The authors would like to acknowledge The Garnett Passe and Rodney Williams Memorial Foundation and the Bosch Institute Animal Behavioural Facility.

1. Animals
<ol>
	<li>Mice (C57/BL6) of ages 1, 9, and 13 months old were obtained from the Animal Resources Centre (Perth, Australia). These mice were housed in standard mouse cages in the Bosch Rodent Facility at the University of Sydney on a 12/12 hr light/dark cycle with access to food and water ad libitum. The procedures outlined below were approved by the University of Sydney Animal Ethics Committee.</li>
	<li>Bring mouse cages into the testing room prior to each test for 10 min to allow mice to acclimatize to the testing environment.</li>
</ol>
2. Rotarod
<ol>
	<li>Set up the rotarod apparatus (Figure 1A):
	<ol>
		<li>Install the dowels in each lane of the rotarod. 
		Note: In this instance rat dowels (70 mm in diameter) are used instead of mouse dowels (32 mm in diameter) to discourage the mice from clinging to the dowel and performing &#8220;passive rotations&#8221;10.</li>
		<li>Position the magnetic landing platforms on the wire situated at the bottom of each lane of the rotarod making sure that they are not tilting to touch the floor of the rotarod and are placed as close as possible to the magnetic right wall of each lane without touching. 
		Note: During rotarod testing mice are required to walk in a forward direction to stay on the rotating and accelerating dowels. When a mouse is no longer able to stay on the dowel, they fall and displace the landing platform that subsequently activates a magnetic sensor. The time taken to fall from the rotating dowel, the dowel rpm at time of falling and the distance traveled is automatically calculated for each mouse and recorded on the display screen at the front of the rotarod.</li>
		<li>Slide 2 clear plastic panels into the front of each rotarod lane with the shorter panels at the bottom and the longer panels above.</li>
		<li>Input the test parameters using the keypad located at the front of the rotarod. Follow steps 2.1.4.1 to 2.1.4.6 for the accelerating rotarod test parameters and steps 2.1.4.7 to 2.1.4.12 for the fixed speed rotarod test parameters.
		<ol>
			<li>Set the maximum duration of the test to 60 sec.</li>
			<li>Set the number of lanes to be used (or the number of mice to be tested).</li>
			<li>Set the starting speed of the test to 5 rpm.</li>
			<li>Set the top speed of the test to 44 rpm.</li>
			<li>Set the ramp speed of the test to 60 sec.</li>
			<li>Set the size of the dowels chosen and the direction of rotation to rat dowels rotating in a forward direction.</li>
			<li>Set the maximum duration of the test to 240 sec.</li>
			<li>Set the number of lanes to be used to 1 as the mice are tested individually.</li>
			<li>Set the starting speed of the test to 15 rpm.</li>
			<li>Set the top speed of the test to 15 rpm.</li>
			<li>Set the ramp speed of the test to 0 sec.</li>
			<li>Set the size of the dowel chosen and the direction of rotation to a rat dowel rotating in a forward direction. 
			Note: The settings above may be altered to suit the needs of different experiments.</li>
		</ol>
		</li>
		<li>Place a camera in front of the rotarod for the fixed speed rotarod test, so that the behavior of the mouse during trials can be recorded and the videos used for later analysis to determine the duration of time the mice were able to stay on the rotarod.</li>
	</ol>
	</li>
	<li>Follow steps 2.2.1 to 2.2.4 for the accelerating rotarod test:
	<ol>
		<li>Place one mouse on each stationary dowel for 5 min to allow mice to acclimatize to the rotarod.</li>
		<li>Gently nudge the mice to face the back of the rotarod and start the rotarod test when all subjects are facing in this direction (see Figure 1B).</li>
		<li>Return all mice to their cages when they have fallen from the rotating dowels and leave them to rest for 10 min with access to food and water.</li>
		<li>Repeat steps 2.2.1 to 2.2.3 to complete a total of 8 trials making sure to clean the dowels, lanes, and landing platforms of the rotarod for urine and feces, and move the landing platforms back to its starting position betwen each trial. 
		Note: The first 3-5 trials are used as training trials to allow the mice to familiarize themselves with the task. The time to fall, distance walked and end rpm of the dowel at the time of the fall for each subsequent trial is recorded for later analysis (Figure 2).</li>
	</ol>
	</li>
	<li>Follow steps 2.3.1 to 2.3.8 for the fixed speed rotarod test:
	<ol>
		<li>Place one mouse on a dowel for 5 min to allow it to acclimatize to the rotarod. Return the mouse back to its cage.</li>
		<li>Start video recording on the camera and press start on the rotarod. Then place the mouse on the rotating dowel ensuring it faces the back of the rotarod.</li>
		<li>Stop video recording on the camera when the mouse falls from the rotating dowels, and return the mouse to its cages for 10 min with access to food and water.</li>
		<li>Repeat steps 2.3.2 and 2.3.3 until a total of 8 trials is acquired making sure to clean the dowels, lanes, and landing platforms of the rotarod for feces and urine, and move the landing platforms back to its starting position between each trial.</li>
		<li>Switch on the custom built rotator at 3 Hz for 20 sec to allow the mice to become familiar with the sound. Stop the rotator after 20 sec by switching off the drill and place hands on either side of the running wheel to stop it from continuing to spin past the initial 20 sec. 
