This research was supported by the Intramural Research Program of the NIH, The National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), Grant 1Z01 DK011006. We wish to thank Michele Allen for providing technical assistance, Eleni Solomos for editorial assistance, and the NIH veterinary and animal care staff for providing care for the mice used in developing this method.

This procedure was approved by the National Institute of Diabetes and Digestive and Kidney Diseases Animal Care and Use Committee.
1. Preparation
<ol>
	<li>To allow for rapid identification of each mouse prior to testing, tattoo the tails of all mice to be trained and tested with identifying marks. 
	NOTE: This step is optional. Permanent marker or other methods of identification can be used as an alternative to tattooing.</li>
	<li>Prior to training and testing mice, ensure that the treadmill is on a flat surface and set to the treadmill to desired angle of inclination (recommended angle of inclination: 10&#176;, to be kept consistent throughout training and testing) and set the electric shock frequency and intensity appropriately (recommended: 2 Hz, 1.22 mA). 
	NOTE: The electric shock used should produce no more than a mild tingling sensation when touched by an ungloved finger and should be delivered in a pulsatile fashion (with each shock lasting 200 msec).</li>
	<li>Place a clean sheet of butcher&#39;s paper or an absorbent pad under the treadmill to collect fecal boli and urine during training and testing.</li>
	<li>Place a sheet of paper or an absorbent pad over the third of the treadmill housing (i.e., the clear plastic lid that covers the treadmill lanes) furthest from the shock grid. 
	NOTE: This step is optional, but will create a darker space and may provide additional encouragement to avoid the lower portion of the treadmill.</li>
	<li>If planning to use a wire brush to provide additional motivation during training, ensure that one is readily available prior to beginning training sessions.</li>
	<li>Ensure that any drug or method for inducing and/or alleviating fatigue is available and can be prepared or performed during Step 2.14.</li>
</ol>
2. Training Mice to Use the Treadmill
NOTE: Training is necessary to ensure that mice are familiar with the treadmill and task and can perform appropriately when tested. If the majority of mice being trained are receiving frequent shocks or otherwise performing poorly during any training session, additional training sessions should be performed. On the first day, most mice will be shocked several times. By the second day of training, mice should be rarely making contact with the grid. If a mouse displays consistently poor training performance, it should be removed from the study. For female C57BL/6NCr mice, this is a rare occurrence (less than 1% have been removed from studies due to poor training performance), but it should be noted that other strains may perform differently during training.
<ol>
	<li>With the treadmill off (and speed set to 0 m/min), individually lift the mice by the tail and place mice into separate lanes of a mouse treadmill. Promptly turn on the corresponding grid after placing each mouse on the treadmill. Ensure that mice are placed directly on the treadmill belt. 
	NOTE: The amount of time and distance each mouse is held by its tail should be minimized by placing the cage near the treadmill prior to transferring mice to the treadmill and/or allowing mice to stand on a solid platform (e.g., a wire cage lid) until they are near the treadmill and the experimenter is ready to place them in the treadmill.</li>
	<li>Allow mice to freely explore the treadmill for 1-3 min or until each mouse has explored its lane and/or received at least one shock from the grid.</li>
	<li>Turn on the treadmill and slowly increase the speed until it begins moving (approximately 1.5 to 3.0 m/min). Monitor all mice to ensure that they begin walking. If a mouse does not begin walking or walks toward the shock grid, be prepared to intervene by tapping the mouse with a wire brush or tail tickling.</li>
