This work was funded in part by the Beatrice H. Barrett endowment for research on neuro-operant relations to the University of North Texas (UNT). We are grateful for the input and assistance of all members of the Neuroplasticity and Repair Laboratory, especially Valerie Rojas, Mary Kate Moore, Cameron Scallon, and Hannah McGee.

All procedures and animal care were approved by the University of North Texas institutional animal care and use committee (IACUC) and adhered to National Institutes of Health guide for the care and use of Laboratory animals. Adult male and female Long-Evans rats (400-800 g, 1.5 years old), used in the present study, were housed in colony caging.
1. Equipment preparation
<ol>
	<li>Obtain or assemble the one-rat turnstile (ORT) according to the design files and instructions for construction (see Supplementary File 1 and Supplementary Coding File 1). Refer to Butcher et al.16 for further details. 
	NOTE: ORTs are specific to rat size, so a colony cage should include animals that are approximately the same size. If one doesn&#39;t wish to self-assemble ORTs, they may be purchased pre-assembled (see Table of Materials).</li>
	<li>Obtain and attach a radio-frequency identification (RFID, see Table of Materials) reader and obtain and inject animals with RFID tags. 
	NOTE: When injecting RFID full duplex (FDX) tags, the orientation must be perpendicular to the RFID antenna as the rat walks through the ORT. In this validation, tags were implanted subcutaneously between the shoulder blade on a plane parallel with the spine.</li>
	<li>Affix the RFID antenna to the tube of the ORT.</li>
	<li>Construct and/or obtain the behavioral apparatus(es) and colony caging appropriate for the experimental question. In this example, custom-built colony caging21,22 was used in conjunction with commercially available operant chambers (see Table of Materials), though any equipment could theoretically be used. 
	NOTE: Competition of colony-housed animals for access to the behavioral apparatus(es) via the ORT should be considered. Anticipate needing one ORT + behavioral apparatus for every 4 to 6 animals.</li>
	<li>Attach the ORT(s) between the behavioral apparatus and colony caging.</li>
	<li>Cut a portal hole in the behavioral apparatus and colony caging using a Dremel rotary tool (see Table of Materials) or similar instrument. The internal diameter should be equal to the outer diameter of the constructed ORT tunnel. 
	&#8203;NOTE: The ORT must be elevated a few inches to operate, so a small platform or stand will be needed to align the colony caging and apparatus heights.</li>
	<li>Install RFID system to read animals as they pass through the ORT and, if desired, integrate it with the behavioral apparatus.</li>
</ol>
2. Presurgical behavioral training
<ol>
	<li>Obtain same-sized cohort of rats and introduce them into the colony caging. 
	NOTE: Animals that have been reared or housed extensively in isolation or with few conspecifics may have more trouble exploring the chamber, especially when it involves traversing social areas of the colony caging. Animals should be exposed to group caging early in life to avoid this pitfall.</li>
	<li>Remove access to any manipulanda within the behavioral apparatus and set the chamber to auto-deliver rewards every 60 s, on average, when occupied. 
	NOTE: This study used sucrose water (30% to 40%) as the reward, but sweetened condensed milk is also effective.</li>
	<li>Train all rats to regularly enter the behavioral apparatus(es) via the ORT.</li>
	<li>At least once per day, check the data to ensure all animals are entering the ORT. If animals are not entering, insert a pen-sized object into the locking mechanism to prevent it from locking temporarily allow animals to explore more freely. If animals are still not entering, remove the turnstile and attach a temporary sidewall to allow free tunnel access to the chamber.</li>
	<li>Once all animals are regularly entering the chamber, return the lock (and turnstile) and reassess. 
	NOTE: Animals may also occupy the ORT and chamber as a temporary reprieve from other rats. One way to preempt this sort of monopolization of the chamber is to attach an additional ORT that bridges to a simple isolation chamber.</li>
	<li>Introduce the manipulandum-the pull handle, in this example case-and set to the highest sensitivity. Insert the handle just inside the box (up to 2 cm) or just outside the box. 
	NOTE: Painter&#39;s tape can evoke reach attempts if affixed to the back of the handle, just out of reach.</li>
	<li>Reduce the frequency that the reward (i.e., 30% sucrose water) is auto-delivered (e.g., every 90-120 s). Remember that any reward can be utilized that fits the needs of the experimenter and the animals&#39; preferences.</li>
	<li>Check the data daily to ensure that all animals have learned to activate the lever. Bait the lever and/or change insertion level until all animals are pulling.</li>
	<li>Discontinue auto-delivery of rewards so that they are only available via activation of the pull handle.</li>
	<li>If previously inserted, retract the lever each day (provided that all rats continue&#160;to pull at that retraction level) by 0.25 mm to 0.5 mm until the lever is in its final position, 1 cm to 1.25 cm outside the chamber. 
	NOTE: The exact position of the lever depends on the size of the rats. Ensure to choose a position which results in the desired reaching topography.</li>
	<li>Initiate a percentile or other training program to progressively increase required pull forces to activate the handle. 
	NOTE: This study used a percentile schedule that sets the criterion for reinforcement at the upper quartile of the previous 15 responses. Alternatively, stepwise increases of the pull criterion can be used7.</li>
	<li>Once animals reliably reach the final criterion range of 120 g pulls, remove the percentile training program and fix the criterion for handle activation at a constant of 120 g.</li>
	<li>Collect baseline data at this force requirement until success rates have been steady (non-trending) for about a week.</li>
</ol>
3. Inducing stroke
<ol>
	<li>Surgically induce stroke in all colony caged animals at the same time. 
	NOTE: To induce the stroke an endothelian-1 model of stroke was used, which has been described elsewhere23.</li>
	<li>Allow animals to recover in traditional caging, isolated individually, for 3-7 days.</li>
</ol>
4. Postsurgical behavioral testing
<ol>
	<li>After recovery, return animals to the colony caging with the ORT-attached skilled reach apparatus.</li>
	<li>Perform the behavioral testing, keeping the pull requirements at the final of 120 g (follow step 2) until sufficient data is collected to evaluate post-stroke deficits (from one to several days).</li>
	<li>Implement any post-stroke or recovery-related independent variables during subsequent days while animals are accessing the chamber.</li>
</ol>