		Note: The rotator itself consists of a rodent running wheel secured to a drill (Figure 3A). At the center of the running wheel is a small chamber with a mesh lid where the mouse is placed (Figure 3B). The rotator spins in a counter-clockwise direction about the vertical axis. The magnitude of the stimulus is in line with previous studies that show rotary stimulations ranging from 0.2 to 3 Hz are sufficient to generate VOR and VCR responses4,11,12.</li>
		<li>Place the mouse inside the chamber at the center of the rotator and replace the lid.</li>
		<li>Switch on the rotator at its lowest setting of 3 Hz for 20 sec. Start the rotarod and begin video recording on the camera during this time in preparation for the upcoming trial. Switch off the drill at the end of the 20 sec and place hands on either side of the running wheel to stop it from spinning. Retest the mouse on the rotarod immediately after by transferring it as quickly as possible to the rotating dowel.</li>
		<li>Stop video recording on the camera when the mouse falls from the dowel and return the mouse to its cage.</li>
	</ol>
	</li>
	<li>Clean the clear plastic panels with a mild detergent/water mix and the cylindrical dowels, lanes and metal landing platforms of the rotarod with 70% ethanol when all mice have been tested.</li>
</ol>
3. Balance Beam with Vestibular Challenge
<ol>
	<li>Set up the balance beam apparatus as seen in Figure 4A. 
	Note: The balance beam apparatus was adapted from an apparatus described in Carter et al. (2001)13. For this test, mice walk from the lower end of the beam, which is 52.5 cm above the ground, to a darkened goal box (13 x 22 cm, with a 5 x 6 cm doorway) situated 60 cm above the ground (Figure 4A). Mice naturally seek out the darkness and protection of the goal box in favor of the exposed beam and are further encouraged to traverse the beam by the slight incline which exploits their natural escape mechanism to run in an upwards direction14. The beam itself is 1 m in length and has a circular cross section with a diameter of 14 mm. A tailored range of beam diameters can be used which allows the experimenter to adjust the sensitivity of the test or accommodate larger subjects. At the lower end of the balance beam a white line indicates the starting line. Another line has been drawn 60 cm from the start line at the higher end of the beam to indicate the finish line (Figure 4A).
	<ol>
		<li>Position 2 cameras, one on either side of the balance beam, at the lower end of the balance beam (Figure 4B). 
		Note: These cameras should be angled to capture the entire length of the balance beam and ensure that start and finish lines marked on the balance beam are clearly visible. These cameras will be used to video record the behavior of mice as they traverse the balance beam, with resulting videos being used for later analysis.</li>
		<li>Line the floor of the goal box with paper towel, to enable easy cleaning of urine and feces after testing each mouse, and place the housing dome from the subjects home cage inside the goal box.</li>
		<li>Place adequate foam or other cushioning material underneath the raised beam to protect any subjects that fall from the apparatus. Mice that fall will be picked up immediately by the experimenter and placed inside the goal box to rest.</li>
	</ol>
	</li>
	<li>Place one mouse in the goal box for 2 min so that it becomes familiar with this environment. Cover the opening to the goal box with a gloved hand for 5 sec if the mouse tries to walk onto the beam during this time to discourage this behavior.</li>
	<li>Train the mouse by placing it on the beam just outside the opening to the goal box and allowing it to walk into the goal box. Continue to train the mouse by placing it on the beam progressively further away from the goal box until the mouse is able to walk from the start line to the goal box with no assistance and minimal hesitation. Leave the mouse to rest in the goal box for 1 min after each run.</li>
	<li>Begin testing the mouse when training is complete.&#160;
	<ol>
		<li>Start video recording on cameras.</li>
		<li>Place the mouse at the start line of the beam and wait while it traverses the beam in the direction of the goal box.</li>
		<li>Stop video recording on cameras when the mouse reaches the box.</li>
		<li>Leave the mouse to rest in the goal box for 1 min. Remove any urine or feces that may have been deposited during the trial while waiting.</li>
		<li>Repeat steps 3.4.1 to 3.4.4 until a total of 5 trials have been completed.</li>
	</ol>
	</li>
	<li>Switch on the custom built rotator at 3 Hz for 20 sec (like in the fixed speed rotarod test) to allow the mice to become familiar with the sound. Stop the rotator after 20 sec by switching off the drill and place hands on either side of the running wheel to stop it from spinning.</li>
	<li>Place the mouse inside the chamber at the center of the rotator and replace the lid.</li>
	<li>Switch on the rotator at its lowest setting of 3 Hz for 20 sec. Start video recording on the cameras during this time, in preparation for the upcoming trial. Switch off the drill at the end of the 20 sec and place hands on either side of the running wheel to stop it from continuing to spin past the initial 20 sec. Transfer the mouse to the start of the balance beam as quickly as possible and wait while the mouse traverses the beam to the goal box.</li>
	<li>Stop video recording on cameras when the mouse reaches the goal box and return the mouse to its cage.</li>
	<li>Clean the balance beam apparatus with 70% ethanol and change the paper towel in the goal box after each mouse has been tested.</li>
</ol>