	<li>Slowly increase the treadmill speed to 8 m/min. Start a timer and continue monitoring behavior.</li>
	<li>Increase treadmill speed to 9 m/min at 5 min, 10 m/min at 7 min, and stop the treadmill at 10 min.</li>
	<li>Allow the mice to briefly explore the treadmill, then remove and return each to its cage.</li>
	<li>Clean the treadmill and grid with alcohol and replace the paper or absorbent pad beneath the treadmill.</li>
	<li>To train additional mice, repeat Steps 2.1 through 2.7. 
	NOTE: Allow alcohol to dry prior to placing new mice on the treadmill.</li>
	<li>On the second day of training, repeat Step 2.1. Turn on the treadmill and increase the speed to 10 m/min. Start a timer. 
	NOTE: Treadmill speed can be increased more rapidly than on the first day of training.</li>
	<li>Increase treadmill speed to 11 m/min at 5 min, 12 m/min at 10 min, and stop the treadmill at 15 min.</li>
	<li>Remove mice and return them to their cages.</li>
	<li>Clean the treadmill and grid with alcohol and replace the paper or absorbent pad beneath the treadmill. To train additional mice, repeat Steps 2.9 through 2.12.</li>
	<li>Perform additional days (3 days) of training in the same manner as the second day. 
	NOTE: This step is optional, but is strongly recommended if most or all mice (of the same sex and strain) being trained display difficulty with the task. Mice can generally perform well in Step 3 when they have been trained for 3 days (i.e., with one additional day of training), although additional or fewer days of training may be appropriate depending on their performance during the second training day and the duration of Step 2.14.</li>
	<li>Allow at least one full day to pass in which the mice have no exposure to the treadmill before proceeding to Step 3. 
	NOTE: Any drug(s) used to induce and/or alleviate fatigue should be administered during this step. 
	NOTE: This time period can be varied in length and used to induce fatigue and/or test interventions to reduce or eliminate fatigue. If testing mice more than 7 days after completing training, a pilot study is recommended to verify that the mice used will perform during testing.</li>
</ol>
3. Treadmill Fatigue Test
NOTE: In this test, fatigue-like behavior is defined as spending 5 consecutive seconds in the &#34;fatigue zone&#34;. The fatigue zone is defined as the region encompassing the portion of the treadmill belt within approximately 1 body length of the shock grid as well as the grid, itself. Prior to testing, ensure that the point delineating this zone is clear to the experimenter, such as by applying a mark to the top or side of the treadmill lanes.
<ol>
	<li>Set the treadmill speed to 12 m/min. Do not start the treadmill. Ensure that shock grids are turned off.</li>
	<li>Individually place mice into separate lanes of the treadmill. Turn on the corresponding grid immediately after placing each mouse on the treadmill.</li>
	<li>Simultaneously start the treadmill and a stopwatch. 
	NOTE: Do not intervene during testing except to remove mice that meet the criterion for removal (see Step 3.5).</li>
	<li>Increase treadmill speed as indicated in Table 1. Carefully observe all mice throughout the test. 
	NOTE: The treadmill speeds listed in Table 1 were selected based on observations from adult female C57BL/6NCr mice. Higher treadmill speeds may be appropriate for larger (e.g., outbred CD-1 mice) or more athletic mice.</li>
	<li>If a mouse remains in the fatigue zone for 5 continuous sec, promptly remove the mouse from the treadmill and record the duration and distance it ran.</li>
	<li>When no mice remain on the treadmill, stop the treadmill. Clean the treadmill and grid with alcohol and replace the paper or absorbent pad beneath the treadmill.</li>
	<li>To test additional mice, repeat Steps 3.1 through 3.6. 
	NOTE: This step is optional.</li>
</ol>

