the two sequential images for a region of interest (Figure 2B) and a relative WormScan Score. This WormScan Score is equivalent to changes in locomotor response based on the light intensity produced by the scanner when set to a pixel threshold of 5 (Figure 2C).</li>
		<li>Export the data from WormScan as a spreadsheet. Save the spreadsheet containing the data to the local computer. Normalize the data as percentage of control (POC) and compare across biological replicate experiments for diverse mutant or treatment conditions. Perform statistical analysis to compare mutant and control means using student t-test.</li>
	</ol>
	</li>
</ol>

M.L., N.D.M., N.S., and E.N.-O. have no relevant financial disclosures. M.J.F. is a co-founder of MitoCUREia, Inc., scientific advisory board member with equity interest in RiboNova, Inc., and scientific board member as paid consultant with Khondrion, and Larimar Therapeutics. M.J.F. has previously been or is currently engaged with several companies involved in mitochondrial disease therapeutic preclinical and/or clinical stage development as a paid consultant (Astellas [formerly Mitobridge] Pharma Inc., Cyclerion Therapeutics, Epirium Bio, Imel Therapeutics, Minovia Therapeutics, NeuroVive, Reneo Therapeutics, Stealth BioTherapeutics, Zogenix, Inc.) and/or a sponsored research collaborator (AADI Therapeutics, Cardero Therapeutics, Cyclerion Therapeutics, Imel Therapeutics, Minovia Therapeutics Inc., Mission Therapeutics, NeuroVive, Raptor Therapeutics, REATA Inc., RiboNova Inc., Standigm Therapeutics, and Stealth BioTherapeutics).

Here, the study summarized detailed information and rationales for studying C. elegans neuromuscular activity at the level of diverse outcomes, including worm thrashing, locomotion, pharyngeal pumping, and chemotaxis. The comparison of 16 different activity analysis methodologies was performed in terms of the relative throughput, advantages, and limitations of quantify nematode activities in a single worm or worm populations at different ages and experimental durations. Among these, two novel adaptations and&#16...