<ol>
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The authors have no conflicts to disclose.

This protocol has multiple uses. First, and most broadly, the ORT was developed for the purpose of enabling automated single-subject behavioral training and data collection in the context of social, enriched housing. While this study tested the idea of collecting typical behavioral measures and elaborating upon them in the context of stroke, the same can be done for other applications and behavioral tasks. Even the measures gathered in this validation can also be adjusted as needed to include alternative reinforcement sc...

Behavioral training and testing with rat models are important in countless research areas, from the exploration of cognitive processes to disease states and more1. Typically, this training and testing is conducted with single animals in one-on-one sessions, with a researcher manually removing the animal from their home caging and temporarily placing them in some kind of apparatus. Unfortunately, there are several difficulties and limitations with this approach. First, behavioral testing can take a great deal of time for researchers, and when training is necessary, that time requirement becomes even greater. Second, this approach automatically affects-or even potentially confounds-the acquired data, as has been established elsewhere2. These confounds are especially salient when considering enrichment-related variables. Specifically, laboratory rats are traditionally housed in small cages that are just big enough for one or two rats3, and if running wheels are not provided, they may go a lifetime without meaningful opportunities to exercise. Additionally, isolated housing can be a major source of stress in a social species such as the rat4. Some of these welfare-related drawbacks likely impact rat physiology5,6, which may preempt the development of species-typical behavioral expression4 and impact the quality of rodent models as applied to human contexts.
Researchers have pursued several types of solutions to these problems in recent years. The simplest type of solution has been to automate behavioral testing and training7,8,9,10, thus removing the requirement for a single researcher to attend to a single animal. An additional solution has been to automate animal transfer to experimental chambers11,12, further removing the need for human involvement. Last, several setups have been explored which allow animals to be housed in colony caging with other animals and with more room for exploration and enrichment13. Despite these advantages, such colony setups can limit or complicate the efforts to gather individually differentiated behavioral data (though see efforts to use computer vision)14,15. If individual behavioral data is required, it can be more difficult or complex to identify and retrieve animals from colony caging for behavioral sessions as well. At present, few systems exist for collecting individual behavioral data from (enriched) colony housing16,17,18.
These drawbacks may specifically impact research on the behavioral effects of acquired brain injury. First, it is clear that the presence and/or sex of humans as well as handling practices affect rodent behavior2,19, and these variables may differentially impact the behavior of rats before vs. after stroke. Second, human behavioral outcomes after stroke can be worsened by voluntarily decreased engagement with the recommended dosage of rehabilitation exercises20. Currently, rodent experiments tend to not model this sort of context, because rats are not free to choose to engage or abstain from behavioral sessions.
