Method Article

Restraint to Induce Stress in Mice and Rats

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DOI:

10.3791/67387

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December 6th, 2024

1Department of Psychological and Brain Sciences, Fairfield University

In This Article

Summary

This article covers the procedures to induce stress responses using physical restraint stress in mice and rats. Additional considerations that should be observed when selecting and using restraint stress in rodent models are discussed.

Abstract

Across all animal species, exposure to stressful conditions induces stress responses. One method to study the effects of stress using rodent models is the restraint stress procedure. Restraint stress has been used for decades to investigate changes in physiology, genetics, neurobiology, immunology, and other systems impacted by stress. Due to the ease of performing the procedure, low cost, and numerous modifications to scale for the intensity and duration of stress exposure, a vast literature of studies has used restraint stress in mice and rats. As one example, this study presents previously published data showing the impact of restraint stress in transgenic mice on plasma corticosterone levels and optogenetically-induced norepinephrine release. Acute restraint stress increased plasma corticosterone levels, yet this effect was blunted in mice following repeat restraint stress. However, stimulated norepinephrine release in the bed nucleus of the stria terminalis was increased only in the repeat restraint stress group. These data highlight important considerations of restraint parameters on dependent measures. Additional descriptions of restraint stress in rats are also included for comparison. Finally, the influence of the parameters of the restraint (e.g., acute vs. chronic) and characteristics of the animal subjects (strain, sex, age) are discussed.

Introduction

Due to the universal experience of stress, investigations into the mechanisms of altered responses following stress exposures have been a consistent area of research for several decades1. These investigations have begun to parse out aspects of stress that may be beneficial to increase adaptability and responsiveness to acute stressors from stress experiences that induce maladaptive alterations of physiological and behavioral functions, often resulting from prolonged exposure to repeated and/or unpredictable stressors. Many of these responses to stressors impact the brain function, the hypothalamic-pituitary-adrenal (HPA) axis, the sympathetic and parasympathetic branches of the autonomic nervous system, and the immune system. Exposure to stressors initiates the increased release of corticotropin-releasing factor (CRF) to increase adrenocorticotropic hormone (ACTH) and glucocorticoid hormones, such as cortisol in primates and many other mammals and corticosterone in rodents2. Additionally, epinephrine and norepinephrine are released throughout the periphery and brain as part of the sympathetic nervous system response3. These chemical signals then interact with a number of physiological systems, such as immune function, metabolism, reproduction, and neuronal reactivity4.

Stressful experiences are associated with a myriad of conditions, including increases in anxiety and depression, alterations in learning and memory, impairments in decision-making and executive function, and susceptibility to substance use disorder, among others. Each of these effects is dependent on both parameters of the stressor and the characteristics of the individual experiencing stress4. The nature of the stressor, as acute or chronic and as controllable or unpredictable, substantially alters the outcomes observed. These aspects of stress appear consistent between human experiences and animal models. Thus, to model stress in animal studies, careful consideration must be used to select parameters of stress exposure that are appropriate for the species, sex, and condition.

Although a number of stressors are available for studies with rodents (including psychosocial stressors like social isolation stress or social defeat, pharmacological stressors like cortisol or yohimbine administration, physical stressors like footshock and restraint, and multimodal stressors that combine or alternate between conditions), the focus of this manuscript will be on the use of restraint stress in mice and rats. Restraint stress might be chosen over other stressors because it is easy to perform, inexpensive, adjustable to various sizes of rodents, scalable to both acute and long-term periods, and rarely results in adverse harm to the subject5. Although this manuscript focuses on the lasting changes induced by restraint stress exposure, some studies use restraint in rodents to examine mechanisms and behaviors that occur during restraint, including active coping behaviors involved in escaping the stressor6,7,8,9. The goal of this manuscript is to provide methodological considerations to assess the impact of restraint stress on mice or rat subjects.

Protocol

The experiments and protocols have been approved by the Institutional Animal Care and Use Committee (IACUC) at the University of North Carolina at Chapel Hill (mice) and Fairfield University (rats) and follow the guidelines of NIH10.