Caenorhabiditis elegans is widely recognized as an outstanding model in neuroscience based on it having 302 neurons that coordinate all worm behaviors, including mating, feeding, egg-laying, defecation, swimming, and locomotion on solid media1. These hermaphroditic nematodes are also widely used to understand a wide array of human disease mechanisms, made possible by its well-characterized genome and high homology of ~80% genes between C. elegans and humans2,3,4. C. elegans have long been used to interrogate human mitochondrial disease5,6,7,8,9,10, which is a highly genetically and phenotypically heterogeneous group of inherited metabolic disorders that share impaired capacity to generate cellular energy and often clinically present with substantially impaired neuromuscular function, exercise intolerance, and fatigue11,12,13,14.To this end, the use of C. elegans models enable preclinical modeling of quantitative aspects of animal activity and neuromuscular function in different genetic subtypes of mitochondrial disease, as well as their response to candidate therapies that may improve their neuromuscular function and overall activity.
Neuromuscular activity in C. elegans is objectively measurable by a range of experimental methodologies, including both manual and semi-automated approaches that allow functional analyses in either solid or liquid media (Table 1)1,15. Accurate quantitation of C. elegans activity has proven important to enable discoveries related to function and development of the muscular and nervous system16,17,18. This study summarizes and compares experimental requirements, advantages, and limitations of 17 different assays that can be performed in research laboratories to evaluate neuromuscular function and activity on four key outcomes in C. elegans diseases models, both at the baseline at a range of developmental stages and ages as well as in response to candidate therapies (Table 1). Indeed, the study provides a detailed overview of the range of available experimental approaches to characterize rates of C. elegans thrashing (body bends per minute), locomotor activity, pharyngeal pumping, and chemotaxis-in each case specifying the experimental and analytic methodology used, the advantages and limitations of each method, the equipment and software needed to perform and analyze each assay, and the throughput capacity of each method to support its use for high-throughput genetic or drug screening purposes. The throughput capacity of each assay is described as low, medium, or high based on the experimental protocol complexity, including worm maintenance, processing time, the use of single or multi-well plates, and/or experimenter time needed to complete the experimental setting and data analyses.
Manual analyses of thrashing19, locomotor activity20, pharyngeal pumping17,21, and chemotaxis22,23 are well-established methodologies to evaluate worm activity that require a stereomicroscope24. While measuring thrashing activity of worms requires analysis in liquid media to determine the frequency of body bends per minute, worm locomotor activity may be measured either on solid media or in liquid media. However, manual analyses of individual worm activity are inherently time-consuming and involves unavoidable user-generated bias. Automation of worm activity analyses minimizes user-generated bias and can greatly increase experimental throughput25. Video recordings of worm thrashing activity in liquid media can be analyzed using wrMTrck, an ImageJ plugin26. However, the original experimental settings that were developed for wrMTrck limited its utility, since too many worms in a single liquid drop led to overlapping of worms that made accurate tracking difficult. While this experimental limitation has been resolved27, the wrMTrck method is not able to support high-throughput screening.
A range of methods exist to quantify worm locomotor activity&#160;at baseline and in response to candidate therapies in C. elegans mitochondrial disease models. These include ZebraLab (ViewPoint Life Sciences), Tierpsy Tracker28, wide field-of-view nematode tracking platform (WF-NTP)29, WormMotel, WormWatcher30, WormLab31, Infinity Chip32, and WMicrotracker One33 (Table 1). These methods enable concurrent analysis of locomotion in multiple worm strains or conditions, typically on multi-well plates, thereby supporting higher-throughput drug screening applications. Some of these methods have unique considerations that may limit or enhance their general utility, such as the need for expensive equipment versus open-access software, and varying ease of performing experimental protocols. Overall, no single experimental system or protocol is ideally suited to all C. elegans locomotor activity experiments. Rather, it is important to carefully choose which method is best suited to the specific investigator&#39;s experimental goals and requirements.
Pharyngeal pumping represents another important outcome to assess neuromuscular activity in C. elegans. The C. elegans pharynx is composed of 20 muscle cells, 20 neurons, and 20 other cells that enable ingestion of Escherichia coli (E. coli) at the anterior end of the worm&#39;s alimentary tract34,35,36. Several manual methods have been established to determine pharyngeal pumping rates17,21,37,38. Most methods are based on the use of a stereomicroscope and camera to visualize and record pharyngeal pumping frequency with direct counting by the experimental observer21. Automated pharyngeal pumping rate analysis is possible by performing an extracellular recording termed electropharyngeogram (EPG), which provides additional information on the duration of each pump39. Pharyngeal pumping rate analysis is also possible in a microfluidic system, WormSpa, where individual worms are confined in chambers40,41. A commercial method available to facilitate analysis of the pharyngeal pump rate is the ScreenChip System (InVivo Biosystems), which measures, visualizes, and analyzes the neuromuscular aspects of feeding behavior in a single worm that is immobilized in a custom chip. This pharyngeal pumping quantitation approach can be used to assess both neuronal and physiological responses to drugs, aging, and other factors42,43,44,45.