This article introduces a protocol designed to facilitate individual behavioral testing within the framework of enriched colony caging. This approach not only addresses the constraints of current practices but also opens avenues for the exploration of innovative measures. A one-rat turnstile (ORT) has been developed and can be affixed to a colony cage, enabling animals to enter behavioral chambers independently and initiate their own training and testing sessions. The system is affordable; each ORT can be assembled at low cost (given access to a 3D printer). In the past, validation of this system was carried out using a basic operant chamber, showing that animals could be consistently trained to perform a simple operant lever press without the presence of an experimenter16. Nevertheless, the question of whether this configuration is applicable to other scenarios remains unresolved. The aim is to validate the effectiveness of the ORT-colony caging setup, which was previously established, for training and quantifying skilled reach behavior relevant to motor impairment following a stroke. The configuration was utilized to generate novel variables that are typically not explored in stroke research. These variables include performance metrics for the skilled reach task and measurements of self-initiation, which could be pertinent to motivation and decision-making. Furthermore, stroke-induced changes in the circadian patterns of daily self-initiation across the entire 24 h period were effectively detected.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3D printer&#160;</td>
			<td></td>
			<td></td>
			<td>Consult with local makerspace</td>
		</tr>
		<tr>
			<td>bolt</td>
			<td>Boltdepot</td>
			<td>1346</td>
			<td>6-32 or 8-32 by&#160; 0.5&#34;</td>
		</tr>
		<tr>
			<td>bolt</td>
			<td>Boltdepot</td>
			<td>1348</td>
			<td>6-32 or 8-32 by&#160; 0.75&#34;</td>
		</tr>
		<tr>
			<td>door hinge</td>
			<td>XJS (Amazon)</td>
			<td>43398-16234</td>
			<td>1&#34; cabinet stainless steel door hinge set; Optional (if &#34;perfect hinge&#34; is not printed)</td>
		</tr>
		<tr>
			<td>drill</td>
			<td></td>
			<td></td>
			<td>Any electric drill works</td>
		</tr>
		<tr>
			<td>extension spring</td>
			<td>Nieko (Amazon)</td>
			<td>50456A</td>
			<td>Choose and adjust spring based on ORT sized and desired tension</td>
		</tr>
		<tr>
			<td>granulated sugar</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>lock nuts</td>
			<td>Boltdepot</td>
			<td>2551</td>
			<td>6-32 or 8-32</td>
		</tr>
		<tr>
			<td>measuring tape</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>microcontroller</td>
			<td>Arduino</td>
			<td>A000066</td>
			<td>Arduino Uno</td>
		</tr>
		<tr>
			<td>microswitch</td>
			<td>Sparkfun</td>
			<td>KW4-Z5F</td>
			<td>mini microswitch (SPDT-roller lever)</td>
		</tr>
		<tr>
			<td>One Rat Turnstile (ORT)</td>
			<td>Vulintus</td>
			<td></td>
			<td>Contact company to request quote if not self-assembling</td>
		</tr>
		<tr>
			<td>Operant Chambers as desired for behavioral assessment: For this experiment we used automated isometric pull chambers from Vulintus&#160;</td>
			<td>Vulintus</td>
			<td>No cat #: contact Vulintus</td>
			<td>Contact Vulintus for quote</td>
		</tr>
		<tr>
			<td>PLA filament&#160;</td>
			<td>OVERTURE (Amazon)</td>
			<td>UK-MATTEPLA17511</td>
			<td></td>
		</tr>
		<tr>
			<td>plexiglass</td>
			<td>Lesnlok (Amazon)</td>
			<td>B09P74K7BR</td>
			<td>clear, 1/8&#34; thickness, Cut to size</td>
		</tr>
		<tr>
			<td>plexiglass cutter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>python program</td>
			<td>Python Software Foundation</td>
			<td></td>
			<td>software available on request</td>
		</tr>
		<tr>
			<td>RFID reader</td>
			<td>Priority 1 Design</td>
			<td>RFIDRW-E-USB</td>
			<td>With antenna</td>
		</tr>
		<tr>
			<td>RFID tag</td>
			<td>Unified Information Devices</td>
			<td>UC-1485-10</td>
			<td></td>
		</tr>
		<tr>
			<td>rod</td>
			<td>Boltdepot</td>
			<td>23632</td>
			<td>cut to &#62; 3.5&#34;</td>
		</tr>
		<tr>
			<td>Rotary tool</td>
			<td></td>
			<td></td>
			<td>Used to bore hole in apparatus and colony caging for ORT; any hardware usable</td>
		</tr>
		<tr>
			<td>sand paper</td>
			<td>HSYMQ (Amazon)</td>
			<td>TOMPOL-1118-1915-11</td>
			<td></td>
		</tr>
		<tr>
			<td>socket wrench set</td>
			<td></td>
			<td></td>
			<td>Any socket wrench set works</td>
		</tr>
		<tr>
			<td>soldering iron</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>super glue</td>
			<td></td>
			<td>234790</td>
			<td></td>
		</tr>
		<tr>
			<td>wire</td>
			<td>Plusivo (Amazon)</td>
			<td>EAN0721248989789</td>
			<td></td>
		</tr>
	</tbody>
</table>

