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 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.
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-field 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.
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
2. Elevator vertical motion device
3. Ferris-wheel rotation device
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.
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.
Figure 3 demonstrates representative defecation count results. For elevator vertical motion, rats were in one of three different rotation groups of 0.8 Hz, 0.4 Hz, and 0.2 Hz vertical motion, in addition to a control group, called the static group. The equivalence to our periods of motion is as follows: frequency = 0.8Hz = 1/0.8 = 0.1250s = 1250 ms, frequency = 0.4Hz = 1/0.4 = 0.2500s = 2500 ms, and frequency = 0.2Hz = 1/0.2 = 0.5000s = 5000 ms. The EVM significantly increased defecation (one-way ANOVA, F(3,31) = 20.2306, p < 0.00001). The change in Hz vertical motion increased defecation for 0.4 Hz (t = 3.4064, df = 14, p = 0.0043) and 0.8 Hz (t = 10.6895, df = 14, p < 0.0001). For Ferris-wheel rotation, rats were rotated in a clockwise-pause-counterclockwise cycle lasting approximately 10 s to reach its initial position. The entire session of rotation lasted for 2 h. The Ferris-wheel rotation group was compared to a control group, called the static group. The Ferris-wheel rotation group increased defecation as determined by a t-test (t = 10.6895, df = 14, p < 0.0001).
Figure 4 demonstrates the open field examination of total distance traveled results. These data were collected using commercial video tracking software for the analysis of open field behavior (Table of Materials)28, but several open source software pipelines exist for behavioral video analysis such as Bonsai30 and one our group has developed based on Matlab29. Also, here, the total distance traveled was assessed as a metric, but frame-by-frame differences can be used for determining other behaviors such as vertical motion. For elevator vertical motion, rats were in one of three different rotation groups of 0.8 Hz, 0.4 Hz, and 0.2 Hz vertical motion, in addition to a control group, called the static group. The EVM significantly decreased open field distance traveled (one-way ANOVA, F(3,31) = 16.5994, p < 0.00001). The change in Hz vertical motion decreased open-field locomotion for 0.4 Hz (t = 3.1354, df = 14, p = 0.0073) and 0.8 Hz (t = 5.8929, df = 14, p < 0.001). For Ferris-wheel rotation, rats were rotated in a clockwise-pause-counterclockwise cycle lasting approximately 10 s to reach its initial position. The entire session of rotation lasted for 2 h. The Ferris-wheel rotation group was compared to a control group, called the static group. The Ferris-wheel rotation group decreased open-field locomotion as determined by a t-test (t = 4.3341, df = 14, p = 0.0007).
A number of published studies have employed the protocols described here6,7,8,9,10,11,12. One recent example from our group studied the mechanisms behind anticholingenics mecamylamine and scopolamine alleviating motion sickness-induced gastrointestinal symptoms12.
Figure 1: Instrumentation used. (a) Balance Beam. The balance beam is a narrow wooden beam (2.5 cm x 130 cm) between the two stools placed 100 cm (approximately 0.75 m in height) apart. A lamp is placed at the start stool and a black plastic box (15 cm x 15 cm x 8 cm) on the finish stool. (b) Elevator vertical motion device. The elevator vertical motion device amplitude is set at 22 cm up and 22 cm down from neutral. The warm-up vertical motion consists of 2500 ms period for 5 min, 2000 ms for 5 min, and 1500 ms for 5 min. The test motion consists of a 1000 ms period for 2 h. The elevator vertical motion device is slowed in reverse using a 1500 ms period for 5 min, 2000 ms for 5 min, and 2500 ms for 5 min. Rats are placed head towards the front of the elevator vertical motion device. (c) Ferris-wheel rotation device. The Ferris-wheel rotates in a clockwise direction at 16°/s2 accelerating to 120°/s, subsequently decelerating at 48°/s2 to reach 0°/s, pausing for 1 s, and then rotating in a counterclockwise (16°/s2 accelerating to 120°/s, subsequently decelerating at 48°/s2 to reach 0°/s). The clockwise-pause-counterclockwise cycle requires ~10 s to reach its initial position. Rats are placed head towards center of the Ferris-wheel rotation device. Please click here to view a larger version of this figure.
Figure 2: Balance beam results. Time taken to transverse the beam (mean ± standard deviation). The y-axis indicates seconds taken to transverse the beam. Rats were trained for three days prior to evaluation in order to achieve stable performance on the balance beam10. Prior evaluation with the elevator vertical motion or Ferris-wheel devices significantly increases crossing time. Statistical testing was performed by two-tailed t-test with Bonferroni correction between control and every other group. *** indicates p < 0.001. Please click here to view a larger version of this figure.
