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>

<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 border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Rotarod</td>
			<td style="width:71px;">IITC Life Science Inc.</td>
			<td style="width:119px;">#755</td>
			<td style="width:167px;">&#34;Rat dowels&#34; = 70 mm diameter. Do not allow ethanol contact perspex.</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:216px;">iPhone</td>
			<td style="width:71px;">Apple</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Can use any type of camera (e.g. Logitech webcam described above). Velcro fixed to the back surface for attachment to the the 3D articulated arm.</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:216px;">3D articulated arm</td>
			<td style="width:71px;">Fisso/Baitella</td>
			<td style="width:119px;">Classic 3300-28</td>
			<td style="width:167px;">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 height="105">
			<td height="105" style="height:105px;width:216px;">Wooden walking beam: 1m long strip of smooth wood with a circular cross-section of 14 mm diameter</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">A range of diameters and cross section shapes can be used to suit experimental parameters</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Wooden goal box (130 x 140 x 220 mm) made from 11 mm thick boards</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="378">
			<td height="378" style="height:378px;width:216px;">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 &#34;feet&#34;, joining the horizontal and vertical beams at each end; a 21 x 21 x 36 mm block hewn at the apical end of the &#34;starting&#34; vertical beam; a 13 x 13 mm aperture cut out of the centre of this block, forming a tunnel which runs perpendicular to the walking beam.&#160;</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Brace all joins with small steel brackets.&#160;</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:216px;">Adjustible metal ring (13 mm wide)</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Pass this through the aperture in the block, pass the starting end of the balance beam through this ring and tighten until the beam is firmly in place.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Black paint (water based)</td>
			<td style="width:71px;">Handycan</td>
			<td style="width:119px;">Acrylic Matt Black</td>
			<td style="width:167px;">2-3 coats for all wooden surfaces of the balance beam apparatus</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Clear finish</td>
			<td style="width:71px;">Wattle Estapol</td>
			<td style="width:119px;">Polyurethane Matt</td>
			<td style="width:167px;">Single coat for all beams. Double coat for all other surfaces of the balance beam apparatus</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Foam, packaging material</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">To cushion any falls from the balance beam</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Electrical tape</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Fix webcam to roof.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">70% Ethanol, paper towels</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Clean beam and goal box between each animal.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Gauze pads/paper towels</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">To line the floor of the goal box</td>
		</tr>
		<tr height="42">
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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 ...

behavioral-assessment-of-the-aging-mouse-vestibular-system

Research

JoVE Journal

Behavior

16.8K Views.  University of Sydney.  Scheda di controllo motore e prestazioni equilibrio sono noti a peggiorare con l'età. Questo documento presenta una serie di test comportamentali non invasivi standard con l'aggiunta di un semplice stimolo rotante per sfidare i cambiamenti del sistema e mostrare vestibolari nelle prestazioni equilibrio in un modello murino di invecchiamento. 

Video: Behavioral Assessment of the Aging Mouse Vestibular System

Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an entire gene expression pathway by inducing destruction and/or inhibiting translation of these targets. Several miRNAs play key roles in maintaining neuronal structure and function and in higher-level brain functions, and methods are sought for manipulating their levels for exploring these functions. Here, we present a direct in vivo method for examining the cognitive consequences of enforced miRNAs excess in mice by stereotactic injection of miRNA-encoding virus particles. Specifically, the current protocol involves injection into the hippocampal CA1 region, which contributes to mammalian memory consolidation, learning, and stress responses, and offers a convenient injection site. The coordinates are measured according to the mouse bregma and virus perfusion is digitally controlled and kept very slow. After injection, the surgery wound is sealed and the animals recover. Lentiviruses encoding silencers of the corresponding mRNA targets serve to implicate the specific miRNA/target interaction responsible for the observed effect, with na&iuml;ve mice, mice injected with saline and mice injected with "empty" lentivirus vectors as controls. One month post-injection, the animals are examined in the Morris Water Maze (MWM) for assessing their navigation learning and memory abilities. The MWM is a round tank filled with colored water with a small platform submerged 1 cm below the water surface. Steady visual cues around the tank allow for spatial navigation (sound and the earth's magnetic field may also assist the animals in navigating). Video camera monitoring enables measuring the route of swim and the time to find and amount the platform. The mouse is first taught that mounting the hidden platform offers an escape from the enforced swimming; it is then tested for using this escape and finally, the platform is removed and probe tests examine if the mouse remembers its previous location. Repeated tests over several consecutive days highlight improved performance of tested mice at shorter latencies to find and mount the platform, and as more direct routes to reach the platform or its location. Failure to show such improvement represents impaired learning and memory and/or anxiety, which may then be tested specifically (e.g. in the elevated plus maze). This approach enables validation of specific miRNAs and target transcripts in the studied cognitive and/or stress-related processes.

