Name: Assessing the Autonomic and Behavioral Effects of Passive Motion in Rats using Elevator Vertical Motion and Ferris-Wheel Rotation (Video) | JoVE Journal
Uploaded: 2020-02-07T12:00:00.000+00:00
Duration: 6 min 18 s
Description: Learn to assess autonomic and behavioral effects of passive motion in rats using elevator vertical motion and Ferris-wheel rotation protocols. These methods evaluate autonomic nervous system integrity and reveal vestibular disorders.

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5 minutes

This work was supported in part by the Hong Kong Research Grants Council, Early Career Scheme, Project #21201217 to C. L. The FWR device has a patent in China: ZL201120231912.1.

The present study and procedures were approved by the Ethics Committee for Animal Experimentation of the Second Military Medical University (Shanghai, China) in accordance with the Guide for the Care and Use of Laboratory Animals (US National Research Council, 1996).
1. Animals
<ol>
	<li>Use Sprague-Dawley (SD) rats of two months (200&#8211;250 g). For each behavioral assay, use a separate group of rats. Always use separate control and experimental groups. 
	NOTE: There were two autonomic tests: EVM and FWR. The EVM had three conditions in addition to a control group (= 4) with three behavioral assays (balance beam, defecation counting and open field = 3) with 8 rats in each for a total of 96 rats (4 x 3 x 8). The FWR had one condition in addition to a control group (= 2) with three behavioral assays (balance beam, defecation counting and open field = 3) with 8 rats in each for a total of 48 rats (2 x 3 x 8). In total, we report 144 rats.</li>
	<li>Cage rodents under a constant 25 &#176;C temperature and 60%&#8211;70% humidity.</li>
	<li>House rodents in 12 h/12 h light/dark cycles with access to food and drinking water ad libitum. 
	NOTE: Since the following protocols are behavioral experiments, rats should be handled gently. Handling animals should be with both hands with body and rear support, so as not to induce anxiety.</li>
	<li>Perform experiments (EVM and FWR) and evaluation assays (balance beam and open field evaluation) in the darkness to minimize visual cues.</li>
</ol>
2. Elevator vertical motion device
<ol>
	<li>Perform the elevator vertical motion procedures in complete darkness to minimize visual cues.</li>
	<li>Place the rodents in the Plexiglas box (22.5 cm x 26 cm x 20 cm). Here the Plexiglas box can accommodate four rodents (custom-made device).</li>
	<li>Ensure the box is fastened shut and securely closed to avoid rodents falling out. Place the Plexiglass box on the elevator pad of the elevator vertical motion device (custom-made device).</li>
	<li>Turn on the elevator vertical motion device to the lowest setting for acclimatization.</li>
	<li>Set the amplitude as 22 cm up and 22 cm down from neutral. Incrementally change elevator vertical motion as follows:
	<ol>
		<li>Set the initial periods as 2,500 ms for 5 min, 2,000 ms for 5 min, and 1,500 ms for 5 min.</li>
		<li>Use a test period of 1000 ms for 2 h.</li>
		<li>Slow the device in reverse using periods of 1500 ms for 5 min, 2000 ms for 5 min, and 2500 ms for 5 min.</li>
	</ol>
	</li>
</ol>
3. Ferris-wheel rotation device
<ol>
	<li>Ferris-wheel rotation device setup
	<ol>
		<li>Place the plexiglass container (22.5 cm x 26 cm x 20 cm) on a wooden bench (custom-made device).</li>
		<li>Place the rodents in the plexiglass container with the long axis of the body perpendicular to the horizontal rotation rod of the Ferris-wheel (custom-made device). 
		NOTE: The placement with body perpendicular to horizontal rod ensures stimulation of otolith organs (anterior-posterior and vertical direction) during rotation.</li>
		<li>Close the plexiglass box securely.</li>
		<li>Place the second set of rodents in the plexiglass container with the long axis of the body perpendicular to the horizontal rotation rod on the second arm of the Ferris-wheel rotation device. Use a second set of rodents with similar mass to balance the Ferris-wheel.</li>
		<li>Securely close the plexiglass box and place on the Ferris-wheel rotation device.</li>
	</ol>
	</li>
	<li>Ferris-wheel rotation procedure
	<ol>
		<li>Perform the Ferris-wheel rotation procedures in complete darkness to minimize visual cues.</li>