Chemotaxis describes the movement of C. elegans in response to an odorant placed away from the worms in a defined area of the nematode growth media (NGM) plate. Assessing the chemotaxis response provides an integrated measure of worm neuronal and neuromuscular activity that is quantifiable by observing and measuring the physical distance traveled by worms toward the odorant in a defined time period46. The Multi-Worm Tracker is an automatic method that can be used to improve the experimental efficiency of quantifying the distance traveled by worms toward an attractant or from a repellant47.
Here, the detailed protocol for two novel, semi-automated methods established for quantifying worm activity is described. The first approach utilizes ZebraLab a commercial software that was originally developed to study swimming activity of Danio rerio (zebrafish), for a novel medium-throughput application to quantify overall locomotor activity in liquid media of C. elegans based on pixel changes during movement (Table 1, Figure 1). Data output is quickly obtained from a large number of concurrent conditions and samples analyzed on a glass slide, although this method is not suitable to a multi-well plate format. The second approach is a novel adaptation of the WormScan methodology48,49 (Figure 2), which uses a flatbed scanner to create a differential image of two sequential scans that can variably be used with open-source software to enable semi-automated quantitative analysis of integrated physiologic outcomes such as fecundity and survival. Here, a novel high-throughput adaptation of the WormScan methodology to quantify worm locomotor activity in liquid media in populations of fifteen larval-stage 4 (L4) worms per well of a 96-well, flat-bottom plate was developed. This semi-automated and low-cost WormScan methodology can be readily adapted to high-throughput drug screens, as well as to analyses of various animal stages and ages48,49.
Here, the protocol and efficacy of analyzing C. elegans locomotor activity using both ZebraLab and WormScan semi-automated methods is demonstrated in a well-established C. elegans model for mitochondrial complex I disease, gas-1(fc21). gas-1 (K09A9.5 gene) is an ortholog of human NDUFS2 (NADH: ubiquinone oxidoreductase core (iron-sulfur protein) subunit 2) (Figure 3). The C. elegans gas-1(fc21) mutant strain carries a homozygous p.R290K missense mutation in the human ortholog of NDUFS250, causing significantly decreased fecundity and lifespan, impaired respiratory chain oxidative phosphorylation (OXPHOS) capacity51, as well as decreased mitochondrial mass and membrane potential with increased oxidative stress5,8. Despite its well-established use over the past two decades to study mitochondrial disease, locomotor activity of gas-1(fc21) mutants was not previously reported. Here,&#160;ZebraLab and WormScan methods were applied to independently quantify the locomotor activity of gas-1(fc21) as compared to wild-type (WT, N2 Bristol) worms, both as a way to validate the methods as well as to demonstrate their comparative utility and efficiency of the experimental protocols and informatics analyses. ZebraLab software allowed rapid quantitation of several concurrent conditions of worm locomotor activity in C. elegans mitochondrial disease models, with potential application for targeted drug screening or validation studies. WormScan analysis, in particular, is well-suited to readily enable high-throughput drug screens of compound libraries and prioritize leads that improve the animal neuromuscular function and the locomotor activity in preclinical C. elegans models of primary mitochondrial disease.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>C. elegans wild isolate</td>
			<td>&#160;Caenorhabditis Genetics Center (CGC)</td>
			<td>N2 Bristol</td>
			<td></td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>Olympus</td>
			<td>DP73</td>
			<td></td>
		</tr>
		<tr>
			<td>gas-1(fc-21)</td>
			<td>CGC</td>
			<td>CW152</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>ThermoFisher</td>
			<td>4951PLUS</td>
			<td></td>
		</tr>
		<tr>
			<td>Nematode Growth Medium (NGM)</td>
			<td>Research Products International Corp.</td>
			<td>N81800-1000.0</td>
			<td></td>
		</tr>
		<tr>
			<td>OP50 Escherichia coli</td>
			<td>CGC</td>
			<td></td>
			<td>Uracil auxotroph E. coli strain</td>
		</tr>
		<tr>
			<td>Petri dishes (60 mm)</td>
			<td>&#160;VWR international</td>
			<td>25373-085</td>
			<td></td>
		</tr>
		<tr>
			<td>S. Basal</td>
			<td>VWR 5.85 g NaCl, 1 g K2 HPO4, 6 g KH2PO4, and 5 mg cholesterol, in 1 l H2O</td>
			<td>VWR 101175-162, 103467-156, EM1.09828.1000, 97061-660</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanner</td>
			<td>EPSON</td>
			<td>V800</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Olympus</td>
			<td>MVX10 microscope</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well flat bottom</td>
			<td>&#160;VWR international</td>
			<td>29442-056</td>
			<td></td>
		</tr>
		<tr>
			<td>WormScan software</td>
			<td>Mathew et al. 45</td>
			<td>S1 Standalone Java platform</td>
			<td>Software for automation of difference image of scanned plates</td>
		</tr>
		<tr>
			<td>ZebraLab software</td>
			<td>ViewPoint</td>
			<td></td>
			<td>Software for automated quantization and tracking of zebrafish behavior, designed by ViewPoint (http://www.viewpoint.fr/en/p/software/zebralab-zebrafish-behavior-screening) and here applied to C. elegans. This system is applicable for high-throughput behavioral analysis</td>
		</tr>
	</tbody>
</table>