MicroRNAs have significant roles in brain structure and function. Here we describe a method to enforce hippocampal miRNA over-expression using stereotactic injection of an engineered miRNA-expressing lentivirus. This approach can serve as a relatively rapid way to assess the in vivo effects of over-expressed miRNAs in specific brain regions.

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an ...

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 ...

Compensatory Limb Use and Behavioral Assessment of Motor Skill Learning Following Sensorimotor Cortex Injury in a Mouse Model of Ischemic Stroke

Mouse models have become increasingly popular in the field of behavioral neuroscience, and specifically in studies of experimental stroke. As models advance, it is important to develop sensitive behavioral measures specific to the mouse. The present protocol describes a skilled motor task for use in mouse models of stroke. The Pasta Matrix Reaching Task functions as a versatile and sensitive behavioral assay that permits experimenters to collect accurate outcome data and manipulate limb use to mimic human clinical phenomena including compensatory strategies (i.e., learned non-use) and focused rehabilitative training. When combined with neuroanatomical tools, this task also permits researchers to explore the mechanisms that support behavioral recovery of function (or lack thereof) following stroke. The task is both simple and affordable to set up and conduct, offering a variety of training and testing options for numerous research questions concerning functional outcome following injury. Though the task has been applied to mouse models of stroke, it may also be beneficial in studies of functional outcome in other upper extremity injury models.

Mouse models have become increasingly popular in studies of behavioral neuroscience. As models advance, it is important to develop sensitive behavioral measures specific to the mouse. This protocol describes the Pasta Matrix Reaching Task, which is a skilled motor task for use in mouse models of stroke.

Mouse models have become increasingly popular in the field of behavioral neuroscience, and specifically in studies of experimental stroke. As models ...

Assessment of Murine Exercise Endurance Without the Use of a Shock Grid: An Alternative to Forced Exercise

Using laboratory mouse models, the molecular pathways responsible for the metabolic benefits of endurance exercise are beginning to be defined. The most common method for assessing exercise endurance in mice utilizes forced running on a motorized treadmill equipped with a shock grid. Animals who quit running are pushed by the moving treadmill belt onto a grid that delivers an electric foot shock; to escape the negative stimulus, the mice return to running on the belt. However, avoidance behavior and psychological stress due to use of a shock apparatus can interfere with quantitation of running endurance, as well as confound measurements of postexercise serum hormone and cytokine levels. Here, we demonstrate and validate a refined method to measure running endurance in na&#239;ve C57BL/6 laboratory mice on a motorized treadmill without utilizing a shock grid. When mice are preacclimated to the treadmill, they run voluntarily with gait speeds specific to each mouse. Use of the shock grid is replaced by gentle encouragement by a human operator using a tongue depressor, coupled with sensitivity to the voluntary willingness to run on the part of the mouse. Clear endpoints for quantifying running time-to-exhaustion for each mouse are defined and reflected in behavioral signs of exhaustion such as splayed posture and labored breathing. This method is a humane refinement which also decreases the confounding effects of stress on experimental parameters.

A method to assess exercise endurance in laboratory mice without the use of a shock grid is demonstrated. This method is a humane refinement that can decrease the confounding effects of stress on experimental parameters.

Using laboratory mouse models, the molecular pathways responsible for the metabolic benefits of endurance exercise are beginning to be defined. The most ...