gathering self-initiated rat behavioral data to characterize post-stroke deficits

Behavioral testing in rat models is frequently utilized for diverse purposes, including psychological, biomedical, and behavioral research. Many traditional approaches involve individual, one-on-one testing sessions between a single researcher and each animal in an experiment. This setup can be very time consuming for the researcher, and their presence may impact the behavioral data in unwanted ways. Additionally, traditional caging for rat research imposes a lack of enrichment, exercise, and socialization that would normally be typical for the species, and this context may also skew the results of behavioral data. Overcoming these limitations may be worthwhile for several research applications, including the study of acquired brain injury. Here, an example method is presented for automatically training and testing individual rat behavior in a colony cage without the presence of humans. Radio frequency identification can be utilized to tailor sessions to the individual rat. The validation of this system occurred in the example context of measuring skilled forelimb motor performance before and after stroke. Traditional characteristics of post-stroke behavioral impairments and novel measures enabled by the system are measured, including success rate, various aspects of pull force, bout analysis, initiation rate and patterns, session duration, and circadian patterns. These variables can be collected automatically with few limitations; though the apparatus removes experimental control of exposure, timing and practice, the validation produced reasonable consistency in these variables from animal to animal.

The animals were trained and tested with four female rats in one colony cage and four male rats in a separate colony cage. All rats learned to pass through the ORTs in four days or less. The four female rats reached &#62;85% successful bouts at the 120 g force requirement in approximately 6 weeks of training and the male rats reached the same criterion in 10 weeks (compared to roughly 3 weeks with standard training with deprived rats)7. This training duration was greatly lengthened due to several ...