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.

Analysis of C. elegans locomotor activity in the liquid media could easily capture an integrated phenotype of mitochondrial disease worm models that may not be easily quantifiable on solid media. ZebraLab was used to quantify locomotor activity of the well-established mitochondrial complex I disease gas-1(fc21) strain relative to WTworms in liquid media at the L4 larval stage. The activity of 5 worms in a single liquid drop was recorded over 1 min, with a total of 19 videos (technical replicate...

Watch this Scientific Journal Video about Comparative Analysis of Experimental Methods to Quantify Animal Activity in Caenorhabditis elegans Models of Mitochondrial Disease at JoVE.com

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.

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 ...

comparative-analysis-experimental-methods-to-quantify-animal-activity

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2.6K Views.  The Children’s Hospital of Philadelphia. 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.

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

Assessment of Sensorimotor Function in Mouse Models of Parkinson's Disease

Sensitive and reliable behavioral outcome measures are essential to the evaluation of potential therapeutic treatments in preclinical trials for many neurodegenerative diseases. In Parkinson's disease, sensorimotor tests sensitive to varying degrees of nigrostriatal dysfunction are fundamental for testing the efficacy of potential therapeutics. Reliable and quite elegant sensorimotor measures exist for rats, however many of these tests measure sensorimotor asymmetry within the rat and are not entirely suitable for the newer genetic mouse models of PD. We have put together a battery of sensorimotor tests inspired by the sensitive tests in rats and adapted for mice. The test battery highlighted in this study is chosen for a) its sensitivity in a wide variety of mouse models of PD, b) its ease in implementing into a study, and c) its low expense. These tests have proven useful in characterizing novel genetic mouse models of PD as well as in testing potential disease-modifying therapies.

Assessment of Sensorimotor Function in Mouse Models of Parkinson&#039;s Disease

In Parkinson's disease and movement disorders in general, sensitive and reliable behavioral assays are essential for testing novel potential therapeutics. Here, we describe a manageable battery of sensorimotor tests for mice that are sensitive to varying degrees of injury to the nigrostriatal system and useful for preclinical studies.

Sensitive and  reliable behavioral outcome measures are essential to the evaluation of  potential therapeutic treatments in preclinical trials for many  ...

Nest Building as an Indicator of Health and Welfare in Laboratory Mice

The minimization and alleviation of suffering has moral and scientific implications. In order to mitigate this negative experience one must be able to identify when an animal is actually in distress. Pain, illness, or distress cannot be managed if unrecognized. Evaluation of pain or illness typically involves the measurement of physiologic and behavioral indicators which are either invasive or not suitable for large scale assessment. The observation of nesting behavior shows promise as the basis of a species appropriate cage-side assessment tool for recognizing distress in mice. Here we demonstrate the utility of nest building behavior in laboratory mice as an ethologically relevant indicator of welfare. The methods presented can be successfully used to identify thermal stressors, aggressive cages, sickness, and pain. Observation of nest building behavior in mouse colonies provides a refinement to health and well-being assessment on a day to day basis.

We demonstrate the utility of nest building behavior in laboratory mice as an indicator of welfare. Nest scoring is a sensitive technique that is altered by temperature, illness, and aggression. The time to integrate into nest test (TINT) is a simple cage-side assessment that can detect postoperative pain.

The minimization and alleviation of  suffering has moral and scientific implications. In order to mitigate this  negative experience one must be able to ...

Measurement of Fronto-limbic Activity Using an Emotional Oddball Task in Children with Familial High Risk for Schizophrenia

Adolescence is a critical developmental period where the early symptoms of schizophrenia frequently emerge. First-degree relatives of people with schizophrenia who are at familial high risk (FHR) may show similar cognitive and emotional changes. However, the neurological changes underlying the emergence of these symptoms remain unclear. This study sought to identify differences in frontal, striatal, and limbic regions in children and adolescents with FHR using functional magnetic resonance imaging. Groups of 21 children and adolescents at FHR and 21 healthy controls completed an emotional oddball task that relied on selective attention and the suppression of task-irrelevant emotional information. The standard oddball task was modified to include aversive and neutral distractors in order to examine potential group differences in both emotional and executive processing. This task was designed specifically to allow for children and adolescents to complete by keeping the difficulty and emotional image content age-appropriate. Furthermore, we demonstrate a technique for suitable fMRI registration for children and adolescent participants. This paradigm may also be applied in future studies to measure changes in neural activity in other populations with hypothesized developmental changes in executive and emotional processing.

