This work was supported by funding from the Jupiter Life Science Initiative at Florida Atlantic University to ERD and ACK. This work was also supported by grants R21NS105071 (awarded to ACK and ERD) and R15MH118625 (awarded to ERD) from the National Institutes of Health.

The protocol has been approved by the Institutional Animal Care and Use Committeeat Florida Atlantic University .
1. Preparation
<ol>
	<li>Designate an isolated room for performing behavioral studies, or close off a section of a room so that it is isolated. 
	NOTE: The room should be undisturbed and have low traffic to avoid disrupting normal behavior of the fish.</li>
	<li>Move the following materials and equipment into the behavioral room: (i) a camera and lens, (ii) an infrared filter which can be attached to the lens, (iii) a camera stand, (iv) a computer with camera acquisition software, (v) a firm and stable table to perform the assay on, (vi) infrared lights (IR lights; 850 nm or 940 nm), (vii) a white acrylic diffuser, which is longer than the length of the recording tank (viii) 1.8 L trapezoidal plastic assay tank (referred to as the &#39;novel tank&#39;; the one used here is 12 x 18 inches), and (ix) a bucket of fish system water. 
	NOTE: For the novel tank, our lab uses commercially available plastic vessels, which are trapezoidal in shape. The dimensions of the tank are roughly 6 in x 9 in (detailed dimensions are provided in Figure 1A). The diffuser board we use is slightly larger than the novel tank (12 in x 18 in). Novel tank experiments have been performed with tanks having differing shapes, such as those which are rectangular or those with different trapezoidal dimensions20,21. Typically, fish behavior is similar in all tanks regardless of their dimensions: for all containers, fish initially prefer the bottom half, yet over time begin exploring top half with greater frequency.</li>
	<li>Attach the infrared filter to the camera lens. The wavelengths of the infrared light strips typically range from 850 nm to 940 nm. The filter is a long pass filter that restricts light of wavelengths less than 720 nm from transmitting through to the camera.</li>
	<li>Select suitable parameters for the camera acquisition software. For most recordings, set the camera acquisition to a rate of 30 frames-per-second, and recording duration to 10-min. 
	NOTE: These parameters may differ, depending on the experiment. For example, to study habituation in a novel tank22,23, longer recordings may be required.</li>
</ol>
2. Setup
NOTE: The steps in this section describe setting up the novel tank assay. A diagram of the end product is given in Figure 1B.
<ol>
	<li>Place the novel tank in the middle of the table.</li>
	<li>Position the infrared lights behind the tank and place the white acrylic sheet or diffuser screen in between the tank and LED light source.
	<ol>
		<li>Place the diffuser so that it maximally spreads the light coming from the LEDs, and the intensity of the light is enough to illuminate the novel tank. The closer the board is to the light source, the brighter the lights will be, yet the less it will diffuse. By contrast, placing the diffuser board away from the light source will reduce light intensity, but spread the light better.</li>
	</ol>
	</li>
	<li>Fill approximately three quarters of the novel tank with fish system water. 
	NOTE: System water is generated using reverse osmosis of tap water, followed by dosing such that conductivity equals 900 &#177; 100 &#181;S, that pH is neutral (7.2), and that the temperature is 27 &#177; 1 &#176;C.</li>
	<li>Attach the camera to the camera stand and connect the camera to the computer. Open up the video acquisition software and adjust the camera to face the front of the tank and ensure the entire novel tank can be seen and that there are no obscured areas in the video. Adjust the tank and the infrared lights such that there is sufficient and even illumination throughout the tank when observed through the camera. 
	NOTE: Before proceeding to experiments, it can often be useful to perform a trial run, in which video of a fish is captured and tracking is performed. This will ensure that the setup is sufficient for experimentation of behavior.</li>
</ol>
3. Novel tank test setup
<ol>