		<li>Start the Ferris-wheel rotating in a clockwise direction at 16&#176;/s2 to reach an angular velocity of 120&#176;/s, and then begin to decelerate at 48&#176;/s2 to reach 0&#176;/s. After a 1 s pause, have the container continue to rotate in a counterclockwise direction in the same manner as above (acceleration at 16&#176;/s2 to reach an angular velocity of 120&#176;/s and then deceleration at 48&#176;/s2 to reach 0&#176;/s). The clockwise-pause-counterclockwise cycle requires approximately 10 s to reach its initial position.</li>
		<li>Continue the clockwise-counterclockwise rotation for 2 h per session for approximately 720 rotations.</li>
	</ol>
	</li>
</ol>
4. Evaluation of EVM and FWR
NOTE: The evaluation of Ferris-wheel rotation device and elevator vertical motion is done by three procedures: balance beam testing, defecation counting, and open-field examination. Identical procedures are used to evaluate elevator vertical motion. These evaluation procedures should be done as soon as possible after Ferris-wheel rotation or elevator vertical motion.
<ol>
	<li>Balance beam
	<ol>
		<li>Balance beam setup
		<ol>
			<li>Set up the balance beam10,11,12 by placing two wooden stools (approximately 0.75 m in height) in the experimental field, approximately 110 cm apart.</li>
			<li>Place a black plastic box (15 cm x 15 cm x 8 cm) on the finish stool.</li>
			<li>Place a narrow wooden beam (2.5 cm x 130 cm) between the two stools, leaving a 100 cm distance between the stool edges, from the start stool to the finish stool. 
			NOTE: The entrance to the black plastic box should be at the finish line of the 100 cm.</li>
			<li>Place a lamp at the start stool. Turn on the lamp.</li>
			<li>Turn off the room lights and ensure that the room is as dark as possible. This ensures the rodent follows the direction of the balance beam from the lighted region to the obscured region.</li>
		</ol>
		</li>
		<li>Balance beam procedures 
		NOTE: The motor coordination assay of the balance beam is assessed by measuring the time taken to traverse the elevated wooden beam.
		<ol>
			<li>Train each rodent daily for 3 consecutive days, before the examination period, in order to achieve stable performance on the balance beam10. Train by introducing the rat to the beam in the lighted corner and prompting it to cross the beam. Eventually the rat will cross of its own volition. Rats in the present protocol took 3.6 &#177; 0.9 seconds. 
			NOTE: Some rodents fail to achieve stable performance during training and should be excluded. Some rodents do not perform the task while others lack motivation to cross the beam. Stable performance was two consecutive trial periods of crossing times less than 4 seconds. If a rat falls off during training or assessment it should be categorized as a rat &#39;fall&#39; and not assessed further.</li>
			<li>For the actual procedure, place the trained rodent on the start stool near the light and simultaneously press start on a stopwatch. The rodent should cross the balance beam rapidly and enter the black box on the finish stool.</li>
			<li>Press start on the stopwatch once the rodent is in place and press stop when the nose enters the dark box on the finish stool. The time to traverse the beam is from start stool to finish stool. 
			NOTE: Once the rodent is trained, you may perform an intervention or manipulation, such as inducing motion sickness, prior to evaluation. You may also obtain a baseline measurement, prior to the intervention, using the time to traverse of the last training session.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Defecation counting
	<ol>
		<li>Place the plexiglass container containing the four rodents on a bench after the Ferris-wheel test period.</li>
		<li>Remove the rodents and place in individual open-field boxes (below).</li>
		<li>Count the number of feces pellets in the plexiglass box attributed to each rodent. 
		NOTE: A baseline measurement can be obtained, for comparison with the evaluation after elevator motion, by counting feces pellets prior to undergoing elevator vertical motion.</li>
	</ol>
	</li>
	<li>Open-field examination
	<ol>
		<li>Place the rodents in the open-field box (40 cm x 40 cm x 45 cm).</li>
		<li>Record open field behavior using an IR-video camera for 3 min28,29,30.</li>
		<li>Determine the total distance traveled. 
		NOTE: It is very important NOT to place the rodent in the open-field box before elevator vertical motion. The environment must be novel to the rodent. Therefore, baseline measurements should NOT be taken for open-field examination.</li>
	</ol>
	</li>
</ol>