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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+video+and+magnetic+search+coil+recordings+of+mouse+eye+movements.">A comparison of video and magnetic search coil recordings of mouse eye movements.</a> J. Neurosci. Methods. 99, 101-110 (2000).</li><li>Takemura, K., King, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vestibulo-collic+reflex+(VCR)+in+mice.">Vestibulo-collic reflex (VCR) in mice.</a> Exp. Brain Res. 167, 103-107 (2005).</li><li>Carter, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+progressive+motor+deficits+in+mice+transgenic+for+the+human+Huntington's+disease+mutation.">Characterization of progressive motor deficits in mice transgenic for the human Huntington's disease mutation.</a> J. Neurosci. 19, 3248-3257 (1999).</li><li>Wallace, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+and+reflexive+behavior+in+the+aging+rat.">Motor and reflexive behavior in the aging rat.</a> J. Gerontol. 35, 364-370 (1980).</li><li>Ingram, D. 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The authors declare they have no competing financial interests.

Critical Steps within the Protocol
Previous work has shown that it is easy to overtrain mice on both the rotarod and balance beam apparatus and as a consequence, the acquisition of accurate measurements can be challenging15. For example, overtraining on the rotarod can lead to mice intentionally jumping off the dowels during both the acclimatization and trial periods, while overtraining on the balance beam can lead to more frequent stopping (exploratory behavior) and travelling in the ...