This paper describes how to use the emotional oddball task and fMRI to measure brain activation in children and adolescents at familial high risk for schizophrenia (FHR).&#160;FMRI was used to measure differences in fronto-striato-limbic regions during an emotional oddball task. Children with FHR exhibited abnormal functional activation during adolescence.

Adolescence is a critical developmental period where the early symptoms of schizophrenia frequently emerge. First-degree relatives of people with ...

A Computational Method to Quantify Fly Circadian Activity

In most animals and plants, circadian clocks orchestrate behavioral and molecular processes and synchronize them to the daily light-dark cycle. Fundamental mechanisms that underlie this temporal control are widely studied using the fruit fly Drosophila melanogaster as a model organism. In flies, the clock is typically studied by analyzing multiday locomotor recording. Such a recording shows a complex bimodal pattern with two peaks of activity: a morning peak that happens around dawn, and an evening peak that happens around dusk. These two peaks together form a waveform that is very different from sinusoidal oscillations observed in clock genes, suggesting that mechanisms in addition to the clock have profound effects in producing the observed patterns in behavioral data. Here we provide instructions on using a recently developed computational method that mathematically describes temporal patterns in fly activity. The method fits activity data with a model waveform that consists of four exponential terms and nine independent parameters that fully describe the shape and size of the morning and evening peaks of activity. The extracted parameters can help elucidate the kinetic mechanisms of substrates that underlie the commonly observed bimodal activity patterns in fly locomotor rhythms.

A method to quantify the main temporal features seen in fly circadian locomotor rhythms is presented. The quantification is achieved by fitting fly activity with a multi-parametric model waveform. The model parameters describe the shape and size of the morning and evening peaks of daily activity.

In most animals and plants, circadian clocks orchestrate behavioral and molecular processes and synchronize them to the daily light-dark cycle. ...

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) ...

Use of a Wireless Video-EEG System to Monitor Epileptiform Discharges Following Lateral Fluid-Percussion Induced Traumatic Brain Injury

The lateral fluid percussion injury (FPI) model is well established and has been used to study TBI and post-traumatic epilepsy (PTE). However, considerable variability has been reported for the specific parameters used in different studies that have employed this model, making it difficult to harmonize and interpret the results between laboratories. For example, variability has been reported regarding the size and location of the craniectomy, how the Luer lock hub is placed relative to the craniectomy, the atmospheric pressure applied to the dura and the duration of the pressure pulse. Each of these parameters can impact injury severity, which directly correlates with the incidence of PTE. This has been manifested as a wide range of mortality rates, righting reflex times and incidence of convulsive seizures reported. Here we provide a detailed protocol for the method we have used to help facilitate harmonization between studies. We used FPI in combination with a wireless EEG telemetry system to continuously monitor for electrographic changes and detect seizure activity.&#160; FPI is induced by creating a 5 mm craniectomy over the left hemisphere, between the Bregma and Lambda and adjacent to the lateral ridge. A Luer lock hub is secured onto the skull over the craniectomy. This hub is connected to the FPI device, and a 20-millisecond pressure pulse is delivered directly to the intact dura through pressure tubing connected to the hub via a twist lock connector. Following recovery, rats are re-anesthetized to remove the hub. Five 0.5 mm, stainless steel EEG electrode screws are placed in contact with the dura through the skull and serve as four recording electrodes and one reference electrode. The electrode wires are collected into a pedestal connector which is secured into place with bone cement. Continuous video/EEG recordings are collected for up to 4 weeks post TBI.

Here we present a protocol to induce severe TBI with the lateral fluid percussion injury (FPI) model in adult, male Wistar rats. We also demonstrate the use of a wireless telemetry system to collect continuous video-EEG recordings and monitor for epileptiform discharges consistent with post-traumatic epileptogenesis.&#160;

The lateral fluid percussion injury (FPI) model is well established and has been used to study TBI and post-traumatic epilepsy (PTE). However, ...