Experimental Assessment of Mouse Sociability Using an Automated Image Processing Approach

Mouse is the preferred model organism for testing drugs designed to increase sociability. We present a method to quantify mouse sociability in which the test mouse is placed in a standardized apparatus and relevant behaviors are assessed in three different sessions (called session I, II, and III).
The apparatus has three compartments (see Figure 1), the left and right compartments contain an inverted cup which can house a mouse (called &#34;stimulus mouse&#34;).
In session I, the test mouse is placed in the cage and its mobility is characterized by the number of transitions made between compartments. In session II, a stimulus mouse is placed under one of the inverted cups and the sociability of the test mouse is quantified by the amounts of time it spends near the cup containing the enclosed stimulus mouse vs. the empty inverted cup. In session III, the inverted cups are removed and both mice interact freely. The sociability of the test mouse in session III is quantified by the number of social approaches it makes toward the stimulus mouse and by the number of times it avoids a social approach by the stimulus mouse.
The automated evaluation of the movie detects the nose of the test mouse, which allows the determination of all described sociability measures in session I and II (in session III, approaches are identified automatically but classified manually). To find the nose, the image of an empty cage is digitally subtracted from each frame of the movie and the resulting image is binarized to identify the mouse pixels. The mouse tail is automatically removed and the two most distant points of the remaining mouse are determined; these are close to nose and base of tail. By analyzing the motion of the mouse and using continuity arguments, the nose is identified.
<img alt="Figure 1" src="/files/ftp_upload/52508/52508fig1.jpg" /> 
Figure 1. Assessment of Sociability During 3 sessions. Session I (top): Acclimation of test mouse to the cage. Session II (middle): Test mouse moving freely in the cage while the stimulus mouse is enclosed in an inverted cup. Session III (bottom): Both test mouse and stimulus mouse are allowed to move freely and interact with each other.

This protocol describes a method to quantify mouse sociability. Mice are videotaped as they move and interact in a special cage. Movie processing allows for the automated quantification of sociability with excellent accuracy and reliability.

Mouse is the preferred model organism for testing drugs designed to increase sociability. We present a method to quantify mouse sociability in which the ...

A Behavioral Test Battery for the Repeated Assessment of Motor Skills, Mood, and Cognition in Mice

Pharmacological and toxicological studies in neurodegeneration require comprehensive behavioral analysis in mice because motor dysfunctions and dysfunctions in mood and cognition are common and often shared symptoms in neurodegenerative diseases. Shown here is a behavioral test battery for motor, mood, and cognition, which can be repeatedly tested in a longitudinal study. This battery assesses the overall behavioral phenotype in mice by examining each domain of behavior with at least two independent well-accepted tests (i.e., open-field test and rotarod test for motor function, social interaction test, elevated plus maze test, and forced swim test for emotional function, and Morris water maze test and novel object recognition test for cognitive function). Therefore, this sensitive and comprehensive test battery is a powerful tool for the study of behavioral alternation in neurodegeneration.

A comprehensive behavioral test battery of motor skills, mood&#8212;including social interaction, depression, and anxiety&#8212;and cognition is designed for the repeated assessment of neurodegeneration-related behavioral changes in mice.

Pharmacological and toxicological studies in neurodegeneration require comprehensive behavioral analysis in mice because motor dysfunctions and ...

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) ...

Using Unidirectional Rotations to Improve Vestibular System Asymmetry in Patients with Vestibular Dysfunction