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The authors have nothing to disclose.

The current protocol describes how to use a mouse treadmill to measure fatigue-like behavior. This method has several advantages over VWRA, a common preclinical assay of fatigue-like behavior. VWRA requires that mice choose to interact with the test apparatus. As a result, some inbred strains of mice rarely interact with the wheel16 and run so little that it may be difficult or impossible to identify a fatigue-induced decrease in activity. In contrast, the treadmill fatigue test eliminates that choice and ther...

Fatigue affects a wide range of people, can markedly reduce quality of life, and frequently has an unclear or unknown pathogenesis. Cancer-related fatigue (CRF), for example, is experienced by the majority of cancer patients undergoing treatment and can persist long after cancer treatment has been completed and in the absence of detectable cancer1. Moreover, fatigue is also a prominent symptom in numerous other diseases and disorders, including chronic fatigue syndrome, depression, diabetes, and fibromyalgia. Fortunately, there are non-pharmacological interventions that are capable of helping some people experiencing fatigue (e.g., exercise can reduce CRF for some breast cancer patients2,3), but many individuals still lack effective treatment. Furthermore, existing drug treatments for CRF have not been found to be broadly, if at all, efficacious4-7.
Despite the therapeutic need and lack of drug treatment options, preclinical assays of fatigue to aid in the discovery and development of novel fatigue treatments are lacking, especially in animal models. One of the only preclinical assays of fatigue for rodent studies is voluntary wheel running activity (VWRA)9-15, in which mice or other rodents are given free access to a running wheel and their daily running activity is recorded. In many studies, VWRA is the only measure of fatigue-like behavior, with fatigue-like behavior defined (in either VWRA or the current protocol) as a decrease in the measured physical activity in the experimental group. Although VWRA can provide a useful longitudinal measure of fatigue-like behavior, it is a relatively low-throughput assay, running varies considerably between inbred mouse strains16, and it requires subjects to be individually housed, which may cause changes in behavior and test performance17-19. Other assays, such as home cage behavioral monitoring and analysis, can also provide continuous data collection and some systems may allow for subjects to be housed in pairs20. These assays have utility, but may be less sensitive as a means of detecting fatigue-like behavior and, like wheel running, are also low-throughput.
In contrast to VWRA, mouse treadmill tests do not rely upon voluntary activity and can be completed in a short time frame, allowing for higher throughput. In comparison to VWRA, these tests employ external motivators. Specifically, there is usually an electrified metal grid located to the rear of the moving belt to provide mice with an electric shock should they cease to run. In addition to this shock grid, mice may be motivated to run on the treadmill via several other methods, including prodding, poking, or touching them with a hand, brush, or other tool and directing short puffs of air at them. Instead of fatigue, mouse treadmill tests are often used to measure aerobic and/or anaerobic exercise capacity21-25. Mice are motivated to run until they are incapable of or unwilling to continue running on the treadmill as a means of escaping further electric shocks. Testing then ends when mice meet the criterion for exhaustion. In these protocols, to ensure that mice reach true physiologic exhaustion, the criterion for exhaustion is often defined as spending five continuous seconds laying on top of the shock grid and failing to continue running in the face of repeated aversive stimuli. Thus, fatigue-like behavior may be masked in typical treadmill tests due to the strong aversive nature of the external motivation and criterion for ending the test. Interestingly, and in contrast to many other studies using rodent treadmills, a recent publication describes another version of a treadmill fatigue test, which was used as part of an examination of the effects of social stress in mice26. Although the method used by this group markedly differed from the current protocol (i.e., they employed a single-lane treadmill and required 10 sec of electric shock as the criterion for ending their test), their study highlights the utility of and interest in developing a quick, simple fatigue test using the mouse treadmill.
Fatigue is likely to be detectable by means other than wheel running and alterations in routine behaviors. CRF makes patients feel exhausted by a lesser amount of muscle fatigue, as determined by electromyographic analysis, than people without CRF27. Additionally, reduced motivation has been noted in and is measured by several scales measuring human fatigue28,29. Thus, a useful preclinical assay of fatigue-like behavior should distinguish between healthy and fatigued mice on the basis of a measure other than physiological capability and should not obscure decreases in motivation. To achieve that end while avoiding limitations of VWRA and other assays, the current method was developed by adapting the mouse treadmill test. This method uses a shock grid as the sole external motivator to make mice run on the treadmill. Mice quickly learn that the grid provides an aversive stimulus and will promptly move away from it when placed on the treadmill and maintain some distance from it when running.
When mice fatigue, they spend progressively more time toward the back of the treadmill instead of maintaining speed toward the front end. Therefore, the criterion for test completion in this protocol is spending five continuous seconds in the designated fatigue zone (i.e., the rear of the treadmill, ranging from approximately one body length from the shock grid to, and including, the shock grid). This takes advantage of the aversive nature of the grid without requiring mice to receive many or any actual shocks after training. By allowing mice to complete testing using the current criterion rather than exhaustion (as defined above), this method provides a means of using the treadmill to measure fatigue-like behavior rather than its maximal (or near-maximal) physiological capability. Thus, this method can provide a simple, high-throughput assay of fatigue-like behavior in mice and can serve either as an independent or complementary measure to other assays of fatigue-like behavior.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Exer 3/6 Animal Treadmill</td>
			<td style="width:167px;">Columbus Instruments</td>
			<td style="width:131px;">1050-RM Exer-3/6</td>
			<td style="width:427px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stopwatch</td>
			<td>Daigger</td>
			<td>EF24490M&#160;</td>
			<td></td>
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the treadmill fatigue test: a simple, high-throughput assay of fatigue-like behavior for the mouse

