This work was supported by the National Natural Science Foundation of China (61327902-6 to W. Wang and 81801318 to F.F. Ding). We acknowledge Dr. Sigrid Veasy for establishing the SF experimental system and kindly providing technical details. We acknowledge Dr. Maiken Nedergaard for instructive comments for related experiments.

This protocol was approved by the Institutional Animal Care and Use Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology.
1. Mice screening and preparation for the experiment
<ol>
	<li>Select wild-type adult (8&#8211;10 weeks old) male mice with weight of 20&#8211;28 g for the whole experiment. 
	NOTE: Wild-type C57BL/6 mice are obtained from the Hubei Research Center for Laboratory Animals, Hubei, China.</li>
	<li>Randomly assign all mice to the CSF and the control group. House 3&#8211;5 mice in each cage to avoid social isolation stress. The number of mice housed in the control cages is matched with that housed in the paired CSF cages. 
	NOTE: Mice in the same group cages are pooled to perform follow-up behavioral experiments.</li>
	<li>Locate the control cages in the same room with the CSF cages, to keep the surrounding environment and labor effects identical.</li>
	<li>Number and mark the mice in each group on their ears using an ear tag for monitoring purposes.</li>
	<li>Maintain ambient temperature and humidity between 21&#8211;23 &#176;C and 35%&#8211;60 %.</li>
	<li>Maintain the ambient environment in 12-hour light-dark cycle (8:00 AM&#8211;8:00 PM light-ON, 8:00 PM&#8211;8:00 AM light-OFF), to avoid biased effect on normal sleep rhythm in mice.</li>
	<li>Minimize the noise and interference while the researcher is present in the modeling room.</li>
	<li>Provide mice with sufficient food and water. Use long nozzles with ball valve tips on water bottles, to prevent water leakage upon the platform movements. Fasten the water bottle on top of the cage with a spring to avoid the dislocation of the bottle during rotor running.</li>
</ol>
2. Preparation and setting of the orbital rotor
<ol>
	<li>Prepare an electrically controlled orbital rotor with enlarged platform (67 cm x 110 cm), on which 10 cages can be placed at most.</li>
	<li>Set the orbital rotor on during light-ON phase (8:00 AM&#8211;8:00 PM) controlled by a program timer, which is the time when mice exhibit the majority of their daily sleep.</li>
	<li>Set the orbital rotor with a speed of 110 rpm and a repetitive cycle of 10 s-on, 110 s-off controlled with a solid-state timer. 
	NOTE: The load capacity of the platform is 50 kg. The fixed amplitude of the rotor horizon vibrating is 2.5 cm.</li>
	<li>Fasten the CSF cages on top of the rotor platform by thick springs to prevent dislocation of cages upon platform rotations.</li>
</ol>
3. Chronic sleep fragmentation modeling and monitoring
<ol>
	<li>Place cages of the CSF and the control mice into the modeling room for one week prior to experiments, to let mice adapt to the ambient environment.</li>
	<li>At the beginning of the modeling, ensure that all mice have free access to food and water during orbital rotations.</li>
	<li>At the beginning of the modeling, observe at least for 1 h to ensure the orbital rotor operating in gear.</li>
	<li>During the period of modeling, check that the orbital rotor is operating properly and mice conditions every 2 days to ensure mice have enough food and water. Change beddings of cages weekly.</li>
	<li>During the period of modeling, weigh the mice weekly at 8:00 AM when changing the bedding. Remove the mice with significant weight loss from the modeling, and&#160;also from the experimental groups. 
	NOTE: Significant weight loss is defined as weighing less than 20 g lasting for 2 weeks.</li>
	<li>During the entire modeling sessions, remove the aggressor, if any, from the cage and, also from the experimental groups.</li>
	<li>After the termination of modeling, continue to maintain and feed the mice in the original room.</li>
</ol>
4. Morris water maze (MWM) test