Watch this Scientific Journal Video about Gathering Self-Initiated Rat Behavioral Data to Characterize Post-Stroke Deficits at JoVE.com

Gathering Self-Initiated Rat Behavioral Data to Characterize Post-Stroke Deficits

A system for acquiring data from self-initiated individual behavior sessions within a social colony cage setting is presented. The efficacy of this system is demonstrated using an automated skilled reach assessment, enabling the characterization of post-stroke motor impairments, potential behavioral alterations related to motivation, circadian variations, and other innovative dependent variables.

Behavioral testing in rat models is frequently utilized for diverse purposes, including psychological, biomedical, and behavioral research. Many ...

gathering-self-initiated-rat-behavioral-data-to-characterize-post

Research

JoVE Journal

Behavior

968 Views.  University of North Texas. A system for acquiring data from self-initiated individual behavior sessions within a social colony cage setting is presented. The efficacy of this system is demonstrated using an automated skilled reach assessment, enabling the characterization of post-stroke motor impairments, potential behavioral alterations related to motivation, circadian variations, and other innovative dependent variables.

Video: Gathering Self-Initiated Rat Behavioral Data to Characterize Post-Stroke Deficits

Behavioral Phenotyping of Murine Disease Models with the Integrated Behavioral Station (INBEST)

Due to rapid advances in genetic engineering, small rodents have become the preferred subjects in many disciplines of biomedical research. In studies of chronic CNS disorders, there is an increasing demand for murine models with high validity at the behavioral level. However, multiple pathogenic mechanisms and complex functional deficits often impose challenges to reliably measure and interpret behavior of chronically sick mice. Therefore, the assessment of peripheral pathology and a behavioral profile at several time points using a battery of tests are required. Video-tracking, behavioral spectroscopy, and remote acquisition of physiological measures are emerging technologies that allow for comprehensive, accurate, and unbiased behavioral analysis in a home-base-like setting. This report describes a refined phenotyping protocol, which includes a custom-made monitoring apparatus (Integrated Behavioral Station, INBEST) that focuses on prolonged measurements of basic functional outputs, such as spontaneous activity, food/water intake and motivated behavior in a relatively stress-free environment. Technical and conceptual improvements in INBEST design may further promote reproducibility and standardization of behavioral studies.

Behavioral Phenotyping of Murine Disease Models with the Integrated Behavioral Station INBEST

Prolonged and comprehensive monitoring of mice in a home-cage environment provides a deeper understanding of aberrant behavior in murine models of brain diseases. This paper describes the Integrated Behavioral Station (INBEST) as the key component of contemporary behavioral analysis.

Due to rapid advances in genetic engineering, small rodents have become the preferred subjects in many disciplines of biomedical research. In studies of ...

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 ...

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 ...

Detecting Behavioral Deficits in Rats After Traumatic Brain Injury

With the increasing incidence of traumatic brain injury (TBI) in both civilian and military populations, TBI is now considered a chronic disease; however, few studies have investigated the long-term effects of injury in rodent models of TBI. Shown here are behavioral measures that are well-established in TBI research for times early after injury, such as two weeks, until two months. Some of these methods have previously been used at later times after injury, up to one year, but by very few laboratories. The methods demonstrated here are a short neurological assessment to test reflexes, a Beam-Balance to test balance, a Beam-Walk to test balance and motor coordination, and a working memory version of the Morris water maze that can be sensitive to deficits in reference memory. Male rats were handled and pre-trained to neurological, balance, and motor coordination tests prior to receiving parasagittal fluid percussion injury (FPI) or sham injury. Rats can be tested on the short neurological assessment (neuroscore), the beam-balance, and the Beam-Walk multiple times, while testing on the water maze can only be done once. This difference is because rats can remember the task, thus confounding the results if repeated testing is attempted in the same animal. When testing from one to three days after injury, significant differences are detected in all three non-cognitive tasks. However, differences in the Beam-Walk task were not detectable at later time points (after 3 months). Deficits were detected at 3 months in the Beam-Balance and at 6 months in the neuroscore. Deficits in working memory were detected out to 12 months after injury, and a deficit in a reference memory first appeared at 12 months. Thus, standard behavioral tests can be useful measures of persistent behavioral deficits after FPI.