Fatigue is a prominent symptom in many diseases and disorders and reduces quality of life for many people. The lack of clear pathogenesis and failure of current interventions to adequately treat fatigue in all patients leaves a need for new treatment options. Despite the therapeutic need and importance of preclinical research in helping identify promising novel treatments, few preclinical assays of fatigue are available. Moreover, the most common preclinical assay used to assess fatigue-like behavior, voluntary wheel running, is not suitable for use with some strains of mice, may not be sensitive to drugs that reduce fatigue, and has relatively low throughput. The current protocol describes a novel, non-voluntary preclinical assay of fatigue-like behavior, the treadmill fatigue test, and provides evidence of its efficacy in detecting fatigue-like behavior in mice treated with a chemotherapy drug known to cause fatigue in humans and fatigue-like behavior in animals. This assay may be a beneficial alternative to wheel running, as fatigue-like behavior and potential interventions can be assessed in a greater number of mice over a shorter time frame, thus permitting faster discovery of new therapeutic options.

This protocol allows fatigue-like behavior to be measured in mice using a treadmill. The data presented in this section was obtained by training and testing 3 separate groups of mice using the current protocol (excluding Figure 1A and 1C). To induce fatigue, 5-fluorouracil (5-FU), a cytotoxic chemotherapy drug known to cause fatigue in humans30 and fatigue-like behavior in mice10,13, was administered.&#160;All data presented are from...

Watch this Scientific Journal Video about The Treadmill Fatigue Test: A Simple, High-throughput Assay of Fatigue-like Behavior for the Mouse at JoVE.com

The Treadmill Fatigue Test: A Simple, High-throughput Assay of Fatigue-like Behavior for the Mouse

Fatigue is a common, undertreated and frequently poorly-understood symptom in many diseases and disorders. New preclinical assays of fatigue may help to improve current understanding and future treatment of fatigue. To that end, the current protocol provides a novel means of measuring fatigue-like behavior in the mouse.

Fatigue is a prominent symptom in many diseases and disorders and reduces quality of life for many people. The lack of clear pathogenesis and failure of ...

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Research

JoVE Journal

Behavior

18.4K Views.  National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health. Fatigue is a common, undertreated and frequently poorly-understood symptom in many diseases and disorders. New preclinical assays of fatigue may help to improve current understanding and future treatment of fatigue. To that end, the current protocol provides a novel means of measuring fatigue-like behavior in the mouse.

Video: The Treadmill Fatigue Test: A Simple, High-throughput Assay of Fatigue-like Behavior for the Mouse

The Resident-intruder Paradigm: A Standardized Test for Aggression, Violence and Social Stress

This video publication explains in detail the experimental protocol of the resident-intruder paradigm in rats. This test is a standardized method to measure offensive aggression and defensive behavior in a semi natural setting. The most important behavioral elements performed by the resident and the intruder are demonstrated in the video and illustrated using artistic drawings. The use of the resident intruder paradigm for acute and chronic social stress experiments is explained as well. Finally, some brief tests and criteria are presented to distinguish aggression from its more violent and pathological forms.

This video shows the resident-intruder paradigm in rats. This test is a standardized method to measure offensive aggression, defensive behavior and violence in a semi-natural setting. The use of the paradigm for social stress experiments is explained as well.

This video publication explains in detail the experimental protocol of the resident-intruder paradigm in rats. This test is a standardized method to ...