1. Restraint stress in mice

NOTE: Mice offer a number of advantages when studying the effects of stress on physiology and neuronal systems. With the prevalence of transgenic mouse lines, mechanistic investigations into the interactions between genes and stress exposures are readily feasible. Whether using transgenic or wild-type mice, consideration of the background strain is recommended. Additionally, due to their size and social housing, mice offer an advantage in space utilization over other species.

  1. Restraint stress exposure
    1. Randomly assign mice to homecage control, acute restraint stress, or chronic restraint stress groups.
    2. Determine the length of stress exposure. For acute stressors, use a single session lasting 5 min up to 12 h confined in the restraint apparatus. For chronic stressors, ensure that the session matches the length of the acute stressor but is repeated daily for as short as 3 days and as long as 21 days.
      NOTE: Because mice will be without access to food and water while in the restraint conditions, consider whether homecage control animals should also lack access to food and water.
    3. Modify 50 mL conical tubes to add ventilation holes.
      1. Use a power drill with a 1/8" bit to evenly space holes around each conical tube to ensure airflow throughout all areas of the tube.
      2. Place these holes to allow the mouse access to air regardless of which direction the mouse is facing in the tube. Ensure no sharp edges remain after modification.
        NOTE: These modified conical tubes should be effective for most adult mice (~20-40 g) but may not restrain small/young mice or larger strains.
    4. In a testing room, separate from animal housing and behavioral testing, place the restraint-assigned animal into the modified conical tube with the cap attached to confine the mouse in the tube for the designated length of restraint.
      NOTE: The specifics of the testing room design can vary based on the space available in the facility; the room could include fume hoods and other laboratory equipment and/or laboratory tables/benches or be an empty procedure room. However, regardless of the room setup, the restraint room environment should remain consistent over repeated restraint exposures to avoid context-induced alterations in stress responses11.
    5. Place the restraint tube horizontally on a flat surface. If needed, secure the tube with laboratory tape or a pipette basin/reagent reservoir so it does not roll with the mouse inside.
      NOTE: Under stress conditions, mice produce ultrasonic vocalizations. Consider whether the study design requires sound isolation from other restrained subjects12. Corticosterone and other markers of stress responses follow circadian rhythms. As such, considerations regarding the lighting conditions and time of stressor should be made. Across stress groups, restraint should occur at a consistent time of day, preferably early in the light cycle13,14,15. For the study presented here, mice were restrained for approximately 3 h following lights on.
    6. Monitor each animal during restraint stress at least every 20 min. Visually determine whether the animal is displaying unusual responses (slow breathing, lack of movement). If observed, remove the mouse from the restraint tube, contact a veterinarian, and exclude the mouse from the study.
    7. Return the mouse to the home cage and provide access to food and water.

2. Restraint stress in rats

NOTE: As larger rodents, rats have advantages to consider when investigating the effects of stress. Like mice, rats have various strains with differential responses to stress exposure16,17. Additionally, the increased size of rats makes rats easier to use with certain behavioral measures, e.g., drug self-administration.

  1. Restraint stress exposure
    1. Randomly assign the rat to homecage control, acute restraint stress, or chronic restraint stress groups.
    2. Determine the length of stress exposure. For acute stressors, use a single session lasting 5 min up to 12 h confined in the restraint apparatus. For chronic stressors, ensure that the session matches the length of the acute stressor but is repeated daily for as short as 3 days and as long as 21 days.
      NOTE: Because rats will be without access to food and water while in the restraint condition, consider whether homecage control animals should also lack access to food and water.
    3. For a commercially available restraint tube, follow the steps described below.
      NOTE: Commercially available restraint tubes for rats are available. These devices have a variety of designs, including metal wire, containers, and plastic/plexiglass tubes with rounded and flat-bottom options. Each option is simple to use.
      1. Insert the rat into the device, then add a plug adjusted to the subject's size, and lock it in place. When placed in the restraint tube, ensure that the rat is snugly confined to prevent head-to-tail turns but can breathe easily.
        NOTE: These devices are designed for restraining rats and do not need modification for respiration. This study uses the tailveiner restrainer (Table of Materials), which includes suction cups on a platform under the tube to secure the device. An additional benefit of this device is that the tail is accessible outside of the device for control of the subject during insertion and removal from the tube or tail vein blood draw during restraint.
    4. In a testing room separate from animal housing and behavioral testing, place restraint-assigned animas into the restraint tube for the designated length of restraint. Place the restraint tube horizontally on a flat surface.
      NOTE: Under stress conditions, rats also produce ultrasonic vocalizations. Consider whether the study design requires sound isolation from other restrained subjects.
    5. Monitor each animal during restraint stress at least every 20 min. Visually determine whether the animal is displaying unusual responses (slow breathing, lack of movement). If observed, remove the rat from the restraint tube, contact a veterinarian, and exclude the rat from the study.
    6. Return the rat to the home cage and provide access to food and water.