	<li>Prepare a 250 mL beaker pre-filled with fish system water, and at least two holding tanks.</li>
	<li>On the morning of the test, transfer at least 10 test adult zebrafish to be used for each experimental condition (controls and experimental adults) from fish facility into a holding tank, transfer them to the behavior room, and allow them to acclimate for at least one hour. 
	NOTE: A power analysis should be performed before experimentation, yet in our hands, an n = 10 is usually sufficient to detect statistical significance. Moreover, the holding tank should contain no more than five individuals per liter of water. An acclimation of one hour is sufficient as zebrafish adults have been shown to habituate within 30 minutes of a new tank22. Also, behavioral rhythms are affected by circadian processes, and thus experimental replicates done on different days should be performed within the same hours. We typically perform all experiments between the hours of 11:00 am and 6:00 pm.</li>
	<li>Label the tanks such that the condition or genotype of the animals is blind to the experimenter. 
	NOTE: Experiments can easily be blinded to the experimenter by labeling tanks using a letter or number system (i.e., one tank is labeled &#39;A&#39;, another &#39;B&#39;, etc.). A party not involved in the experiments labels the tanks with such a system, and masks the identities from the experimenter until after post-analysis is complete.</li>
	<li>Using a net, gently place a single adult in the pre-filled beaker from step 3.1. Allow the adult fish to acclimate in the beaker for 10 minutes. 
	NOTE: Record the sex of the adult, as it might be important post-analysis to look for sex-specific differences.</li>
	<li>After acclimation in the beaker, introduce the fish into the novel tank (set up in section 1) by gently pouring out the water and adult from the beaker.</li>
	<li>After introducing the adult into the novel tank, start the camera recording, and move away from the setup to prevent additional distress to the fish.</li>
	<li>After the recording has finished, remove the individual from the novel tank and place into a new holding tank. 
	NOTE: A different holding tank from the one in step 3.2 should be used to prevent repeated testing on the same individuals.</li>
	<li>Repeat steps 3.4 to 3.7 for each adult until all animals have been tested. 
	NOTE: In addition to blinding conditions or genotypes, randomize trials. Use a random number generator or any tool that allows one to randomize between the trials. This should be done before experimentation so that each trial is determined before the experiments begin.</li>
	<li>At the end of all tests, return the fish back to the fish facility.</li>
</ol>
4. Pretreatment with drug
NOTE: The aim of the following steps is to compare the behavior of an individual before and after the use of drugs. This comparison is achieved by first performing a novel tank test as in step 3.4 to 3.6, followed by drug treatment, and then a second novel tank test (Figure 3A).
<ol>
	<li>Prepare a stock solution of the drug, including positive and negative controls. 
	NOTE: If the drug has previously been used in the literature, find an appropriate working dose and use this. For example, for buspirone in the representative results, we make a 100x stock solution and use 0.05 mg/mL as the final concentration, as described in the literature13,20. If suggested dose is unknown, a dose response curve should be performed by examining several concentrations. Set up more beakers with serial dilutions of drug. If the drug is not dissolvable in water, use dimethyl sulfoxide (DMSO) as a solvent.</li>
	<li>Dilute drugs to working concentration in 250 mL beakers with system water. For example, if a 100x solution was made, dilute 1:100 in system water. Set up a beaker with only system water as a control. 
	NOTE: If DMSO was used as a solvent in step 3.1, use an equal volume of DMSO in the control beaker.</li>
	<li>With the help of another researcher, mask the identities of the drug and control beakers to ensure that the tester is blind to the treatment conditions until post-analysis. 
	NOTE: A number or letter system may be used.</li>
	<li>Perform a novel tank test by following steps 3.1 to 3.6 to obtain a baseline behavioral stress response.</li>