<ol>
	<li>Preparation for the test
	<ol>
		<li>Prepare the apparatus of a circular tank filled with warm water (20&#8211;23 &#176;C).</li>
		<li>Suspend four signs with different shapes and colors on the curtain surrounding the tank in four quadrant directions as the distant vision reference. Make the water to appear opaque by the addition of powdered milk.</li>
		<li>Locate a platform in the middle of the southwest quadrant.</li>
	</ol>
	</li>
	<li>The training test
	<ol>
		<li>Subject mice to four consecutive trials between 8:00 AM and 12:00 AM each day over a 5-day training period.</li>
		<li>Release each mouse into the water facing the sidewall at one of four quadrants in four trials. In each trial, allow the mouse to swim for 60 s to find the platform. If the mouse is unable to arrive at the platform within 60 s, guide it to the platform and to remain there for 15 s.</li>
		<li>Use a video tracking system to automatically record the escape latency of mice to find the hidden platform.</li>
	</ol>
	</li>
	<li>The probe test
	<ol>
		<li>Conduct the probe test on the sixth day after 5 training days.</li>
		<li>Remove the platform. Release each mouse from the northeast quadrant and allow it to swim for 60 s</li>
		<li>Use a video tracking system to automatically record the track data of mice.</li>
	</ol>
	</li>
</ol>
5. Novel object recognition (NOR) test
<ol>
	<li>The familiar phase
	<ol>
		<li>Place mice in a tank (length 30 cm, width 28 cm, height 35 cm) in sequence, which contains two copies of objects (A1 and A2). Allow the mice to explore freely (10 min per trial).</li>
		<li>Use a video tracking system to automatically record the track data of mice.</li>
	</ol>
	</li>
	<li>The test phase
	<ol>
		<li>Conduct the test trial after a 1 h delay of the familiar phase. Replace one of the original objects by a novel object (&#8220;novel&#8221;) in the tank keeping the other one unchanged. Return the mice to the tank and allow it to explore for 5 min per trial.</li>
		<li>Use a video tracking system to automatically record the time spent in exploration of each object by each mouse. 
		NOTE: The exploration of the object is determined by licking, sniffing, chewing, or moving vibrissae while orienting the nose toward and less than 1 cm from the object. The Discrimination Index (DI) is calculated with the equation (TN &#8722; TF)/(TN + TF), where TN = time spent exploring the &#8220;novel&#8221; object and TF = time spent exploring the &#8220;familiar&#8221; object.</li>
	</ol>
	</li>
</ol>
6. Open field test (OFT)
<ol>
	<li>Prepare the apparatus of a tank (30 cm x 28 cm x 35 cm).</li>
	<li>During the test, place each mouse into the center of the tank and allow it to explore freely for 5 min. Clean the tank with 75% ethanol after each trial to avoid the leftover effects of the previous mouse.</li>
	<li>Use a video tracking system to automatically record the track data of mice.</li>
</ol>
7. Forced swimming test (FST)
<ol>
	<li>Prepare the apparatus of an open cylindrical vessel, which contains water (20&#8211;23 &#176;C) that is 15 cm deep.</li>
	<li>During the test, place each mouse into the cylinder and allow it to remain there for 6 min.</li>
	<li>Use a video track system to automatically record the immobility time during the last 4 min of the test by each mouse. 
	NOTE: The mouse is determined to be immobile when it stops struggling and floats in the water, making only movements which are necessary to keep its head above water.</li>
</ol>
8. Data Analysis
<ol>
	<li>Analyze data using statistical analysis software (e.g., GraphPad Prism 6.0).</li>
	<li>Express all data as the mean &#177; SEM.</li>
	<li>Compare the escape latency in MWM test between two groups using two-way ANOVA with repeated measures followed by Bonferroni posttests. Other comparisons between the CSF and the control groups are determined by unpaired t tests.</li>
	<li>Consider differences significant if P &#60; 0.05 in all tests.</li>
</ol>