Paw-Dragging: a Novel, Sensitive Analysis of the Mouse Cylinder Test

The cylinder test is routinely used to predict focal ischemic damage to the forelimb motor cortex in rodents. When placed in the cylinder, rodents explore by rearing and touching the walls of the cylinder with their forelimb paws for postural support. Following ischemic injury to the forelimb sensorimotor cortex, rats rely more heavily on their unaffected forelimb paw for postural support resulting in fewer touches with their affected paw which is termed forelimb asymmetry. In contrast, focal ischemic damage in the mouse brain fails to result in comparable consistent deficits in forelimb asymmetry. While forelimb asymmetry deficits are infrequently observed, mice do demonstrate a novel behaviour post stroke termed &#8220;paw-dragging&#8221;. Paw-dragging is the tendency for a mouse to drag its affected paw along the cylinder wall rather than directly push off from the wall when dismounting from a rear to a four-legged stance. We have previously demonstrated that paw-dragging behaviour is highly sensitive to small cortical ischemic injuries to the forelimb motor cortex. Here we provide a detailed protocol for paw-dragging analysis. We define what a paw-drag is and demonstrate how to quantify paw-dragging behaviour. The cylinder test is a simple and inexpensive test to administer and does not require pre-training or food deprivation strategies. In using paw-dragging analysis with the cylinder test, it fills a niche for predicting cortical ischemic injuries such as photothrombosis and Endothelin-1 (ET-1)-induced ischemia &#8211; two models that are ever-increasing in popularity and produce smaller focal injuries than middle cerebral artery occlusion. Finally, measuring paw-dragging behaviour in the cylinder test will allow studies of functional recovery after cortical injury using a wide cohort of transgenic mouse strains where previous forelimb asymmetry analysis has failed to detect consistent deficits.

Classical forelimb asymmetry analysis of the cylinder test is routinely used to assess behavioural deficits in rats following brain injury or stroke; however, it fails to detect consistent deficits in mice. This study demonstrates that quantifying paw-dragging behaviour is a more sensitive analysis of brain injury in mice.

The cylinder test is routinely used to predict focal ischemic damage to the forelimb motor cortex in rodents. When placed in the cylinder, rodents explore ...

Morris Water Maze Test: Optimization for Mouse Strain and Testing Environment

The Morris water maze (MWM) is a commonly used task to assess hippocampal-dependent spatial learning and memory in transgenic mouse models of disease, including neurocognitive disorders such as Alzheimer&#8217;s disease. However, the background strain of the mouse model used can have a substantial effect on the observed behavioral phenotype, with some strains exhibiting superior learning ability relative to others. To ensure differences between transgene negative and transgene positive mice can be detected, identification of a training procedure sensitive to the background strain is essential. Failure to tailor the MWM protocol to the background strain of the mouse model may lead to under- or over- training, thereby masking group differences in probe trials. Here, a MWM protocol tailored for use with the F1 FVB/N x 129S6 background is described. This is a frequently used background strain to study the age-dependent effects of mutant P301L tau (rTg(TauP301L)4510 mice) on the memory deficits associated with Alzheimer&#8217;s disease. Also described is a strategy to re-optimize, as dictated by the particular testing environment utilized.

This manuscript describes a Morris water maze (MWM) protocol tailored for use with a commonly used mouse model of Alzheimer's disease. The MWM is widely used in transgenic mouse models. Implementation of a procedure sensitive to the background strain of the mouse model is essential for detecting group differences.

 The Morris water maze (MWM) is a commonly used task to assess hippocampal-dependent spatial learning and memory in transgenic mouse models of disease, ...