Our sense of balance is perhaps one of the most overlooked yet vital components of even the most basic motor activities including walking and turning. Balance is influenced by numerous factors including muscle strength, motor coordination and vestibular function, and it is only in the presence of vestibular neuropathies or during normal aging that the importance of a fully functioning balance system is appreciated. Disturbances to the vestibular system are often associated with experiences of vertigo or dizziness and disequilibrium resulting in an increased risk of falls and subsequent injuries1. This is particularly critical in older populations where falls are one of the leading causes of injury2.
Vestibular function tests are commonly based on the vestibular reflexes, in particular, the vestibulo-ocular (VOR) or the vestibulo-collic reflex (VCR). The VOR and VCR are essential for the stabilization of images on the retina and head position during movements of the head and body respectively. Commonly, VOR measurements require invasive implantation of search coils to measure eye movements or video tracking of eye movement3. This is challenging in mice due to the small nature of the mouse eye and the difficulty in detecting the pupil for video analysis3. As an alternative, the VCR has been used to measure stabilization of the head in response to body movements in mice without the need for invasive surgery4. Despite this, few studies focus specifically on how the vestibular system performs as a whole and more importantly how it changes during aging.
To assess overall balance performance simply and noninvasively we modified two commonly used behavioral tests. The rotarod and inclined balance beam tests assess different aspects of motor performance in rodents and in previous studies have been used in a test battery to acquire a complete profile of motor capability. This capability can be affected by disease or genetic modification, and is also sensitive to processes associated with normal development and aging5-7. Earlier work using the rotarod has shown that motor coordination in mice declines after 3 months of age8. In addition, rats show noticeable balance deficits with increasing age on the balance beam test9.
This paper describes the use of the rotarod and balance beam tests in conjunction with a vestibular stimulus in order to challenge the vestibular system and characterize the subsequent impact on balance performance in young and older mice. While the simple and noninvasive methods described are not designed as stand-alone measures of peripheral vestibular function, they do provide a useful and simple behavioral measure to compare cellular and subcellular changes at multiple stages of vestibular processing during normal aging in mice.

<table><tbody><tr><td>Rotarod</td><td>IITC Life Science Inc.</td><td>#755</td><td>&quot;Rat dowels&quot; = 70 mm diameter. Do not allow ethanol to contact perspex.</td></tr><tr><td>iPhone</td><td>Apple</td><td></td><td>Can use any type of camera. Velcro fixed to the back surface for attachment to the the 3D articulated arm.</td></tr><tr><td>3D articulated arm</td><td>Fisso/Baitella</td><td>Classic 3300-28</td><td>Any type of stable vertical stand would be adequate. Velcro is fixed to the apical end of the arm for iPhone attachment.</td></tr><tr><td>Wooden walking beam: 1 m long strip of smooth wood with a circular cross-section of 14 mm diameter</td><td></td><td></td><td>A range of diameters and cross section shapes can be used to suit experimental parameters</td></tr><tr><td>Wooden goal box (130 x 140 x 220 mm) made from 11 mm thick boards</td><td></td><td></td><td></td></tr><tr><td>Support stand made of 41 x 41 mm beams: 2 vertical beams 525 and 590 mm from ground at the start and goal ends respectively; 803 mm horizontal beam that runs along the ground directly under the walking beam; two 20 mm long beams act as &quot;feet&quot;, joining the horizontal and vertical beams at each end; a 21 x 21 x 36 mm block hewn at the apical end of the &quot;starting&quot; vertical beam; a 13 x 13 mm aperture cut out of the center of this block, forming a tunnel which runs perpendicular to the walking beam.</td><td></td><td></td><td>Brace all joins with small steel brackets.</td></tr><tr><td>Black paint (water based)</td><td>Handycan</td><td>Acrylic Matt Black</td><td>2-3 coats for all wooden surfaces of the balance beam apparatus</td></tr><tr><td>Clear finish</td><td>Wattle Estapol</td><td>Polyurethane Matt</td><td>Single coat for all beams. Double coat for all other surfaces of the balance beam apparatus</td></tr><tr><td>Foam, packaging material</td><td></td><td></td><td>To cushion any falls from the balance beam</td></tr><tr><td>70% Ethanol, paper towels</td><td></td><td></td><td>Clean beam and goal box between each animal.</td></tr><tr><td>Gauze pads/paper towels</td><td></td><td></td><td>To line the floor of the goal box</td></tr><tr><td>Mouse house (from home cage)</td><td></td><td></td><td></td></tr></tbody></table>