Low-Cost Gait Analysis for Behavioral Phenotyping of Mouse Models of Neuromuscular Disease

Measurement of animal locomotion is a common behavioral tool used to describe the phenotype of a given disease, injury, or drug model. The low-cost method of gait analysis demonstrated here is a simple but effective measure of gait abnormalities in murine models. Footprints are analyzed by painting a mouse&#8217;s feet with non-toxic washable paint and allowing the subject to walk through a tunnel on a sheet of paper. The design of the testing tunnel takes advantage of natural mouse behavior and their affinity for small dark places. The stride length, stride width, and toe spread of each mouse is easily measured using a ruler and a pencil. This is a well-established and reliable method, and it generates several metrics that are analogous to digital systems. This approach is sensitive enough to detect changes in stride early in phenotype presentation, and due to its non-invasive approach, it allows for testing of groups across life-span or phenotypic presentation.

Footprint analysis is a low-cost alternative to digitized gait analysis programs for researchers quantifying movement abnormalities in mice. Because of its speed, simplicity, and longitudinal potential, it is ideal for behavioral phenotyping of mouse models.

Measurement of animal locomotion is a common behavioral tool used to describe the phenotype of a given disease, injury, or drug model. The low-cost method ...

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. ...

Animal Models of Depression - Chronic Despair Model (CDM)

Major depressive disorder is one of the most prevalent forms of mental illnesses and causes tremendous individual suffering and socioeconomic burden. Despite its importance, current pharmacological treatment is limited, and novel treatment options are urgently needed. One key factor in the search for potential new drugs is evaluating their anti-depressive potency in appropriate animal models. The classical Porsolt forced swim test was used for this purpose for decades to induce and assess a depressive-like state. It consists of two short periods of forced swimming: the first to induce a depressed state and the second on the following day to evaluate the antidepressant effect of the agent given in between the two swim sessions. This model might be suitable as a screening tool for potential antidepressive agents but ignores the delayed onset of action of many antidepressants. The CDM was recently established and represented a modification of the classical test with notable differences. Mice are forced to swim for 5 consecutive days, following the idea that in humans, depression is induced by chronic rather than by acute stress. In a resting period of several days (1-3 weeks), animals develop sustained behavioral despair. The standard read-out method is the measurement of immobility time in an additional delayed swim session, but several alternative methods are proposed to get a broader view of the mood status of the animal. Multiple analysis tools can be used targeting behavioral, molecular, and electrophysiological changes. The depressed phenotype is stable for at least 4 weeks, providing a time window for rapid but also subchronic antidepressant treatment strategies. Furthermore, alterations in the development of a depressive-like state can be addressed using this approach. CDM, therefore, represents a useful tool to better understand depression and to develop novel treatment interventions.

Animal Models of Depression - Chronic Despair Model CDM

The chronic despair mouse model (CDM) of depression consists of repetitive forced swim sessions and another delayed swim phase as a read-out. It represents a suitable model for induction of a chronic depressive-like state stable for at least 4 weeks, amendable to evaluate subchronic and acute treatment interventions.

Major depressive disorder is one of the most prevalent forms of mental illnesses and causes tremendous individual suffering and socioeconomic burden. ...

A Simple Technique to Assay Locomotor Activity in Drosophila

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and diverse behavioral features. Identifying behavioral deficiency in animal models is a crucial measure of disease severity, for example, in neurodegenerative diseases where patients often experience impairments in motor function. However, with the availability of various systems to track and assess motor deficits in fly models, such as drug-treated or transgenic individuals, an economical and user-friendly system for precise evaluation from multiple angles is still lacking. A method based on the AnimalTracker application programming interface (API) is developed here, which is compatible with the Fiji image processing program, to systematically evaluate the movement activities of both adult and larval individuals from recorded video, thus allowing for the analysis of their tracking behavior. This method requires only a high-definition camera and a computer peripheral hardware integration to record and analyze behavior, making it an affordable and effective approach for screening fly models with transgenic or environmental behavioral deficiencies. Examples of behavioral tests using pharmacologically treated flies are given to show how the techniques can detect behavioral changes in both adult flies and larvae in a highly repeatable manner.

A Simple Technique to Assay Locomotor Activity in Drosophila

The present protocol assesses the locomotor activity of Drosophila by tracking and analyzing the movement of flies in a hand-made arena using open-source software Fiji, compatible with plugins to segment pixels of each frame based on high-definition video recording to calculate parameters of speed, distance, etc.

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and ...