The Social Dimension of Stress: Experimental Manipulations of Social Support and Social Identity in the Trier Social Stress Test

In many situations humans are influenced by the behavior of other people and their relationships with them. For example, in stressful situations supportive behavior of other people as well as positive social relationships can act as powerful resources to cope with stress. In order to study the interplay between these variables, this protocol describes two effective experimental manipulations of social relationships and supportive behavior in the laboratory. In the present article, these two manipulations are implemented in the Trier Social Stress Test (TSST)&#8212;a standard stress induction paradigm in which participants are subjected to a simulated job interview. More precisely, we propose (a) a manipulation of the relationship between different protagonists in the TSST by making a shared social identity salient and (b) a manipulation of the behavior of the TSST-selection committee, which acts either supportively or unsupportively. These two experimental manipulations are designed in a modular fashion and can be applied independently of each other but can also be combined. Moreover, these two manipulations can also be integrated into other stress protocols and into other standardized social interactions such as trust games, negotiation tasks, or other group tasks.

Previous research on the social dimension of stress has focused on two important variables: social identity and social support. This protocol introduces an effective experimental manipulation of these two social variables and describes their implementation in a standard stress induction paradigm (Trier Social Stress Test).

In many situations humans are influenced by the behavior of other people and their relationships with them. For example, in stressful situations ...

The 4 Mountains Test: A Short Test of Spatial Memory with High Sensitivity for the Diagnosis of Pre-dementia Alzheimer's Disease

This protocol describes the administration of the 4 Mountains Test (4MT), a short test of spatial memory, in which memory for the topographical layout of four mountains within a computer-generated landscape is tested using a delayed match-to-sample paradigm. Allocentric spatial memory is assessed by altering the viewpoint, colors and textures between the initially presented and target images.
Allocentric spatial memory is a key function of the hippocampus, one of the earliest brain regions to be affected in Alzheimer&#39;s disease (AD) and impairment of hippocampal function predates the onset of dementia. It was hypothesized that performance on the 4MT would aid the diagnosis of predementia AD, which manifests clinically as Mild Cognitive Impairment (MCI).
The 4MT was applied to patients with MCI, stratified further based on cerebrospinal fluid (CSF) AD biomarker status (10 MCI biomarker positive, 9 MCI biomarker negative), and with mild AD dementia, as well as healthy controls. Comparator tests included tests of episodic memory and attention widely accepted as sensitive measures of early AD. Behavioral data were correlated with quantitative MRI measures of the hippocampus, precuneus and posterior cingulate gyrus.
4MT scores were significantly different between the two MCI groups (p = 0.001), with a test score of &#8804;8/15 associated with 100% sensitivity and 78% specificity for the classification of MCI with positive AD biomarkers, i.e., predementia AD. 4MT test scores correlated with hippocampal volume (r = 0.42) and cortical thickness of the precuneus (r = 0.55).
In conclusion, the 4MT is effective in identifying the early stages of AD. The short duration, easy application and scoring, and favorable psychometric properties of the 4MT fulfil the need for a simple but accurate diagnostic test for predementia AD.

This article describes the 4 Mountains Test (4MT), a hippocampus-dependent test of working allocentric spatial memory. The hippocampus is affected early in Alzheimer's disease (AD) and this article outlines the 4MT methodology and results of patient testing, which demonstrates the value of the 4MT in the diagnosis of pre-dementia AD.

This protocol describes the administration of the 4 Mountains Test (4MT), a short test of spatial memory, in which memory for the topographical layout of ...

The Rodent Psychomotor Vigilance Test (rPVT): A Method for Assessing Neurobehavioral Performance in Rats and Mice

The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article describes a rodent version of the PVT&#8212;termed the &#34;rPVT&#34;&#8212;that measures similar aspects of attention (i.e., performance accuracy, motor speed, premature responding, and lapses in attention). Data are presented that demonstrate both the short- and long-term usefulness of the rPVT when employed with laboratory rats. Rats easily learn the rPVT, and learning to perform the basic procedure takes less than two weeks of training. Once acquired, rat performances in the rPVT show a high degree of similarity to these same performance measures in the human PVT, including similarities in, lapses in attention, reaction times, vigilance decrements across session time (i.e., the human &#34;time-on-task&#34; effects), and the response-stimulus interval (RSI) effect described for humans. Thus the rPVT can be an extremely valuable tool for assessing the effects of a wide range of variables on sustained attention quite similar to human PVT performances, and thus can be useful for developing novel treatments for neurobehavioral dysfunctions.