behavioral assessment of the aging mouse vestibular system

Age related decline in balance performance is associated with deteriorating muscle strength, motor coordination and vestibular function. While a number of studies show changes in balance phenotype with age in rodents, very few isolate the vestibular contribution to balance under either normal conditions or during senescence. We use two standard behavioral tests to characterize the balance performance of mice at defined age points over the lifespan: the rotarod test and the inclined balance beam test. Importantly though, a custom built rotator is also used to stimulate the vestibular system of mice (without inducing overt signs of motion sickness). These two tests have been used to show that changes in vestibular mediated-balance performance are present over the murine lifespan. Preliminary results show that both the rotarod test and the modified balance beam test can be used to identify changes in balance performance during aging as an alternative to more difficult and invasive techniques such as vestibulo-ocular (VOR) measurements.

Rotarod
The motor performance of mice was described as the Time To Fall (TTF) recorded for each mouse over 8 trials. Using these measurements of TTF, training curves for each mouse can be plotted. Figure 2 shows examples of the motor performance of one 1 month-old mouse and one 9 month-old mouse over the course of 8 trials. These training curves show an increase in TTF during the first 3-5 trials followed by a subsequent plateau. Measurements of TTF recorded before the plateau we...

Watch this Scientific Journal Video about Behavioral Assessment of the Aging Mouse Vestibular System at JoVE.com

Behavioral Assessment of the Aging Mouse Vestibular System

Motor control and balance performance are known to deteriorate with age. This paper presents a number of standard noninvasive behavioral tests with the addition of a simple rotary stimulus to challenge the vestibular system and show changes in balance performance in a murine model of aging.

Age related decline in balance performance is associated with deteriorating muscle strength, motor coordination and vestibular function. While a number of ...

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16.8K Views.  University of Sydney. Motor control and balance performance are known to deteriorate with age. This paper presents a number of standard noninvasive behavioral tests with the addition of a simple rotary stimulus to challenge the vestibular system and show changes in balance performance in a murine model of aging.

Video: Behavioral Assessment of the Aging Mouse Vestibular System

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

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

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

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

Assessment of Motor Balance and Coordination in Mice using the Balance Beam

Brain injury, genetic manipulations, and pharmacological treatments can result in alterations of motor skills in mice. Fine motor coordination and balance can be assessed by the beam walking assay. The goal of this test is for the mouse to stay upright and walk across an elevated narrow beam to a safe platform. This test takes place over 3 consecutive days: 2 days of training and 1 day of testing. Performance on the beam is quantified by measuring the time it takes for the mouse to traverse the beam and the number of paw slips that occur in the process. Here we report the protocol used in our laboratory, and representative results from a cohort of C57BL/6 mice. This task is particularly useful for detecting subtle deficits in motor skills and balance that may not be detected by other motor tests, such as the Rotarod.

Deficits in fine motor coordination can be assessed with the balance beam test. Performance on the beam is quantified by the speed at which the beam is traversed and the number of times the mouse slips on the beam.

Brain injury, genetic manipulations, and pharmacological treatments can result in alterations of motor skills in mice.  Fine motor coordination and ...

Neuroscience

Battery of Behavioral Tests Assessing General Locomotion, Muscular Strength, and Coordination in Mice

Behavioral testing is used in pre-clinical trials to assess the phenotypic effects and outcomes that a particular disease or treatment has on the animal&#39;s wellbeing and health. There are numerous behavioral tests that may be applied. We selected a test for general locomotion, the open field test (OFT); a test for muscular strength, the mesh test (MT); and a test for coordination, the rotarod test (RR). Testing can be accomplished on a weekly or monthly basis. As a test for general locomotion, the OFT works by objectively monitoring movement parameters while the mouse is in an open field apparatus. The field is generally a 2&#39; x 2&#39; box, and the movements are recorded through laser sensing or through video capture. The mouse is placed in the center of the open field and allowed to move freely for the test. The MT uses the latency for a mouse to fall off an inverted screen as a measure of muscular strength. A mouse is placed on a screen, which is inverted over a clear box, and is timed for their latency to fall. Three trials are performed, with the best of the three trials scored for that day. A score of 60 s is the maximum time a mouse is left inverted. Mice are given a 5-min rest period between mesh test trials. Lastly, an accelerated protocol on the RR assesses motor coordination and endurance. During a trial, a mouse walks on a rotating rod as it increases in speed from 4 rpm to 40 rpm over 5 min. The trial ends when the mouse touches the magnetized pressure sensor upon falling. Each mouse undergoes three trials, and the best trial is scored for that day. This combined behavioral data allows for the global assessment of mobility, coordination, strength, and movement of the test animals. At least two out of the three behavioral testing measures must show improvement for an animal to qualify as having overall improved motor function.