The authors declare that they have no competing financial interests.

Critical steps in the current protocol include setting up sleep fragmentation machines with the optimized parameters according to the study purpose and maintaining the mice in comfortable and quiet living environment throughout the entire modeling sessions. It is also crucial to decide the proper timing to interrupt or stop sleep fragmentation and arrange behavioral tests for those mice. Like other sleep manipulation models, it is important to perform the protocol in a dedicated room with controlled light cycles and void...

Sleep disturbance is increasingly common both in patients with sleep-disturbing conditions and healthy people with sleep-disturbing events. It has been observed that&#160;patients with neurodegenerative diseases, chronic pain, emotional stress, respiratory system diseases, urinary system diseases, etc., usually complain about unpleasant sleep experiences1,2,3,4,5. Obstructive sleep apnea (OSA), periodic limb movements in sleep (PLMS), sleep maintenance insomnia among other sleep disorders are the most common causes, which induce sleep fragmentation6,7. In developed countries, OSA has over 5% to 9% prevalence in adult population and 2% in child population8,9,10. Meanwhile, there is an increasing proportion of the healthy population experiencing sleep disturbance due to the overuse of smart phones, irregular sleep habits, annoying noises, and work duties, such as night shifts for caregivers. Sleep is acknowledged to be important for brain waste clearance11,12, memory consolidation13,14, metabolic balance15,16, among many other physiological processes. Yet, it still remains largely unknown whether long-term sleep disturbance gives rise to irreversible pathogenesis alterations in healthy human beings, and whether it is the etiology or a contributing factor of developing central nervous system diseases, such as neurodegenerative diseases in a couple of years down the road. Our goal is to report an experimental model that generates stable and evident cognitive deficit and anxiety-like behavior in young wild-type mice after a 2-month sleep fragmentation treatment. This model would be applied for answering the scientific questions listed above.
Sleep disturbance is listed as a potential risk factor for developing Alzheimer&#8217;s disease (AD) or dementia. Kang et al. first found and described the exacerbation of AD pathology by 6 h acute sleep deprivation17. Thereafter, many other studies reported that sleep deprivation or fragmentation could aggravate pathogenesis in transgenic AD mice models18,19,20. However, very few researchers have studied the consequence of sleep disturbance in young wild type mice; that is,&#160;whether sleep disturbance gives rise to AD-like behavior or pathological changes in young wild-type mice. In our recent publication, we reported that 2 months of sleep fragmentation induced evident spatial memory deficit and anxiety-like behavior, as well as increased intracellular Amyloid-&#946; (A&#946;) accumulation both in cortex and hippocampus in 2&#8211;3 month-old wild-type mice21. We also observed altered expression levels of endosome-autophagosome-lysosome pathway markers and microglia activation, which was similar to the pathological changes reported in APP/PS1 mice21,22.
This presented sleep fragmentation (SF) protocol was validated by Sinton et al.23 and modified by Li et al.24. In brief, an orbital rotor vibrating at 110 rpm interrupts sleep for 10 s every 2 min during light-ON phase (8:00 AM&#8211;8:00 PM). Sleep structure alteration in this model was previously characterized with electrophysiological sleep recordings and reported by Li et al.24, indicating a significant increase in the wake time and decrease in rapid eye movement (REM) sleep during the light-ON phase, with the total sleep and wake times (in 24 hour) unaffected after more than 4 weeks&#8217; modeling24. Currently, total sleep or partial sleep deprivation are the most commonly used sleep manipulation models. Total sleep deprivation is usually performed by sustained gentle handling or exposing the animal to novel objects, alternatively by continuously rotating a bar or a running treadmill25,26,27,28,29. Due to ethical reasons, total sleep deprivation is usually shorter than 24 h. The most commonly applied partial sleep deprivation model is the water platform method, which primarily ablating REM sleep30,31,32. Other approaches using either a treadmill or a bar that sweeps along the bottom of the cage, could induce sleep fragmentation when set on at fixed intervals33,34,35,36,37,38. It is noteworthy that SF interrupts sleep and intermittently causes arousals across all sleep stages24. One of the prominent advantages of this CSF model applying orbital rotor is that it can be performed continuously for months automatically controlled by machines, which avoids frequent processing labor daily except for regular monitoring. Furthermore, the apparatus would allow to simultaneously model multiple cages of mice under uniformed interventions. During entire modeling sessions, mice are housed in their home cages with usual bedding and nesting materials, while some other methods require exposure to diversified environments and inevitable stress.
Sleep fragmentation was previously characterized by the sleep manipulation method, which mimics frequent arousals during the sleep phase and substantial sleep rebound during the wake phase. In some literatures, CSF was regarded as the animal model for OSA39,40. In this study, the rationale of the chosen frequency of arousal to be 30 times per hour is based on the observation of arousal indices in patients with moderate-to-severe sleep apnea. It was observed that 4 weeks&#8217; sleep fragmentation significantly increased hypercapnic arousal latency and the tactile arousal threshold, which could at least last 2 weeks after recovery24. This phenotype was explained by revealing c-fos activation reduction in noradrenergic, orexinergic, histaminergic, and cholinergic wake-active neurons in response to hypercapnia, as well as reduced catecholaminergic and orexinergic projections into the cingulate cortex24. However, it is necessary to note that the most important feature in OSA is hypoxia caused by airway obstruction, which results in sleep disruption41,42. Sleep disturbance and repetitive hypoxia reciprocally interact with each other in OSA pathogenesis. Therefore, sleep fragmentation alone might not be able to fully demonstrate all key features of OSA in mice.
Herein, we present a standardized protocol to model chronic sleep fragmentation in young wild-type mice. Cognitive deficit and anxiety-like as well as depression-like behaviors after CSF treatment were evaluated by Morris water maze, Novel object recognition, Open field test, and Forced swimming test. It is important to note that this model should be taken as a whole that generates phenotypes of dysregulated sleep pattern, cognitive deficit, and anxiety-like behavior. The current model could potentially be applied, but not be limited, to the following purposes: 1) Further investigating the functional or molecular pathogenesis mechanisms induced by chronic sleep disturbance in young mice without genetic predisposition, 2) Identifying the direct pathway leading to neurodegeneration initiated by sleep disturbance, 3) Exploring the therapeutics for improving phenotypes induced by chronic sleep disturbance, 4) Studying the intrinsic protective/compensatory mechanisms in wild-type mice upon chronic sleep disturbance, 5) To be applied for studying sleep-wake regulation and state-transition mechanisms.