Figure 3: Defecation count results. Elevator vertical motion results (a) Left panel – Defecation count (mean ± standard deviation) by group for 0.8 Hz, 0.4 Hz, and 0.2 Hz vertical motion, in addition to a control group, called the static group at 0 Hz. Note the significant increase in defecation for 0.8 Hz and 0.4 Hz as indicated by the asterisks. Ferris-wheel rotation results (b) Right panel – Defecation count (mean ± standard deviation) for Ferris-wheel rotation rat group (see description for angular velocity paradigm) and a control group (0 Hz), called the static group. Note the significant increase in defecation for the rotation group as indicated by the asterisks. Please click here to view a larger version of this figure.
Figure 4: Total distance traveled. (a) Elevator vertical motion results. This panel consists of total distance traveled (mean ± standard deviation) by cm in the open field locomotion test by group for 0.8 Hz, 0.4 Hz, and 0.2 Hz vertical motion, in addition to a control (static) group. Note the significant decrease in total distance traveled for 0.8 Hz and 0.4 Hz as indicated by the asterisks. Statistical testing was performed by two-tailed t-test with Bonferroni correction between control and every other group. ** indicates p < 0.01 and *** indicates p < 0.001. (b) Ferris-wheel rotation results. This panel consists of total distance traveled (mean ± standard deviation) by cm in the open-field locomotion test for Ferris-wheel rotation rat group and a control (static) group. Note the significant decrease in total distance as indicated by the asterisks. Statistical testing was performed by two-tailed t-test between control and Ferris-wheel group. *** indicates p < 0.001. Please click here to view a larger version of this figure.
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 receiving visual information that differs from the anticipated internal model of the environment2,3 leads to autonomic reaction eliciting symptoms such epigastric discomfort, nausea and/or vomiting1. Further theories have outlined that postural instability, as would occur on a yawing ship4,5, elicits autonomic reaction. Despite these significant advances, questions remain that can be aided by evaluation protocols such as elevator vertical motion and Ferris-wheel rotation.
A critical step for balance beam is training. Rats must be motivated and have confidence to cross the beam; otherwise, balance (i.e., vestibular integrity) is not measured in an evaluation period. For researchers interested in examining anxiety14,17 or traumatic injury15,16,17, other behaviors during training or balance beam crossing may be relevant. For example, in anxiety research using the balance beam, defecation, urination, falls, and missteps can be enumerated14. Also in some research areas, rodents that lack motivation to cross the beam may be evaluated differently13,14,15,16,17. It is critical during elevator vertical motion and Ferris-wheel rotation to ensure that the box is fastened shut and securely closed, as rodents in an unsecured box may be propelled and injured. Also, ensure that rodents are evaluated in the open-field box28,29,30 only once and immediately after the elevator vertical motion and Ferris-wheel to ensure rapid evaluation of vestibular effects.
The above-mentioned protocols use quantitative measures. Therefore, the limitations for balance beam include rodents that lack motivation to cross the beam, as balance is the behavior being evaluated. Limitations for the elevator vertical motion and Ferris-wheel rotation defecation assays include requiring a well-fed rodent. This is necessary; otherwise, the rodent may not experience a robust autonomic reaction to vestibular stimulation. It is good practice to observe baseline defecation count for a normal/control period of 2.5 h duration for comparative purposes.
Another important consideration when using the protocols, and interpreting results, is differences in motion sickness responses across species. In humans, and also other species like cats and dogs, retching and vomiting are two common symptoms31,32,33,34. Rats, on the other hand, cannot vomit. However, rats display motion sickness symptoms such as pica35,36, defecation response37, and spontaneous locomotion reduction35,38. Also, humans rely primarily on vision for sensory input and motion sickness is likely related to sensory conflict with the vestibular system2,39. In rats, especially albino rats (e.g., Sprague-Dawley), vision is not typically the primary sense, but rather somatosensory (whiskers). This may lead to inter-species differences in the relative contributions of different sensory inputs to the conflict. Lastly, there are inter-rodent species differences in the motion sickness response. For example, the shrew mouse (Suncus murinus) is able to have an emetic response40,41.
Collectively the procedures described form a short battery of assessments for the examination and evaluation of autonomic reactions in rodents during motion sickness6,7,8,9,10,11. The present techniques coupled to more physiological measures such as electrophysiology to determine the cortical consequences during vestibular stimulation would be of great interest.
The authors have nothing to disclose.
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.
Elevator vertical motion device | Custom | Custom-made Elevator vertical motion device to desired specifications | |
Ethovision | Noldus Information Technology | Video tracking software | |
Ferris-wheel rotation device | Custom | Custom-made Ferris-wheel rotation device to desired specifications | |
Latex, polyvinyl or nitrile gloves | AMMEX | Use unpowdered gloves 8-mil | |
Open field box | Custom | Darkened plexiglass box with IR camera | |
Rat or mouse | JAX labs | Any small rodent | |
Small rodent cage | Tecniplast | 1284L | |
Wooden beam and stools | Custom | Custom-made wooden beam and stools to specifications indicated |