Behavioral testing is used in pre-clinical trials to assess the phenotypic effects of a disease or treatment on an animal's wellbeing. In order to globally assess motor functioning, we selected tests for general locomotion, muscular strength, and coordination: the open field test, the mesh test, and the rotarod test, respectively.

Behavioral testing is used in pre-clinical trials to assess the phenotypic effects and outcomes that a particular disease or treatment has on the ...

Gait Analysis of Age-dependent Motor Impairments in Mice with Neurodegeneration

Motor behavior tests are commonly used to determine the functional relevance of a rodent model and to test newly developed treatments in these animals. Specifically, gait analysis allows recapturing disease relevant phenotypes that are observed in human patients, especially in neurodegenerative diseases that affect motor abilities such as Parkinson&#39;s disease (PD), Alzheimer&#39;s disease (AD), amyotrophic lateral sclerosis (ALS), and others. In early studies along this line, the measurement of gait parameters was laborious and depended on factors that were hard to control (e.g., running speed, continuous running). The development of ventral plane imaging (VPI) systems made it feasible to perform gait analysis at a large scale, making this method a useful tool for the assessment of motor behavior in rodents. Here, we present an in-depth protocol of how to use kinematic gait analysis to examine the age-dependent progression of motor deficits in mouse models of neurodegeneration; mouse lines with decreased levels of endophilin, in which neurodegenerative damage progressively increases with age, are used as an example.

In this study, we demonstrate the use of kinematic gait analysis based on ventral plane imaging to monitor the subtle changes in motor coordination as well as the progression of neurodegeneration with advancing age in mouse models (e.g., endophilin mutant mouse lines).

Motor behavior tests are commonly used to determine the functional relevance of a rodent model and to test newly developed treatments in these animals. ...

Developmental Biology

Assessing the Autonomic and Behavioral Effects of Passive Motion in Rats using Elevator Vertical Motion and Ferris-Wheel Rotation

The overall goal of this study is to assess the autonomic and behavioral effects of passive motion in rodents using the elevator vertical motion and Ferris-wheel rotation devices. These assays can help confirm the integrity and normal functioning of the autonomic nervous system. They are coupled to quantitative measures based on defecation counting, open-field examination, and balance beam crossing. The advantages of these assays are their simplicity, reproducibility, and quantitative behavioral measures. The limitations of these assays are that the autonomic reactions could be epiphenomena of non-vestibular disorders and that a functioning vestibular system is required. Examination of disorders such as motion sickness will be greatly aided by the detailed procedures of these assays.

Protocols are presented to assess the autonomic and behavioral effects of passive motion in rodents using elevator vertical motion and Ferris-wheel rotation.

The overall goal of this study is to assess the autonomic and behavioral effects of passive motion in rodents using the elevator vertical motion and ...

An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy

Understanding vestibular hair cells function under normal conditions, or how trauma, disease, and aging disrupt this function is a vital step in the development of preventative approaches and/or novel therapeutic strategies. However, the majority of studies looking at abnormal vestibular function have not been at the cellular level but focused primarily on behavioral assays of vestibular dysfunction such as gait analyses and vestibulo-ocular reflex performance. While this work has yielded valuable data about what happens when things go wrong, little information is gleaned regarding the underlying causes of dysfunction. Of the studies that focus on the cellular and subcellular processes that underlie vestibular function, most have relied on acutely isolated hair cells, devoid of their synaptic connections and supporting cell environment. Therefore, a major technical challenge has been access to the exquisitely sensitive vestibular hair cells in a preparation that is least disrupted, physiologically. Here we demonstrate a semi-intact preparation of the mouse vestibular sensory epithelium that retains the local micro-environment including hair cell/primary afferent complexes.