<table>
	<tbody>
		<tr>
			<td>Any-maze behavior tracking system</td>
			<td>Stoelting,Inc,USA</td>
			<td>-</td>
			<td>A video-tracking system which was used to record the behavior track of mice.</td>
		</tr>
		<tr>
			<td>C57BL/6J mice</td>
			<td>Hubei Research Center for Laboratory Animals, Hubei, China.</td>
			<td>-</td>
			<td>healthy male C57BL/6J mice aged 10-12 weeks were purchased from Hubei Research Center for Laboratory Animals</td>
		</tr>
		<tr>
			<td>Graphpad Prism 6.0 Software</td>
			<td>Graphpad Software,Inc.USA</td>
			<td>-</td>
			<td>Graphpad Prism 6.0 software was used to draw statistical graphs.</td>
		</tr>
		<tr>
			<td>Morris water maze system</td>
			<td>Shanghai XinRuan Information Technology Co.,Ltd,China</td>
			<td>XR-XM101</td>
			<td>The system was used to perform Morris water maze test</td>
		</tr>
		<tr>
			<td>Orbial rotor</td>
			<td>Shanghai ShiPing Laboratory Equipment Co.,Ltd,China</td>
			<td>SPH-331</td>
			<td>The orbital rotor was used to establish the chronic sleep fragmentation model</td>
		</tr>
		<tr>
			<td>Solid state timer</td>
			<td>OMRON Corporation, Kyoto, Japan</td>
			<td>H3CR-F8-300</td>
			<td>The solid state time was used to control the frequency and time of the rotor running</td>
		</tr>
		<tr>
			<td>Wooden Lusterless Tank</td>
			<td>-</td>
			<td>-</td>
			<td>length 30 cm, width 28 cm, height 35 cm The tank was used to perform open field test and novel object recognition test</td>
		</tr>
	</tbody>
</table>

a chronic sleep fragmentation model using vibrating orbital rotor to induce cognitive deficit and anxiety-like behavior in young wild-type mice