3. Assessments of stress

  1. Measurement of HPA activation
    1. If appropriate, following restraint stress exposure or removal from homecage, rapidly decapitate subjects to collect trunk blood into heparinized tubes.
    2. Isolate plasma via centrifugation and store plasma samples at -80 °C. Analyze blood corticosterone using an enzyme-linked immunosorbent assay (ELISA) kit.
      NOTE: Although corticosterone is often used as a readout of the severity of stress exposure, especially acute stress, corticosterone levels do not always correlate with stress-induced behavioral changes6. Furthermore, the HPA axis response regulating corticosterone release habituates with repeated exposure to the same stressor18. Alternatively, adrenocorticotrophic hormone (ACTH) may be a more reliable measure of the intensity of an acute stressor4. It remains elevated for approximately an hour following the cessation of stressor exposure, but severe and prolonged (approximately > 2 h) stress may fatigue its production.
  2. Behavioral effects
    1. If appropriate, following restraint stress exposure or removal from homecage, assess the behavioral performance of the rodent with the appropriate behavioral task.
      ​NOTE: Further information regarding the effects of restraint on various behavioral models can be found in the discussion section.
  3. Neurobiology
    1. Alternatively or additionally, assess neurobiological function following restraint stress exposure or control. A wide range of assays can investigate restraint stress effects on cellular, synaptic, and circuitry. Depending on the assay, implant an in vivo measurement device or harvest brain tissue to perform the selected analysis.
      NOTE: The effects of stress on cell-type specific changes in protein synthesis and epigenetic modifications have been thoroughly reviewed by McEwen and colleagues1. These genetic and protein modifications induce plasticity to alter synaptic connections and circuitry. In order to conduct these studies, harvest brain tissue following stress exposure and perform genomic, proteomic, ex vivo physiology, and other analyses. Alternatively, assess neurobiological changes through in vivo measurements such as microdialysis, fast-scan cyclic voltammetry (FSCV), fiber photometry, and others. These approaches record neurochemical release and neuronal activation, both following restraint stress exposure2,19 and now during restraint8,9,20. The toolbox to assess the neurobiological mechanisms underlying the effects of restraint stress is rapidly expanding allowing new insights into the complexities of stress.

Results

In a previously published study21, the restraint stress procedures described in this manuscript were used to assess the impact of restraint stress on the regulation of cortisol and norepinephrine, falling under the broad possibilities of changes in neurobiology induced by stress mentioned in protocol 3.3. In this study, we utilized the protocol described above for the restraint of mice. Additional details that are beyond the scope of the restraint focus of this article, e.g., the rationale for brain region, optogenetic stimulation parameters, or FSCV, can be found in the referenced manuscript.

We used male and female transgenic mice (DBH:Cre+/-::Ai32+/-) expressing channelrhodopsin selectively in noradrenergic neurons and measured stimulated norepinephrine release in the bed nucleus of the stria terminalis (BNST) with ex vivo FSCV21. Mice were restrained as described in protocol 1 acutely (2 h) and chronically (2 h/day for 5 days) or home cage control. Following exposure, blood was collected from some subjects for corticosterone analysis, as described in protocol 3.1. Immediately following acute restraint stress, mice showed elevated corticosterone levels, but the response was blunted following repeated stress (see Figure 1). Additional mice were used to assess the impact of restraint stress on norepinephrine release. 24-h following the final stress exposure, brain tissue was rapidly harvested and sliced to record norepinephrine release in the BNST. Interestingly, the acute restraint did not impact stimulated norepinephrine release, but repeated stress exposure caused a significant increase in stimulated norepinephrine release across stimulation parameters (see Figure 2). These results converge with previous investigations of the impact of restraint stress on norepinephrine release in rats using the approaches described in protocol 217,22.