Analysis of vestibular hair cell function is complicated by their location deep within the hardest part of the skull, the petrous temporal bone. Most functional hair cell studies have used acutely isolated hair cells. Here we describe a semi-intact preparation of mouse vestibular epithelium for electrophysiological and two-photon microscopy studies.

Understanding vestibular hair cells function  under normal conditions, or how trauma, disease, and aging disrupt this  function is a vital step in the ...

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.

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 ...

Behavioral Training Procedures for Head-fixed Virtual Reality in Mice

Virtual reality (VR) combined with head-fixation is increasingly being utilized in behavioral neuroscience studies as it allows complex behavioral assays to be performed in head-fixed mice. This enables precise behavioral recordings while incorporating various neurophysiological techniques that require head-fixation to minimize movement-related signal noise during neural recordings. However, despite the growing use of VR, there is little published data on the detailed methodology of how to implement it. In this study, a training protocol is developed whereby male and female C57B16/J mice are trained to run down a virtual linear corridor, the length of which is increased from 1-3 m over multiple training sessions. Building upon this foundation, this study investigated the feasibility of mice performing complex behaviors within VR using a Y-maze paradigm. The task required navigating to the arm with black walls from the choice point in the Y-maze. After reaching a criterion of two consecutive days equal to or greater than 70% correct, the mice progressed to increasingly difficult sensory discrimination. The findings provide important details on the methodologies useful for the successful training of mice in VR and demonstrate that mice exhibit learning capabilities in navigating the Y-maze. The methodology presented not only offers insights into training duration in VR-based assays but also underscores the potential for probing intricate behaviors in mice, opening avenues for more comprehensive neuroscience investigations.

The article describes the experimental procedures for the commonly used linear track virtual reality (VR) paradigm in mice as well as determining the feasibility of running complex VR tasks by testing a Y-shaped signal discrimination task.

Virtual reality (VR) combined with head-fixation is increasingly being utilized in behavioral neuroscience studies as it allows complex behavioral assays ...

Long-term Sensory Conflict in Freely Behaving Mice

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning in mice. By permanently wearing a device fixed on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages. Therefore, this protocol readily enables the study of the visual system and multisensory interactions over an extended timeframe that would not be accessible otherwise. In addition to lowering the experimental costs of long-term sensory learning in naturally behaving mice, this approach accommodates the combination of in vivo and in vitro experiments. In the reported example, video-oculography is performed to quantify the vestibulo-ocular reflex (VOR) and optokinetic reflex (OKR) before and after learning. Mice exposed to this long-term sensory conflict between visual and vestibular inputs presented a strong VOR gain decrease but exhibited few OKR changes. Detailed steps of device assembly, animal care, and reflex measurements are hereby reported.

The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning. By permanently wearing a fixed device on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages.

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for ...

Surgical Labyrinthectomy of the Rat to Study the Vestibular System

To study the vestibular system or the vestibular compensation process, a number of methods have been developed to cause vestibular damage, including surgical or chemical labyrinthectomy and vestibular neurectomy. Surgical labyrinthectomy is a relatively simple, reliable, and rapid method. Here, we describe the surgical technique for rat labyrinthectomy. A postauricular incision is made under general anesthesia to expose the external auditory canal and the tympanic membrane, after which the tympanic membrane and the ossicles are removed without the stapes. The stapes artery, which is located between the stapes and the oval window, is a vulnerable structure and must be preserved to obtain a clear surgical field. A hole to fenestrate the vestibule is made with a 2.1-mm drill bur superior to the stapes. Then, 100% ethanol is injected through this hole and aspirated several times. Meticulous dissection under a microscope and careful bleeding control are essential to obtain reliable results. Symptoms of vestibular loss, such as nystagmus, head tilting, and a rolling motion, are seen immediately after surgery. The rotarod or rotation chair test can be used to objectively and quantitatively evaluate the vestibular function.

This protocol describes the surgical labyrinthectomy of a rat, which is a useful method for studying the vestibular system.

To study the vestibular system or the vestibular compensation process, a number of methods have been developed to cause vestibular damage, including ...