Sleep disturbance is generally common in populations as a chronic disease or a complained event. Chronic sleep disturbance is proposed to be closely linked to the pathogenesis of diseases, especially neurodegenerative diseases. We recently found that 2 months of sleep fragmentation initiated Alzheimer&#8217;s disease (AD)-like behavioral and pathological changes in young wild-type mice. Herein, we present a standardized protocol to achieve chronic sleep fragmentation (CSF). Briefly, CSF was induced by an orbital rotor vibrating at 110 rpm and operating with a repetitive cycle of 10 s-on, 110 s-off, during light-ON phase (8:00 AM&#8211;8:00 PM) continuously for up to 2 months. Impairments of spatial learning and memory, anxiety-like but not depression-like behavior in mice as consequences of CSF modeling, were evaluated with Morris water maze (MWM), Novel object recognition (NOR), Open field test (OFT) and Forced swimming test (FST). In comparison with other sleep manipulations, this protocol minimizes the handling labors and maximizes the modeling efficiency. It produces stable phenotypes in young wild-type mice and can be potentially generated for a variety of research purposes.

All the representative results and figures were reproduced from our recent publication21. The reuse of the figures was permitted by the original journal.
The entire experimental design is illustrated in the order of time, which indicates the timing of CSF modeling, behavioral tests of MWM, NOR, OFT, and FST (Figure 1A). We obtained weights of mice every week from the CSF and the control groups, to monitor their general conditions during the...

Watch this Scientific Journal Video about A Chronic Sleep Fragmentation Model using Vibrating Orbital Rotor to Induce Cognitive Deficit and Anxiety-Like Behavior in Young Wild-Type Mice at JoVE.com

A Chronic Sleep Fragmentation Model using Vibrating Orbital Rotor to Induce Cognitive Deficit and Anxiety-Like Behavior in Young Wild-Type Mice

Presented here is a protocol for chronic sleep fragmentation (CSF) model achieved by an electrically controlled orbital rotor, which could induce confirmed cognitive deficit and anxiety-like behavior in young wild-type mice. This model can be applied to explore the pathogenesis of chronic sleep disturbance and related disorders.

Sleep disturbance is generally common in populations as a chronic disease or a complained event. Chronic sleep disturbance is proposed to be closely ...

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5.2K Views.  Huazhong University of Science and Technology, Wuhan 430000,  China. Presented here is a protocol for chronic sleep fragmentation (CSF) model achieved by an electrically controlled orbital rotor, which could induce confirmed cognitive deficit and anxiety-like behavior in young wild-type mice. This model can be applied to explore the pathogenesis of chronic sleep disturbance and related disorders.

Video: A Chronic Sleep Fragmentation Model using Vibrating Orbital Rotor to Induce Cognitive Deficit and Anxiety-Like Behavior in Young Wild-Type Mice

Using Chronic Social Stress to Model Postpartum Depression in Lactating Rodents

Exposure to chronic stress is a reliable predictor of depressive disorders, and social stress is a common ethologically relevant stressor in both animals and humans. However, many animal models of depression were developed in males and are not applicable or effective in studies of postpartum females. Recent studies have reported significant effects of chronic social stress during lactation, an ethologically relevant and effective stressor, on maternal behavior, growth, and behavioral neuroendocrinology. This manuscript will describe this chronic social stress paradigm using repeated exposure of a lactating dam to a novel male intruder, and the assessment of the behavioral, physiological, and neuroendocrine effects of this model. Chronic social stress (CSS) is a valuable model for studying the effects of stress on the behavior and physiology of the dam as well as her offspring and future generations. The exposure of pups to CSS can also be used as an early life stress that has long term effects on behavior, physiology, and neuroendocrinology.

This article describes the use of chronic resident intruder social stress as an ethologically relevant paradigm to model postpartum depression and anxiety in lactating rodents.

Exposure to chronic stress is a reliable predictor of depressive disorders, and social stress is a common ethologically relevant stressor in both animals ...