The goal of the behavioral tests presented here is to detect functional deficits in rats after traumatic brain injury. Four specific tests are presented that detect deficits in behaviors to reflect the damage to specific brain areas at times extending to one year after injury.

With the increasing incidence of traumatic brain injury (TBI) in both civilian and military populations, TBI is now considered a chronic disease; however, ...

Modeling Verbal Behavior Deficits with the Stimulus Control Ratio Equation, SCoRE

Verbal operant analyses are the extension of functional analysis technology to the field of verbal behavior, of particular relevance to autism spectrum disorders and associated developmental disabilities. Similarly, a verbal operant analysis carefully controls specific environmental factors that influence language, and measures strength of responding across four verbal operant classes: tact, mand, echoic, and sequelic. The frequency of each independent verbal operant is then measured against one another using the stimulus control ratio equation (SCoRE) to summarize the relative strength of the speaker&#8217;s repertoire. The verbal behavior SCoRE yields a statistic that can be compared against itself (e.g., for the purposes of pre/post testing) or against others (e.g., for the purposes of randomized controlled trials). The results of this evaluation provide an individualized hierarchy for diverging stimulus control across the verbal operants, from which a treatment plan for errorless language learning may be prescribed.

This article describes the procedures for conducting a verbal operant analysis (VOA), calculating the stimulus control ratio equation (SCoRE), and developing individualized verbal behavior treatment plans, specifically significant to address the deficits characteristic of autism.

Verbal operant analyses are the extension of functional analysis technology to the field of verbal behavior, of particular relevance to autism spectrum ...

A Middle Cerebral Artery Occlusion Technique for Inducing Post-stroke Depression in Rats

Post-stroke depression (PSD) is the most recurrent of all psychiatric complications resulting from an ischemic stroke. A greater majority (about 60%) of all ischemic stroke patients suffer from PSD, a disorder considered to be an ischemic stroke-related precursor for increased death and degradation in health. The pathophysiology of PSD is still obscure. To study the mechanism of development and occurrence of PSD further, and to find out a therapy, we attempted to develop a new protocol that requires occluding the middle cerebral artery (MCA) via the internal carotid artery (ICA) in rats. This protocol describes a model of PSD induced in rats through the middle cerebral artery occlusion (MCAO). Also used in the experiment are the Porsolt forced swim test and the sucrose preference test to confirm and evaluate the depressive mood of the rats under investigation. Rather than inserting the catheter through the external carotid artery (ECA), as stipulated for the original procedure, this MCAO technique has the monofilament passing directly through the ICA. This MCAO technique was developed a few years ago and leads to a reduction in mortality and variability. It is generally accepted that the criteria used are preferred in the selection of biological models. The data obtained with this protocol show that this model of MCAO could be a way of inducing PSD in rats and could potentially lead to the understanding of the pathophysiology and the future development of new drugs and other neuroprotective agents.

Here we present a protocol to induce post-stroke depression in rats by occluding the middle cerebral artery via the internal carotid artery. We use the Porsolt forced swim test and the sucrose preference test to confirm and evaluate induced depressive moods.

Post-stroke depression (PSD) is the most recurrent of all psychiatric complications resulting from an ischemic stroke. A greater majority (about 60%) of ...

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.

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 ...

An Instrumented Pull Test to Characterize Postural Responses

Impairment of postural reflexes, termed postural instability, is a common and disabling deficit in Parkinson's disease. To assess postural reflexes, clinicians typically employ the pull test to grade corrective responses to a backward perturbation at the shoulders. However, the pull test is prone to issues with reliability and scaling (score/4). Here, we present an instrumented version of the pull test to more precisely quantify postural responses. Akin to the clinical test, pulls are manually administered except pull force is also recorded. Displacements of the trunk and feet are captured by a semi-portable motion tracking system. Raw data represent distance traveled (in millimeter units), making subsequent interpretation and analysis intuitive. The instrumented pull test also detects variabilities influencing pull test administration, such as pull force, thereby identifying and quantifying potential confounds that can be accounted for by statistical techniques. The instrumented pull test could have application in studies seeking to capture early abnormalities in postural responses, track postural instability over time, and detect responses to therapy.