	<li>After the baseline recording, use a net to immediately remove the adult fish from the novel tank and place it in the beaker dosed with drug or vehicle, as described in step 4.2. Allow the adult to stay in the beaker for 10 min. 
	NOTE: Ensure that the net does not touch the water in the beakers to prevent cross-contamination of drugs. Ensure proper dosage and administration time depending on the drug used. A 10 min treatment time might not work for all drugs.</li>
	<li>After treatment, use a net to remove the adult from the beaker in step 4.5 and place it in another beaker filled with fresh system water only. This is the washout period to minimize further dosing during the second novel tank test. Allow the adult to stay in the wash out beaker for an additional 10 min. 
	NOTE: Separate nets should be used for each drug condition to prevent any unwanted cross treatment with drug. The washout period may be skipped if the experimenter wishes.</li>
	<li>Perform the novel tank diving test a second time by removing that adult from the beaker in previous step, place it in a new novel tank, and follow steps 3.5 to 3.6.</li>
	<li>After the second novel tank test, remove the individual into a separate holding tank. Pour away the system water in the second novel tank and fill it with fresh system water for the next test. This step prevents cross-contamination of any drug. 
	NOTE: Depending on the half-life of the drugs, fresh beakers containing drugs should be made every 3 hours. For buspirone, make fresh solutions every 3 hours. In addition, following the note in step 3.8, the trials should be randomized between controls and drug treatments.</li>
	<li>At the end of all trials, return individuals back into the fish facility. 
	NOTE: Depending on type of drug used, the effects of these treatments on individuals can be long lasting. Therefore, do not use these individuals in other experiments.</li>
</ol>
5. Video analysis
<ol>
	<li>After all trials, load video files into tracking software of choice. 
	NOTE: We typically use commercially available tracking software, yet several freely available software packages can be used. The steps to achieve tracking may differ according to the software package used.</li>
	<li>Using a still frame from the video, define imaginary boundaries around (i) the entire novel tank arena that is filled with water, and boundaries around (ii) the top third, (iii) middle third, and (iv) bottom third of the tank. Use these to establish time that the fish spent in each portion of the novel tank.</li>
	<li>Calculate x-y displacement per frame for each arena defined in step 5.2.</li>
	<li>Define top, middle, and bottom areas of the tank. Each region should be similar in size. A brief method for determining these regions is to calculate the length of the tank in the y-direction, and divide this by 3. 
	NOTE: Variations to the general protocol do exist. For example, some labs use halves instead of thirds14.</li>
	<li>Determine the time spent in each arena, the distance, and the velocity. 
	NOTE: Most tracking packages will automatically calculate these for the user. However, if the tracking software does not, these can be calculated easily from the x-y displacement values. For example, distance can be determined using the formula: 
	<img alt="Equation 1" src="/files/ftp_upload/59092/59092eq1.jpg" /> 
	and velocity can be determined following the formula: 
	<img alt="Equation 2" src="/files/ftp_upload/59092/59092eq2.jpg" /></li>
	<li>Repeat steps 5.2 to 5.4 to acquire tracks and measurements for all trials. 
	NOTE: Variations to this general protocol</li>
</ol>
6. Testing for normality
<ol>
	<li>Perform statistics before proceeding to calculate statistical differences. Check to see if the data is normally distributed using a Shapiro-Wilk test.</li>
	<li>If the null hypothesis that the data is normally distributed is rejected (i.e., the data does not follow a Gaussian distribution), perform all tests using non-parametric tests. Conversely, if the data is found to follow a normal distribution, proceed to use parametric tests. 
	NOTE: We use commercially available software to perform statistics; however, the R programming language can also be used. A Shapiro-Wilk analysis can be performed using R&#39;s shapiro.test function.</li>
</ol>