The Rodent Psychomotor Vigilance Test rPVT: A Method for Assessing Neurobehavioral Performance in Rats and Mice

A rat version of the human Psychomotor Vigilance Test (PVT) is described that measures aspects of attention similar to those measured with the human PVT, including aspects of human vigilance such as performance accuracy, motor speed, premature responding, and lapses in attention.

The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article ...

A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors remains poorly understood. In Drosophila melanogaster, environmental cues, such as food availability, affect mating activity offering a tractable system to investigate the mechanisms modulating sexual behavior. In D. melanogaster, environmental cues are often sensed via the chemosensory gustatory and olfactory systems. Here, we present a method to test the effect of environmental chemical cues on mating behavior. The assay consists of a small mating arena containing food medium and a mating couple. The mating frequency for each couple is continuously monitored for 24 h. Here we present the applicability of this assay to test environmental compounds from an external source through a pressurized air system as well as manipulation of the environmental components directly in the mating arena. The use of a pressurized air system is especially useful to test the effect of very volatile compounds, while manipulating components directly in the mating arena can be of value to ascertain a compound's presence. This assay can be adapted to answer questions about the influence of genetic and environmental cues on mating behavior and fecundity as well as other male and female reproductive behaviors.

A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

We demonstrate an assay to analyze the environmental and genetic cues that influence mating behavior in the fruit fly Drosophila melanogaster.

An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors ...

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 Simple Non-invasive Method for Temporary Knockdown of Upper Limb Proprioception

Proprioception may be the least well measured of all contributors to the neural control of movement. New precise, reliable measures of proprioception are needed for clinical diagnosis of impairment, and to measure outcomes of proprioceptive training. The purpose of this simple, non-invasive method is to temporarily knockdown upper limb proprioception in healthy adults, to an extent that would be useful in the development and testing of upper limb proprioception measures. Knockdown models have two main advantages over studying humans with impaired proprioception: participant availability and the ability to control the extent of impairment across participants. Current published methods of temporary proprioception knockdown of the upper limb, such as ischemic nerve blocks and cryotherapy, are invasive, impractical, or uncomfortable for the participant. Here, vibration over the ulnar groove was used to reduce upper limb proprioception. High frequency vibration may reduce proprioceptive acuity by inhibiting pacinian corpuscle-induced input. The effect of vibration used in this protocol was confirmed using two quantitative measures. This method was simple to administer, comfortable for participants, and practical.

The goal of this protocol is to demonstrate a practical method to temporarily interfere with proprioception in the upper limb of healthy humans.

Proprioception may be the least well measured of all contributors to the neural control of movement. New precise, reliable measures of proprioception are ...

Measuring Active and Passive Tameness Separately in Mice

Domesticated animals such as dogs and laboratory mice show a high level of tameness, which is important for humans to handle them easily. Tameness has two behavioral components: a reluctance to avoid humans (passive tameness) and a motivation to approach humans (active tameness). To quantify these components in mice, we previously developed behavioral tests for active tameness, passive tameness, and the willingness to stay on a human hand, each designed to be completed within 3 min. The data obtained were used for selective breeding, with a large number of mice analyzed per generation. The active tameness test measures the movement of the mouse toward a human hand and the contact it engages in. The passive tameness test measures the duration of time that a mouse tolerates human touch. In the stay-on-hand test, a mouse is placed on a human hand and touched slowly using the thumb of that hand; the duration of time that the animal remains on the hand is measured. Here, we describe the test set-up and apparatus, explain the procedures, and discuss the appropriate data analysis. Finally, we explain how to interpret the results.

Tameness in animals includes a reduction of avoidance responses to humans (passive tameness) and an increase in active approaches to humans (active tameness). Here, we describe detailed protocols for three behavioral tests (active tameness, passive tameness, and stay-on-hand tests) to separately measure the active and passive tameness of mice.

Domesticated animals such as dogs and laboratory mice show a high level of tameness, which is important for humans to handle them easily. Tameness has two ...