The Crossmodal Congruency Task as a Means to Obtain an Objective Behavioral Measure in the Rubber Hand Illusion Paradigm

The rubber hand illusion (RHI) is a popular experimental paradigm. Participants view touch on an artificial rubber hand while the participants' own hidden hand is touched. If the viewed and felt touches are given at the same time then this is sufficient to induce the compelling experience that the rubber hand is one's own hand. The RHI can be used to investigate exactly how the brain constructs distinct body representations for one's own body. Such representations are crucial for successful interactions with the external world. To obtain a subjective measure of the RHI, researchers typically ask participants to rate statements such as "I felt as if the rubber hand were my hand". Here we demonstrate how the crossmodal congruency task can be used to obtain an objective behavioral measure within this paradigm.
The variant of the crossmodal congruency task we employ involves the presentation of tactile targets and visual distractors. Targets and distractors are spatially congruent (i.e. same finger) on some trials and incongruent (i.e. different finger) on others. The difference in performance between incongruent and congruent trials - the crossmodal congruency effect (CCE) - indexes multisensory interactions. Importantly, the CCE is modulated both by viewing a hand as well as the synchrony of viewed and felt touch which are both crucial factors for the RHI.
The use of the crossmodal congruency task within the RHI paradigm has several advantages. It is a simple behavioral measure which can be repeated many times and which can be obtained during the illusion while participants view the artificial hand. Furthermore, this measure is not susceptible to observer and experimenter biases. The combination of the RHI paradigm with the crossmodal congruency task allows in particular for the investigation of multisensory processes which are critical for modulations of body representations as in the RHI.

We demonstrate how an objective measure can be employed in the widely employed rubber hand illusion paradigm. This measure is obtained by modifying the well-established crossmodal congruency task. This task allows the investigation of multisensory processes which are critical for modulations of body representations as in the rubber hand illusion.

The rubber hand illusion (RHI) is a popular experimental paradigm. Participants view touch on an artificial rubber hand while the participants' own hidden ...

Uncovering Beat Deafness: Detecting Rhythm Disorders with Synchronized Finger Tapping and Perceptual Timing Tasks

A set of behavioral tasks for assessing perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) is presented here with the goal of uncovering rhythm disorders, such as beat deafness. Beat deafness is characterized by poor performance in perceiving durations in auditory rhythmic patterns or poor synchronization of movement with auditory rhythms (e.g., with musical beats). These tasks include the synchronization of finger tapping to the beat of simple and complex auditory stimuli and the detection of rhythmic irregularities (anisochrony detection task) embedded in the same stimuli. These tests, which are easy to administer, include an assessment of both perceptual and sensorimotor timing abilities under different conditions (e.g., beat rates and types of auditory material) and are based on the same auditory stimuli, ranging from a simple metronome to a complex musical excerpt. The analysis of synchronized tapping data is performed with circular statistics, which provide reliable measures of synchronization accuracy (e.g., the difference between the timing of the taps and the timing of the pacing stimuli) and consistency. Circular statistics on tapping data are particularly well-suited for detecting individual differences in the general population. Synchronized tapping and anisochrony detection are sensitive measures for identifying profiles of rhythm disorders and have been used with success to uncover cases of poor synchronization with spared perceptual timing. This systematic assessment of perceptual and sensorimotor timing can be extended to populations of patients with brain damage, neurodegenerative diseases (e.g., Parkinson&#8217;s disease), and developmental disorders (e.g., Attention Deficit Hyperactivity Disorder).

Behavioral tasks that allow for the assessment of perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) are presented. Synchronization of finger tapping to the beat of an auditory stimuli and detecting rhythmic irregularities provides a means of uncovering rhythm disorders.

A set of behavioral tasks for assessing perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) is presented here ...

Use of the Open Field Maze to Measure Locomotor and Anxiety-like Behavior in Mice

Animal models have proven to be invaluable to researchers trying to answer questions regarding the mechanisms of behavior. The Open Field Maze is one of the most commonly used platforms to measure behaviors in animal models. It is a fast and relatively easy test that provides a variety of behavioral information ranging from general ambulatory ability to data regarding the emotionality of the subject animal. As it relates to rodent models, the procedure allows the study of different strains of mice or rats both laboratory bred and wild-captured. The technique also readily lends itself to the investigation of different pharmacological compounds for anxiolytic or anxiogenic effects. Here, a protocol for use of the open field maze to describe mouse behaviors is detailed and a simple analysis of general locomotor ability and anxiety-related emotional behaviors between two strains of C57BL/6 mice is performed. Briefly, using the described protocol we show Wild Type mice exhibited significantly less anxiety related behaviors than did age-matched Knock Out mice while both strains exhibited similar ambulatory ability.