Corticosterone concentration bar graph; naive, single, repeat stress comparison; statistical analysis.
Figure 1: Corticosterone in the plasma of home cage controls (naïve) and following acute (single) and chronic (repeat) restraint stress. Following a single 2 h session of acute restraint stress, plasma corticosterone levels were elevated compared to both naïve and repeat stress subjects. Results are expressed as mean ± SEM. N = 5-6 mice per group. **p < 0.05, ***p < 0.005. This figure has been modified with permission from Schmidt et al.21. Please click here to view a larger version of this figure.

Graph comparing norepinephrine [NE]max under varying light pulse frequencies and stress exposures.
Figure 2: Optogenetically stimulated norepinephrine release is increased following chronic (repeated) restraint stress. The day following the final stress exposure, ex vivo fast-scan cyclic voltammetry was performed to measure optogenetically stimulated norepinephrine release in the bed nucleus of the stria terminalis of mice. (A) Variations of frequency (Hz) of 20 pulses (20 P) stimulations released more norepinephrine in the repeated stress group. (B, C) Variations in the number of pulses (2-20) at a 5 Hz (B) or 10 Hz (C) frequency of stimulation indicated more norepinephrine release in the repeated stress group. Mean ± SEM; n = 5−6 mice/group providing 7−8 slices/group. *p < 0.05, **p < 0.05, ***p < 0.005, and ****p < 0.0001. This figure has been modified with permission from Schmidt et al.21. Please click here to view a larger version of this figure.

Discussion

To investigate the effects of stress on physiological, neurobiological, and behavioral functions in rodent models, conditions that produce stress for rodents must be used. Among these considerations are the conditions that produce stress responses in the subject. Restraint is a validated procedure to produce physiological and behavioral responses in rodents matching stress effects in humans. Using a methodology similar to this protocol, restraint has been used to assess a number of molecular, cellular, endocrine, and physiological responses related to models of psychiatric disorders, including anxiety, depression, pain, and substance use disorder1,19. Here, data from one published study is presented as an example of restraint in rodents, but the effects of restraint extend beyond this particular example to a vast range of dependent measures. Restraint might be selected for use as a stressor because it is easy to perform in mice and rats, inexpensive to establish, and adaptable to induce various levels of stress responses depending on the parameters used.

When compared to other stress-inducing approaches, restraint has some limitations to consider during study design4,23. Notably, restraint stress does not always induce behavioral changes observed with other physical stressors, like intermittent footshock24. These inconsistencies may be a result of restraint's advantage that it can occur in a variety of environments, whereas footshock is more limited to a testing chamber equipped with shock grid floors. Stress contexts greatly influence the resulting effects11. Additionally, confinement inside a traditional restraint tube or device is a disadvantage because it limits access to the subject during the restraint. Newer restraint devices have been developed, e.g., "REcording Signal TRansients ACCessible IN a Tube (RESTRAINT)", to overcome this limitation and allow tethering for neurobiological data collection during restraint sessions8,9,20. Another approach to address this obstacle uses immobilization stress as a similar technique to restraint - often, immobilization and restraint are used synonymously but with important differences25. Immobilization stress involves securing an animal's arms, legs, and head to a surface, whereas restraint involves placement inside a restrictive device. As such, immobilization stress restricts movement while providing access to the subject for the collection of blood, cerebrospinal fluid, pupillary responses, or other physiologic effects that may be of interest to stress research. Additional stressors that have been used to induce stress in rodents include environmental cold stress, administrations of pharmacological agents like corticosterone or yohimbine, ethologically relevant experiences of social defeat or predator odor exposure, and multimodal stress like chronic unpredictable stress (CUS). These alternative stress modalities are selected for use due to their advantages in modeling specific aspects of stress. For example, despite restraint being an aspect of CUS models in mice and rats26, the effects of CUS can be distinct from those produced by an equivalent exposure to chronic restraint stress27,28. The predictability of repeated restraint reduces the response to future restraint episodes, which limits the effectiveness of homotypic restraint stress. As an alternative, CUS and other heterotypic stress models rotate stressors or contexts to minimize habituation11,18. These unpredictable stress models may better model aspects of human psychiatric conditions to fully encapsulate loss of control and expectancy. Another disadvantage of restraint that other stress modalities are better at addressing is the social contribution to stress susceptibility and stress buffering29,30,31. Rodents and humans are social animals with conspecific relationships and hierarchies. Although restraint stress does not incorporate socialization, psychosocial stressors such as social defeat stress provide an ethologically relevant stressor for rodents32,33,34.