The authors declare that they have no competing or financial interests.

Zebrafish exhibit a robust stress response in a novel tank 
Here, we describe a simple behavioral approach for examining stress responses in adult zebrafish, and validate the approach as a simple measure of stress using pharmacology.
The novel tank test is a widely used test for examining innate stress in zebrafish and other species of fish12,14,21,35<s...

Stress responses are altered behavioral and physiological states resulting from potentially harmful or aversive stimuli. Stress responses are conserved throughout the animal kingdom, and are critical for the survival of an organism1. Decades of research have greatly expanded our knowledge of some of the genetic and neuronal mechanisms underlying stress states. Today, areas of the brain such as the amygdala and the striatum2, and genetic factors such as corticotropin releasing hormone (crh), and the glucocorticoid (gr) and mineralocorticoid receptors (mr) have been studied extensively3,4,5,6. Despite these critical findings, much remains unknown about genetic and neuronal regulation of stress. As such, many stress related disorders suffer from a lack of therapeutics.
Genetically amendable model organisms provide a useful tool in the study of genetic and neuronal control of behavior. Fish models, in particular, are extremely powerful: they are small organisms with short generation times, their use in a laboratory setting is facile, their genomes are easily modified, and, as a vertebrate, they share not only genetic, but also neuroanatomical homology with their mammalian counterparts7,8. Standard assays for measuring stress can be paired with zebrafish lines harboring genetic mutations, or those in which manipulation of precise neuronal subsets is possible, and the effects of single genes or defined neurons can be assessed rapidly and efficiently.
Behaviorally, stress responses can be characterized in fish as periods of hyper-activity or prolonged periods of inactivity (akin to &#39;freezing&#39;)9, reduced exploration10, rapid breathing, reduced food intake11, and a place-preference for the bottom of a tank12. For example, when placed into an unfamiliar tank, adult zebrafish and other small fish models show an initial preference for the bottom half of the tank, yet, over time, the fish begin exploring top and bottom halves with near-equal frequency12. Treatment of adults with drugs known to reduce anxiety cause fish to explore immediately the top half10,13. Conversely, drugs that increase anxiety cause fish to show strong preference for the bottom half of the tank12,14,15. Thus, reduced exploration and preference for the bottom half of the tank are simple and reliable indicators of stress.
Like most vertebrates, stress responses in fish are driven by activation of hypothalamic-pituitary-inter-renal axis (HPI; analogous to the hypothalamic-pituitary-adrenal [HPA] axis in mammals)14,16. Hypothalamic neurons expressing the hormone corticotropin-releasing hormone (CRH) signal to the pituitary, which in turn releases adrenocorticotropic releasing hormone (ACTH). ACTH then signals to the inter-renal gland to produce and secrete cortisol, which has a number of downstream targets16, one of them being negative feedback of the crh-producing hypothalamic neurons3,17,18,19.
Here, we describe a method to assess behavioral measures of innate stress. For the behavior, we detail protocols using the novel tank diving test12,14. We then demonstrate, as an example, that a known anxiolytic drug, buspirone, reduces behavioral measures of stress.

<table border="0" cellpadding="0" cellspacing="0" style="width:1043px;" width="1042">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:217px;">Camera</td>
			<td style="width:264px;"></td>
			<td style="width:181px;"></td>
			<td style="width:380px;">We use Point Grey Grasshopper3 USB camera with lens from Edmund Optics.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Infrared filter</td>
			<td style="width:264px;">Edmund Optics</td>
			<td style="width:181px;"></td>
			<td style="width:380px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:217px;">Video Acquisition Program</td>
			<td style="width:264px;"></td>
			<td style="width:181px;"></td>
			<td style="width:380px;">Use programs such as Virtualdub or FlyCapture because the acquisition framerate can be set.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Infrared LED lights</td>
			<td style="width:264px;"></td>
			<td style="width:181px;"></td>
			<td style="width:380px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Assay tank</td>
			<td style="width:264px;">Aquaneering</td>
			<td style="width:181px;">Part number ZT180</td>
			<td style="width:380px;">Size: M3 1.8 liter</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:217px;">Stand and clamp, or standard tripod for camera</td>
			<td style="width:264px;"></td>
			<td style="width:181px;"></td>
			<td style="width:380px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">250mL beaker</td>
			<td style="width:264px;"></td>
			<td style="width:181px;"></td>
			<td style="width:380px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:217px;">Tracking software</td>
			<td style="width:264px;"></td>
			<td style="width:181px;"></td>
			<td style="width:380px;">We use Ethovision XT 13 from Noldus Information Technology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Buspirone chloride</td>
			<td style="width:264px;">Sigma-Aldrich</td>
			<td style="width:181px;">B7148</td>
			<td style="width:380px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Randomized trial generator</td>
			<td></td>
			<td></td>
			<td>We use the RANDBETWEEN function in Microsoft Excel</td>
		</tr>
	</tbody>
</table>

behavioral approaches to studying innate stress in zebrafish

Responding appropriately to stressful stimuli is essential for survival of an organism. Extensive research has been done on a wide spectrum of stress-related diseases and psychiatric disorders, yet further studies into the genetic and neuronal regulation of stress are still required to develop better therapeutics. The zebrafish provides a powerful genetic model to investigate the neural underpinnings of stress, as there exists a large collection of mutant and transgenic lines. Moreover, pharmacology can easily be applied to zebrafish, as most drugs can be added directly to water. We describe here the use of the 'novel tank test' as a method to study innate stress responses in zebrafish, and demonstrate how potential anxiolytic drugs can be validated using the assay. The method can easily be coupled with zebrafish lines harboring genetic mutations, or those in which transgenic approaches for manipulating precise neural circuits are used. The assay can also be used in other fish models. Together, the described protocol should facilitate the adoption of this simple assay to other laboratories.