Impairment of postural reflexes, termed postural instability, is difficult to quantify. Clinical assessments such as the pull test suffer issues with reliability and scaling. Here, we present an instrumented version of the pull test to objectively characterize postural responses.

Impairment of postural reflexes, termed postural instability, is a common and disabling deficit in Parkinson's disease. To assess postural reflexes, ...

Nest Building Behavior as an Early Indicator of Behavioral Deficits in Mice

Nest building is an innate behavior in male and female rodents, even when raised in laboratory settings. As such, many researchers provide rodents synthetic and/or natural materials (such as twine, tissue, cotton, paper, and hay) as a gauge of their overall well-being and as an ancillary assessment to predict the possible decline in cognition. Typically, changes in nesting behaviors, such as failure to create a nest, indicate a change in health or welfare. In addition, nesting behavior is sensitive to many environmental and physiological challenges, as well as many genetic mutations underlying pathological disease states. The following protocol describes a nesting behavior paradigm that explores the usage of four types of nesting material. In addition, the protocol utilizes intraclass correlations to demonstrate that inter-rater reliability is higher when nests are constructed out of shredded paper compared to other common nesting materials such as cotton squares, paper twists, and soft cob bedding. The chosen methodology and statistical considerations (i.e., intraclass correlation) for this assay may be of interest for those conducting experiments assessing the quality of living of mice.&#160;

Here, we present a protocol to quantify nest building behavior in mice, which is known to be impaired in several neurological disorders and diseases. This protocol examines the utility of four materials and offers the opportunity to quantify the rater agreement in scoring, improving the validity and reliability of the assay.

Nest building is an innate behavior in male and female rodents, even when raised in laboratory settings. As such, many researchers provide rodents ...

Flypub To Study Ethanol Induced Behavioral Disinhibition and Sensitization

Alcohol use disorder (AUD) remains a serious problem in our society. To develop effective interventions for addiction, it is important to understand the underlying neurobiological mechanisms, for which diverse experimental approaches and model systems are needed. The main ingredient of alcoholic beverages is ethanol, which causes adaptive changes in the central nervous system and behavior upon chronic intake. Behavioral sensitization (i.e., escalated responses) in particular represents a key adaptive change underlying addiction. Most ethanol-induced behavioral sensitization studies in animal models have been conducted on the locomotor activating effect of ethanol. A prominent effect of ethanol is behavioral disinhibition. Behavioral sensitization to the disinhibition effect of ethanol, however, is underrepresented. To address this issue, we developed the Flypub assay that allows measuring the escalated increase in disinhibited courtship activities upon recurring ethanol exposure in Drosophila melanogaster.&#160;Here, we report the step-by-step Flypub assay including assembly of ethanol exposure chambers, setup of the assay station, criteria for fly care and collection, ethanol delivery, quantification of disinhibited courtship activities, data processing and statistical analysis. Also provided are how to troubleshoot critical steps, overcome limitations and expand its utility to assess additional ethanol-induced behaviors. The Flypub assay in combination with powerful genetic tools in Drosophila melanogaster will facilitate the task of discovering the mechanism underlying ethanol-induced behavioral sensitization.

The Flypub assay measures the behaviors that the fruit fly Drosophila melanogaster displays under the influence of ethanol. The assay can be readily mastered by experimenters at all levels and applied to various vaporized stimuli, facilitating substance abuse and addiction studies.

Alcohol use disorder (AUD) remains a serious problem in our society. To develop effective interventions for addiction, it is important to understand the ...