A protocol is provided to use an Open Field Maze to access general locomotor activity, anxiety and emotionality in a laboratory mouse model.

Animal models have proven to be invaluable to researchers trying to answer questions regarding the mechanisms of behavior. The Open Field Maze is one of ...

Introducing Clicker Training as a Cognitive Enrichment for Laboratory Mice

Establishing new refinement strategies in laboratory animal science is a central goal in fulfilling the requirements of Directive 2010/63/EU. Previous research determined a profound impact of gentle handling protocols on the well-being of laboratory mice. By introducing clicker training to the keeping of mice, not only do we promote the amicable treatment of mice, but we also enable them to experience cognitive enrichment. Clicker training is a form of positive reinforcement training using a conditioned secondary reinforcer, the "click" sound of a clicker, which serves as a time bridge between the strengthened behavior and an upcoming reward. The effective implementation of the clicker training protocol with a cohort of 12 BALB/c inbred mice of each sex proved to be uncomplicated. The mice learned rather quickly when challenged with tasks of the clicker training protocol, and almost all trained mice overcame the challenges they were given (100% of female mice and 83% of male mice). This study has identified that clicker training for mice strongly correlates with reduced fear in the mice during human-mice interactions, as shown by reduced anxiety-related behaviors (e.g., defecation, vocalization, and urination) and fewer depression-like behaviors (e.g., floating). By developing a reliable protocol that can be easily integrated into the daily routine of the keeping of laboratory mice, the lifetime experience of welfare in the mice can be improved substantially.

The development of new refinement strategies for laboratory mice is a challenging task that contributes towards fulfilling the 3R principle. This protocol introduces clicker training as a cognitive enrichment program for laboratory mice.

Establishing new refinement strategies in laboratory animal science is a central goal in fulfilling the requirements of Directive 2010/63/EU. Previous ...

Chronic Sleep Deprivation in Mouse Pups by Means of Gentle Handling

Sleep is critical for proper development and neural plasticity. Moreover, abnormal sleep patterns are characteristic of many neurodevelopmental disorders. Studying how chronic sleep restriction during development can affect adult behavior may add to our understanding of the emergence of behavioral symptoms of neurodevelopmental disorders. While there are many methods that can be used to restrict sleep in rodents including forced locomotion, constant disruption, presentation of an aversive stimulus, or electric shock, many of these methods are very stressful and cannot be used in neonatal mice. Here, we describe gentle handling, a sleep deprivation technique that can be used chronically throughout development and into adulthood to achieve sleep restriction. Gentle handling involves close observation of the mice throughout the sleep deprivation period and requires the researcher to gently prod the animals whenever they are inactive or display behaviors associated with sleep. Coupled with EEG recordings, gentle handling could be used to selectively disrupt a specific phase of sleep such as rapid eye movement (REM) sleep. The technique of gentle handling is a powerful tool for the study of the effects of chronic sleep restriction even in neonatal mice that circumvents many of the more stressful procedures used for sleep deprivation.

We describe a sleep deprivation technique known as gentle handling where investigators gently prod mice any time sleep behavior is observed. This method is a powerful tool that allows researchers to study the effects that chronic sleep restriction throughout development can have on future brain physiology and behavior.

Sleep is critical for proper development and neural plasticity. Moreover, abnormal sleep patterns are characteristic of many neurodevelopmental disorders. ...