Examining stress in zebrafish 
To examine stress behavior over time in wild-type zebrafish, we tested adult fish from the AB strain24 in the novel tank test. AB adults were subjected to the protocol as described above. Briefly, fish were given a 1-h acclimation period in a tank in the behavior room. An individual was placed in a beaker for 10-min, and then placed gently in an unfamiliar tank (novel tank) filled with fresh system water. Locomotor activ...

Watch this Scientific Journal Video about Behavioral Approaches to Studying Innate Stress in Zebrafish at JoVE.com

Behavioral Approaches to Studying Innate Stress in Zebrafish

This manuscript describes a simple method to measure stress behaviorally in adult zebrafish. The approach takes advantage of the innate tendency that zebrafish prefer the bottom half of a tank when in a stressful state. We also describe methods for coupling the assay with pharmacology.

Responding appropriately to stressful stimuli is essential for survival of an organism. Extensive research has been done on a wide spectrum of ...

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8.8K Views.  Florida Atlantic University. This manuscript describes a simple method to measure stress behaviorally in adult zebrafish. The approach takes advantage of the innate tendency that zebrafish prefer the bottom half of a tank when in a stressful state. We also describe methods for coupling the assay with pharmacology.

Video: Behavioral Approaches to Studying Innate Stress in Zebrafish

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 ...

A Procedure to Observe Context-induced Renewal of Pavlovian-conditioned Alcohol-seeking Behavior in Rats

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate drug-seeking behavior and prompt relapse in abstinent drug users. We have developed a procedure to study the behavioral and neural processes that mediate the impact of context on alcohol-seeking behavior in rats. Following acclimation to the taste and pharmacological effects of 15% ethanol in the home cage, male Long-Evans rats receive Pavlovian discrimination training (PDT) in conditioning chambers. In each daily (Mon-Fri) PDT session, 16 trials each of two different 10 sec auditory conditioned stimuli occur. During one stimulus, the CS+, 0.2 ml of 15% ethanol is delivered into a fluid port for oral consumption. The second stimulus, the CS-, is not paired with ethanol. Across sessions, entries into the fluid port during the CS+ increase, whereas entries during the CS- stabilize at a lower level, indicating that a predictive association between the CS+ and ethanol is acquired. During PDT each chamber is equipped with a specific configuration of visual, olfactory and tactile contextual stimuli. Following PDT, extinction training is conducted in the same chamber that is now equipped with a different configuration of contextual stimuli. The CS+ and CS- are presented as before, but ethanol is withheld, which causes a gradual decline in port entries during the CS+. At test, rats are placed back into the PDT context and presented with the CS+ and CS- as before, but without ethanol. This manipulation triggers a robust and selective increase in the number of port entries made during the alcohol predictive CS+, with no change in responding during the CS-. This effect, referred to as context-induced renewal, illustrates the powerful capacity of contexts associated with alcohol consumption to stimulate alcohol-seeking behavior in response to Pavlovian alcohol cues.

A procedure to study the capacity of an alcohol associated environmental context to trigger the renewal of alcohol-seeking behavior in rats is described.

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate ...

Mindfulness in Motion (MIM): An Onsite Mindfulness Based Intervention (MBI) for Chronically High Stress Work Environments to Increase Resiliency and Work Engagement