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<li>Ossenkopp, K. P., Rabi, Y. J., Eckel, L. A., Hargreaves, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reductions+in+body+temperature+and+spontaneous+activity+in+rats+exposed+to+horizontal+rotation%3A+abolition+following+chemical+labyrinthectomy%5BTitle%5D)+AND+%22Physiology+%26+Behavior%22%5BJournal%5D)">Reductions in body temperature and spontaneous activity in rats exposed to horizontal rotation: abolition following chemical labyrinthectomy.</a> Physiology & Behavior. 56, 319-324 (1994).</li>
<li>Oman, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Motion+sickness%3A+a+synthesis+and+evaluation+of+the+sensory+conflict+theory%5BTitle%5D)+AND+%22Canadian+Journal+of+Physiology+and+Pharmacology%22%5BJournal%5D)">Motion sickness: a synthesis and evaluation of the sensory conflict theory.</a> Canadian Journal of Physiology and Pharmacology. 68, 294-303 (1990).</li>
<li>Hu, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Emesis+in+the+shrew+mouse+%28Suncus+murinus%29+induced+by+peroral+and+intraperitoneal+administration+of+staphylococcal+enterotoxin+A%5BTitle%5D)+AND+%22Journal+of+Food+Protection%22%5BJournal%5D)">Emesis in the shrew mouse (Suncus murinus) induced by peroral and intraperitoneal administration of staphylococcal enterotoxin A.</a> Journal of Food Protection. 62, 1350-1353 (1999).</li>
<li>Ueno, S., Matsuki, N., Saito, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Suncus+murinus+as+a+new+experimental+model+for+motion+sickness%5BTitle%5D)+AND+%22Life+Sciences%22%5BJournal%5D)">Suncus murinus as a new experimental model for motion sickness.</a> Life Sciences. 43, 413-420 (1988).</li>
</ol>

The authors declare no financial or non-financial conflicts of interests. The FWR device has a patent in China: ZL201120231912.1.

The present study describes assessing autonomic responses to passive motion in rodents using elevator vertical motion and Ferris-wheel rotation. These equipment and procedures can be easily adopted to other rodents and several modifications of the assays exist to confirm vestibular functioning in different circumstances, such as during in pharmacological challenge or surgical interventions. Research in MS elicited by vestibular stimulation has led to the theory that sensory conflict or neuronal mismatch caused by receivi...