Novel Object Recognition and Object Location Behavioral Testing in Mice on a Budget

Ethologically relevant behavioral testing is a critical component of any study that uses mouse models to study the cognitive effects of various physiological or pathological changes. The object location task (OLT) and the novel object recognition task (NORT) are two effective behavioral tasks commonly used to reveal the function and relative health of specific brain regions involved in memory. While both of these tests exploit the inherent preference of mice for the novelty to reveal memory for previously encountered objects, the OLT primarily evaluates spatial learning, which relies heavily on hippocampal activity. The NORT, in contrast, evaluates non-spatial learning of object identity, which relies on multiple brain regions. Both tasks require an open-field-testing arena, objects with equivalent intrinsic value to mice, appropriate environmental cues, and video recording equipment and the software. Commercially available systems, while convenient, can be costly. This manuscript details a simple, cost-effective method for building the arenas and setting up the equipment necessary to perform the OLT and NORT. Furthermore, the manuscript describes an efficient testing protocol that incorporates both OLT and NORT and provides typical methods for data acquisition and analysis, as well as representative results. Successful completion of these tests can provide valuable insight into the memory function of various mouse model systems and appraise the underlying neural regions that support these functions.

Here we provide a protocol which includes comprehensive instructions for the economical establishment of murine object location and novel object recognition behavioral testing, including the design, cost, and construction of required equipment as well as execution of behavioral testing, data collection, and analysis.

Ethologically relevant behavioral testing is a critical component of any study that uses mouse models to study the cognitive effects of various ...

Using the Race Model Inequality to Quantify Behavioral Multisensory Integration Effects

Multisensory integration research investigates how the brain processes simultaneous sensory information. Research on animals (mainly cats and primates) and humans reveal that intact multisensory integration is crucial for functioning in the real world, including both cognitive and physical activities. Much of the research conducted over the past several decades documents multisensory integration effects using diverse psychophysical, electrophysiological, and neuroimaging techniques. While its presence has been reported, the methods used to determine the magnitude of multisensory integration effects varies and typically faces much criticism. In what follows, limitations of previous behavioral studies are outlined and a step-by-step tutorial for calculating the magnitude of multisensory integration effects using robust probability models is provided.

The current study aims to provide a step-by-step tutorial for calculating the magnitude of multisensory integration effects in an effort to facilitate the production of translational research studies across diverse clinical populations.

Multisensory integration research investigates how the brain processes simultaneous sensory information. Research on animals (mainly cats and primates) ...

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 ...

The Adventures of Fundi Intervention Based on the Cognitive and Emotional Processing in Attention Deficit Hyperactive Disorder Patients

Many children with attention deficit hyperactive disorder (ADHD) are known to have executive dysfunction, which weakens their abilities to learn and behave in daily living. This protocol describes the methodology that is required for the intervention (psychotherapy) based on planning, attention, successive, and simultaneous (PASS theory) cognitive processing and fear emotional processing. It provides guiding principles and practical recommendations. A disproportionately high level of fear (dysregulation) increases the vulnerability for dysfunction in learning and behavior. We explain the interplay between emotion and cognition at the neurological level.
A go/no go task (The Adventures of Fundi), which involves decision making, is administered in a PC- mode to a sample of 66 ADHD subjects. The Adventures of Fundi, a computer program, was constructed to induce successive or simultaneous processing when involving the training of planning and selective attention. It aims to improve the executive function with planning and selective attention. If executive function improves, learning improves, and behavior ameliorates. After intervention over 6 months, remission was achieved in 70% of subjects.
The instructor encourages the use of appropriate strategies and points out the ways in which the strategies can be useful in finding the solution to the problem (go/no go). The emphasis is not on rehearsing and adult instructed verbal sequence. The verbalization may reveal the conscious verbalized strategy to solve a task that is not really the strategy being unconsciously used in that case. A self-verbal report is unreliable. This is an inductive learning rather than deductive rule-learning approach central to cognitive PASS training. This inductive training has proved to produce not only near transfer but also far transfer.
Noncognitive factors (emotional factors) must be considered to maximize the benefit of cognitive training. Indirect and metaphorical communication considers the emotional factor.

This protocol illustrates a remedial therapy based on inductive learning and indirect communication (Ericksonian metaphorical hypnosis) that can be applied to children with Attention Deficit Hyperactive Disorder (ADHD). Particularly, this protocol is intended to test the effectiveness of a go/no-go decision task. Neuroscientific rationale is presented throughout the text.

Many children with attention deficit hyperactive disorder (ADHD) are known to have executive dysfunction, which weakens their abilities to learn and ...