A pragmatic mindfulness intervention to benefit personnel working in chronically high-stress environments, delivered onsite during the workday, is timely and valuable to employee and employer alike. Mindfulness in Motion (MIM) is a Mindfulness Based Intervention (MBI) offered as a modified, less time intensive method (compared to Mindfulness-Based Stress Reduction), delivered onsite, during work, and intends to enable busy working adults to experience the benefits of mindfulness. It teaches mindful awareness principles, rehearses mindfulness as a group, emphasizes the use of gentle yoga stretches, and utilizes relaxing music in the background of both the group sessions and individual mindfulness practice. MIM is delivered in a group format, for 1 hr/week/8 weeks. CDs and a DVD are provided to facilitate individual practice. The yoga movement is emphasized in the protocol to facilitate a quieting of the mind. The music is included for participants to associate the relaxed state experienced in the group session with their individual practice. To determine the intervention feasibility/efficacy we conducted a randomized wait-list control group in Intensive Care Units (ICUs). ICUs represent a high-stress work environment where personnel experience chronic exposure to catastrophic situations as they care for seriously injured/ill patients. Despite high levels of work-related stress, few interventions have been developed and delivered onsite for such environments. The intervention is delivered on site in the ICU, during work hours, with participants receiving time release to attend sessions. The intervention is well received with 97% retention rate. Work engagement and resiliency increase significantly in the intervention group, compared to the wait-list control group, while participant respiration rates decrease significantly pre-post in 6/8 of the weekly sessions. Participants value institutional support, relaxing music, and the instructor as pivotal to program success. This provides evidence that MIM is feasible, well accepted, and can be effectively implemented in a chronically high-stress work environment.

Mindfulness in Motion MIM: An Onsite Mindfulness Based Intervention MBI for Chronically High Stress Work Environments to Increase Resiliency and Work Engagement

The Mindfulness in Motion (MIM) protocol offers a pragmatic Mindfulness Based Intervention (MBI) on-site, for persons working in chronically high-stress work environments that significantly increases resiliency and work engagement. The protocol has proven feasible, beneficial, and is easily adaptable to other high-stress workplaces.

A pragmatic mindfulness intervention to benefit personnel working in chronically high-stress environments, delivered onsite during the workday, is timely ...

Asymmetric Walkway: A Novel Behavioral Assay for Studying Asymmetric Locomotion

Behavioral assays are commonly used for the assessment of sensorimotor impairment in the central nervous system (CNS). The most sophisticated methods for quantifying locomotor deficits in rodents is to measure minute disturbances of unconstrained gait overground (e.g., manual BBB score or automated CatWalk). However, cortical inputs are not required for the generation of basic locomotion produced by the spinal central pattern generator (CPG). Thus, unconstrained walking tasks test locomotor deficits due to motor cortical impairment only indirectly. In this study, we propose a novel, precise foot-placement locomotor task that evaluates cortical inputs to the spinal CPG. An instrumented peg-way was used to impose symmetrical and asymmetrical locomotor tasks mimicking lateralized movement deficits. We demonstrate that shifts from equidistant inter-stride lengths of 20% produce changes in the forelimb stance phase characteristics during locomotion with preferred stride length. Furthermore, we propose that the asymmetric walkway allows for measurements of behavioral outcomes produced by cortical control signals. These measures are relevant for the assessment of impairment after cortical damage.

Here, we present a protocol to quantify precise stepping in rodents. Cortical and the spinal central pattern generator signals are required for precise foot-placement during obstructed locomotion. We report here the novel constrained walking task that directly examines precise stepping behavior.

Behavioral assays are commonly used for the assessment of sensorimotor impairment in the central nervous system (CNS). The most sophisticated methods for ...

A Mouse Model of Subchronic and Mild Social Defeat Stress for Understanding Stress-induced Behavioral and Physiological Deficits

Stressful life events often increase the incidence of depression in humans. To study the mechanisms of depression, the development of animal models of depression is essential. Because there are several types of depression, various animal models are needed for a deeper understanding of the disorder. Previously, a mouse model of subchronic and mild social defeat stress (sCSDS) using a modified chronic social defeat stress (CSDS) paradigm was established. In the paradigm, to reduce physical injuries from aggressors, the duration of physical contact between the aggressor and a subordinate was reduced compared to in the original CSDS paradigm. sCSDS mice showed increased body weight gain, food intake, and water intake during the stress period, and their social behaviors were suppressed after the stress period. In terms of the face validity of the stress-induced overeating and overdrinking following the increased body weight gain, the sCSDS mice may show some features related to atypical depression in humans. Thus, a mouse model of sCSDS may be useful for studying the pathogenic mechanisms underlying depression. This protocol will help establish the sCSDS mouse model, especially for studying the mechanisms underlying stress-induced weight gain and polydipsia- and hyperphagia-like symptoms.