The vestibular system provides information about head movement and mediates reflexes that contribute to balance control and gaze stabilization during daily activities. Vestibular sensors are located in the inner ear on both sides of the head and project to the vestibular nuclei in the brainstem. Vestibular dysfunction is often due to an asymmetry between input from the two sides. This results in asymmetrical neural inputs from the two ears, which can produce an illusion of rotation, manifested as vertigo. The vestibular system has an impressive capacity for compensation, which serves to rebalance how asymmetrical information from the sensory end organs on both sides is processed at the central level. To promote compensation, various rehabilitation programs are used in the clinic; however, they primarily use exercises that improve multisensory integration. Recently, visual-vestibular training has also been used to improve the vestibulo-ocular reflex (VOR) in animals with compensated unilateral lesions. Here, a new method is introduced for rebalancing the vestibular activity on both sides in human subjects. This method consists of five unidirectional rotations in the dark (peak velocity of 320&#176;/s) toward the weaker side. The efficacy of this method was shown in a sequential, double-blinded clinical trial in 16 patients with VOR asymmetry (measured by the directional preponderance in response to sinusoidal rotations). In most cases, VOR asymmetry decreased after a single session, reached normal values within the first two sessions in one week, and the effects lasted up to 6 weeks. The rebalancing effect is due to both an increase in VOR response from the weaker side and a decrease in response from the stronger side. The findings suggest that unidirectional rotation can be used as a supervised rehabilitation method to reduce VOR asymmetry in patients with longstanding vestibular dysfunction.

A new rehabilitation method is presented for rebalancing the vestibular system in patients with asymmetric responses, which consists of unidirectional rotations toward the weaker side. By directly modifying the vestibular pathway rather than enhancing the multisensory aspects of compensation, asymmetry can be normalized within 1-2 sessions and show lasting effects.

The vestibular system provides information about head movement and mediates reflexes that contribute to balance control and gaze stabilization during ...

Assessment of Memory Function in Pilocarpine-induced Epileptic Mice

Cognitive impairment is one of the most common comorbidities in temporal lobe epilepsy. To recapitulate epilepsy-associated cognitive decline in an animal model of epilepsy, we generated pilocarpine-treated chronic epileptic mice. We present a protocol for three different behavioral tests using these epileptic mice: novel object location (NL), novel object recognition (NO), and pattern separation (PS) tests to evaluate learning and memory for places, objects, and contexts, respectively. We explain how to set the behavioral apparatus and provide experimental procedures for the NL, NO, and PS tests following an open field test that measures the animals&#8217; basal locomotor activities. We also describe the technical advantages of the NL, NO, and PS tests with respect to other behavioral tests for assessing memory function in epileptic mice. Finally, we discuss possible causes and solutions for epileptic mice failing to make 30 s of good contact with the objects during the familiarization sessions, which is a critical step for successful memory tests. Thus, this protocol provides detailed information about how to assess epilepsy-associated memory impairments using mice. The NL, NO, and PS tests are simple, efficient assays that are appropriate for the evaluation of different kinds of memory in epileptic mice.

This article presents experimental procedures for assessing memory impairments in pilocarpine-induced epileptic mice. This protocol can be used to study the pathophysiologic mechanisms of epilepsy-associated cognitive decline, which is one of the most common comorbidities in epilepsy.

Cognitive impairment is one of the most common comorbidities in temporal lobe epilepsy. To recapitulate epilepsy-associated cognitive decline in an animal ...

The Three-Chamber Choice Behavioral Task using Zebrafish as a Model System

Neurodegenerative diseases are age-dependent, debilitating, and incurable. Recent reports have also correlated hyperglycemia with changes in memory and/or cognitive impairment. We have modified and developed a three-chamber choice cognitive task similar to that used with rodents for use with hyperglycemic zebrafish. The testing chamber consists of a centrally located starting chamber and two choice compartments on either side, with a shoal of conspecifics used as the reward. We provide data showing that once acquired, zebrafish remember the task at least 8 weeks later. Our data indicate that zebrafish respond robustly to this reward, and we have identified cognitive deficits in hyperglycemic fish after 4 weeks of treatment. This behavioral assay may also be applicable to other studies related to cognition and memory.

We present a behavioral chamber designed to assess cognitive performance. We provide data showing that once acquired, zebrafish remember the task 8 weeks later. We also show that hyperglycemic zebrafish have altered cognitive performance, indicating that this paradigm is applicable to studies assessing cognition and memory.

Neurodegenerative diseases are age-dependent, debilitating, and incurable. Recent reports have also correlated hyperglycemia with changes in memory and/or ...