Motion sickness (MS) due to abnormal visuo-vestibular stimulation leads to autonomic reaction, eliciting symptoms such epigastric discomfort, nausea and/or vomiting1. According to current theories, motion sickness may be caused by a sensory conflict or neuronal mismatch from receiving integrated motion information that differs from the anticipated internal model of the environment2,3 or postural instability as would occur on a yawing ship4,5. Despite significant advances in the field of motion sickness and vestibular autonomic functioning6,7,8,9,10,11,12, future research can be aided by standardized evaluation protocols. Assessing the autonomic effects of standard passive motions will greatly benefit investigations into the causes and prevention of motion sickness. The overall goal of this study is to assess the autonomic and behavioral effects of passive motion in rodents. Animal models, such as rodents, allow easy experimental manipulation (e.g., passive motion and pharmaceutical) and behavioral evaluation, which can be used to study the etiology of motion sickness. Here, we present a detailed battery for testing the effects of passive motion and the integrity of vestibular functioning.
The present study details two assays, elevator vertical motion (EVM) and Ferris-wheel rotation (FWR), that induce autonomic reactions to the passive motion. The assays are coupled to three quantitative behavioral measures, the balance beam (on mice13 and rats14,15,16,17), open-field examination, and defecation counting. The EVM (similar to the pitch and roll of a ship encountering a wave) assesses vestibular functioning by stimulating the otolith sensory organs that encode linear accelerations (i.e., the saccule that responds to movements in the vertical plane)18. The FWR (centrifugal rotation or sinusoidal motion) device stimulates the otolith organs by linear acceleration and the semicircular canals by angular acceleration19,20. The Ferris-wheel/centrifugal rotation device is unique in its autonomic assessment. To date, the only similar device in the literature is the off-vertical axis rotation (OVAR) turntable, which is used to examine the vestibulo-ocular reflex (VOR)18,21,22, conditioned avoidance23,24, and the effects of hypergravity25,26,27. The EVM assay and the FWR device assay induce vestibular stimulation leading to autonomic reactions. We couple the EVM and FWR to quantitative measurements such as balance beam, defecation counting, and open-field analysis28,29,30, to ensure robust and reproducible results. Similar to those previously described in mice13 and rats14,15,16,17, the balance beam assay is a 1.0 m long beam suspended 0.75 m from the ground between two wooden stools using a simple black-box modification at the goal end (finish). The balance beam has been used to assess anxiety (obscure black box)14,17, traumatic injury15,16,17, and here, autonomic reactions affecting balance. We have performed defecation counting for assessing the autonomic response in the motion sickness model previously, and it is a reliable quantitative measurement that is easily performed and unequivocally assessed6,8,9,11. The open-field analysis employs a simple black box open-&#64257;eld behavior assessment using Ethovision28, Bonsai30, or a simple video analysis in Matlab29 to quantify behavior such as motion. In the current protocol, we use the total distance traveled, but we note several different paradigms exist (e.g., elongation, zone of movement, velocity, etc.)28,29,30. Collectively, these procedures form a short battery of assessments for the examination and evaluation of autonomic reactions to passive motion, for example in motion sickness6,7,8,9,10,11. The present assays can be adapted to a variety of animal models.

<table><tbody><tr><td>Elevator vertical motion device</td><td>Custom</td><td></td><td>Custom-made Elevator vertical motion device to desired specifications</td></tr><tr><td>Ethovision</td><td>Noldus Information Technology</td><td></td><td>Video tracking software</td></tr><tr><td>Ferris-wheel rotation device</td><td>Custom</td><td></td><td>Custom-made Ferris-wheel rotation device to desired specifications</td></tr><tr><td>Latex, polyvinyl or nitrile gloves</td><td>AMMEX</td><td></td><td>Use unpowdered gloves 8-mil</td></tr><tr><td>Open field box</td><td>Custom</td><td></td><td>Darkened plexiglass box with IR camera</td></tr><tr><td>Rat or mouse</td><td>JAX labs</td><td></td><td>Any small rodent</td></tr><tr><td>Small rodent cage</td><td>Tecniplast</td><td>1284L</td><td></td></tr><tr><td>Wooden beam and stools</td><td>Custom</td><td></td><td>Custom-made wooden beam and stools to specifications indicated</td></tr></tbody></table>

assessing the autonomic and behavioral effects of passive motion in rats using elevator vertical motion and ferris-wheel rotation

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.

Figure 2 demonstrates representative balance beam results of time taken to transverse. Rats were trained for 3 consecutive days in order to achieve stable performance on the balance beam10. The subsequent day, rats were evaluated for balance beam performance. In the y-axis of the figure, we have the number of seconds taken for rodents to cross the balance beam for Ferris-wheel, elevator vertical motion, and control groups for demonstrative purposes.
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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 ...

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Learn to assess autonomic and behavioral effects of passive motion in rats using elevator vertical motion and Ferris-wheel rotation protocols. These methods evaluate autonomic nervous system integrity and reveal vestibular disorders.

Assessing the Autonomic and Behavioral Effects of Passive Motion in Rats using Elevator Vertical Motion and Ferris-Wheel Rotation (Video) | JoVE Journal

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

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