Here, methods for developing a mouse model of subchronic and mild social defeat stress are described and used to investigate the pathogenic features of depression including hyperphagia- and polydipsia-like symptoms following increased body weight.

Stressful life events often increase the incidence of depression in humans. To study the mechanisms of depression, the development of animal models of ...

An Unpredictable Chronic Mild Stress Protocol for Instigating Depressive Symptoms, Behavioral Changes and Negative Health Outcomes in Rodents

Chronic, unresolved stress is a major risk factor for the development of clinical depression. While many preclinical models of stress-induced depression have been reported, the unpredictable chronic mild stress (UCMS) protocol is an established translationally-relevant model for inducing behavioral symptoms commonly associated with clinical depression, such as anhedonia, altered grooming behavior, and learned helplessness in rodents. The UCMS protocol also induces physiological (e.g., hypercortisolemia, hypertension) and neurological (e.g., anhedonia, learned helplessness) changes that are clinically associated with depression. Importantly, UCMS-induced depressive symptoms can be ameliorated through chronic, but not acute, treatment with common SSRIs. As such, the UCMS protocol offers many advantages over acute stress protocols or protocols that utilize more extreme stressors. Our protocol involves randomized, daily exposures to 7 distinct stressors: damp bedding, removal of bedding, cage tilt, alteration of light/dark cycles, social stresses, shallow water bath, and predator sounds/smells. By subjecting rodents 3-4 hr daily to these mild stressors for 8 weeks, we demonstrate both significant behavioral changes and poor health outcomes to the cardiovascular system. This approach allows for in-depth interrogation of the neurological, behavioral, and physiological alterations associated with chronic stress-induced depression, as well as for testing of new potential therapeutic agents or intervention strategies.

The unpredictable chronic mild stress (UCMS) protocol is a validated method for studying behavioral and physiological changes associated with chronic stress and depressive symptoms. Eight weeks of imposition of the UCMS protocol induces behavioral changes and poor health outcomes in rodents of either gender.

Chronic, unresolved stress is a major risk factor for the development of clinical depression. While many preclinical models of stress-induced depression ...

Behavioral Disturbances: An Innovative Approach to Monitor the Modulatory Effects of a Nutraceutical Diet

In dogs, diets are often used to modulate behavioral disturbances related to chronic anxiety and stress caused by intense and restless activity. However, the traditional ways to monitor behavioral changes in dogs are complicated and not efficient. In the current clinical evaluation, a new, simple monitoring system was used to assess the effectiveness of a specific diet in positively modulating the intense and restless activity of 24 dogs of different ages and breeds. This protocol describes how to easily and rapidly evaluate improvement in a set of symptoms related to generalized anxiety by using a specific sensor, a mobile phone app, a wireless router, and a computer. The results showed that dogs treated with specific diets showed significant improvement in the times spent active and at rest after 10 days (p &lt; 0.01 and p &lt; 0.05, respectively). These dogs also showed an overall significant improvement in clinical and behavioral symptoms. A specific sensor, along with its related hardware, was demonstrated to successfully monitor behavioral changes relating to movement in dogs.

We report a simple approach to evaluate the effectiveness of a specific diet in positively modulating the daily activity and clinical and behavioral symptoms of dogs with evident behavioral disturbances.

In dogs, diets are often used to modulate behavioral disturbances related to chronic anxiety and stress caused by intense and restless activity. However, ...

The Unpredictable Chronic Mild Stress Protocol for Inducing Anhedonia in Mice

Depression is a highly prevalent and debilitating condition, only partially addressed by current pharmacotherapies. The lack of response to treatment by many patients prompts the need to develop new therapeutic alternatives and to better understand the etiology of the disorder. Pre-clinical models with translational merits are rudimentary for this task. Here we present a protocol for the unpredictable chronic mild stress (UCMS) method in mice. In this protocol, adolescent mice are chronically exposed to interchanging unpredictable mild stressors. Resembling the pathogenesis of depression in humans, stress exposure during the sensitive period of mice adolescence instigates a depressive-like phenotype evident in adulthood. UCMS can be used for screenings of antidepressants on the variety of depressive-like behaviors and neuromolecular indices. Among the more prominent tests to assess depressive-like behavior in rodents is the sucrose preference test (SPT), which reflects anhedonia (core symptom of depression). The SPT will also be presented in this protocol. The ability of UCMS to induce anhedonia, instigate long-term behavioral deficits and enable reversal of these deficits via chronic (but not acute) treatment with antidepressants strengthens the protocol&#39;s validity compared to other animal protocols for inducing depressive-like behaviors.

Here we present the unpredictable chronic mild stress protocol in mice. This protocol induces a long-term depressive-like phenotype and enables to assess the efficacy of putative antidepressants in reversing the behavioral and neuromolecular depressive-like deficits.

Depression is a highly prevalent and debilitating condition, only partially addressed by current pharmacotherapies. The lack of response to treatment by ...

Stress-Enhanced Fear Learning, a Robust Rodent Model of Post-Traumatic Stress Disorder

Fear behaviors are important for survival, but disproportionately high levels of fear can increase the vulnerability for developing psychiatric disorders such as post-traumatic stress disorder (PTSD). To understand the biological mechanisms of fear dysregulation in PTSD, it is important to start with a valid animal model of the disorder. This protocol describes the methodology required to conduct stress-enhanced fear learning (SEFL) experiments, a preclinical model of PTSD, in both rats and mice. SEFL was developed to recapitulate critical aspects of PTSD, including long-term sensitization of fear learning caused by an acute stressor. SEFL uses aspects of Pavlovian fear conditioning but produces a distinct and robust sensitized fear response far greater than normal conditional fear responses. The trauma procedure involves placing a rodent in a conditioning chamber and administering 15 unsignaled shocks randomly distributed over 90 minutes (for rat experiments; for mouse experiments, 10 unsignaled shocks randomly distributed over 60 minutes are used). On day 2, rodents are placed in a novel conditioning context where they receive a single shock; then, on day 3 they are placed back in the same context as on day 2 and tested for changes in freezing levels. Rodents that previously received the trauma display enhanced levels of freezing on the test day compared to those that received no shocks on the first day. Thus, with this model, a single highly stressful experience (the trauma) produces extreme fear of the stimuli associated with the traumatic event.

Here we describe the detailed methodology required to conduct stress-enhanced fear learning (SEFL) experiments, a preclinical model of post-traumatic stress disorder, in both rats and mice. The model utilizes aspects of Pavlovian fear conditioning and freezing as an index of enhanced fear in rodents.

Fear behaviors are important for survival, but disproportionately high levels of fear can increase the vulnerability for developing psychiatric disorders ...

A Visual Guide for Studying Behavioral Defenses to Pathogen Attacks in Leaf-Cutting Ants

The complex lifestyle, evolutionary history of advanced cooperation, and disease defenses of leaf-cutting ants are well studied. Although numerous studies have described the behaviors connected with disease defense, and the associated use of chemicals and antimicrobials, no common visual reference has been made. The main aim of this study was to record short clips of behaviors involved in disease defense, both prophylactically and directly targeted towards an antagonist of the colony following infection. To do so we used an infection experiment, with sub-colonies of the leaf-cutting ant species Acromyrmex echinatior, and the most significant known pathogenic threat to the ants&#39; fungal crop (Leucoagaricus gongylophorus), a specialized pathogenic fungus in the genus Escovopsis. We filmed and compared infected and uninfected colonies, at both early and more advanced stages of infection. We quantified key defensive behaviors across treatments and show that the behavioral response to pathogen attack likely varies between different castes of worker ants, and between early and late detection of a threat. Based on these recordings we have made a library of behavioral clips, accompanied by definitions of the main individual defensive behaviors. We anticipate that such a guide can provide a common frame of reference for other researchers working in this field, to recognize and study these behaviors, and also provide greater scope for comparing different studies to ultimately help better understand the role these behaviors play in disease defense.

We present a visual guide to disease defense behaviors in leaf-cutting ants, with individual clips and accompanying definitions, illustrated in an experimental infection scenario. Our main aim is to help other researchers recognize key defensive behaviors and to provide a common understanding for future research in this field.

The complex lifestyle, evolutionary history of advanced cooperation, and disease defenses of leaf-cutting ants are well studied. Although numerous studies ...