Within the use of restraint stress, the intensity and repetition of the administration is a parameter that could be manipulated5. To create a more robust restraint stress, study design can require a longer length of time in the restraint device. As short as 5 min or as long as 12 h are possible parameters for either acute or prolonged restraint sessions, respectively35,36. Once the length of the restraint exposure is determined, the repetition of the stressor can also be a factor in inducing various levels of stress35,37. It should be noted that repeated restraint stress does not necessarily mean an increase in the effects observed. Often repeated restraint stress habituates or otherwise reduces stress responses18. In the data presented here, we observed habituation with the corticosterone response to repeated restraint stress, while at the same time, we measured differences in stimulated norepinephrine release only in the repeat stress exposure group. Therefore, the effects of repeated restraint must be carefully investigated before determining habituation effects35.

Additionally, the consequences of the housing conditions used in studies using restraint stress must be considered. Interaction with stressed conspecifics impacts stress responses in rodents30,38,39. Thus, to maintain consistent responses across experimental groups, subjects should either be individually housed or housed as a group with all cage mates experiencing the same stress condition37. Critically, a drawback to this social isolation of all subjects approach is that social isolation housing is a stressor as well40,41. However, regardless of housing conditions, stress exposure should occur in isolation, if possible, to prevent social buffering of stress impacting the outcome measurements30. Along with these housing conditions, the timing of the stress exposure in relation to environmental light cycles should be considered14,15. As some studies note37, corticosteroid levels follow circadian rhythms, so administering stressors at different times in the light cycle can produce inconsistent effects. Therefore, establishing and maintaining a uniform start time for restraint is essential. For example, one paper examining exercise stress effects on the circadian control of corticosterone shows that in control mice corticosterone peaks late in the light cycle, but in stressed mice, corticosterone remains elevated without a stable peak13. In light of these findings, stressing rodents early in the light cycle is recommended to produce the largest effects.

Beyond the parameters of the restraint conditions, the characteristics of the animal subject have also been important to consider in studies using restraint stressors. For example, numerous studies reported sex differences using restraint stress12,35,37,42,43,44. Understanding the mechanisms underlying these differences is worthy of investigation and might lead to better outcomes for equitable treatments when people experience stress. Perhaps females and males experience and process stressors differently, which leads to the increase in anxiety and depression in women44. For example, restraint stress decreases learning performance in males but increases learning performance in females45. Historically, restraint stress studies in rodents have limited their subjects to males, but moving forward we should design experiments to include both sexes. Another subject characteristic to consider when designing experiments using restraint (and other) stressors in either mouse or rat species is the strain46,47,48,49,50,51. Strain effects have been observed across a number of paradigms, with some strains displaying exacerbated responses to restraint stress. Lastly, subject age should be considered when utilizing restraint stress. From prenatal exposure to stress by restraining a pregnant mother to restraint in aged populations, restraint stress can be adapted to any time point. Differences in the stress response have been shown across the lifespan45,52,53,54,55,56. In addition to requiring different restraining devices to accommodate changes in body size as aging occurs, age-related experiences of stress should be considered when using restraint stressors.

The selection of the appropriate outcome measurement, whether a behavioral task described in 3.2 or a neurobiological variable described in 3.3, requires consideration. For one, acute, mild stress exposure has been shown to enhance performance in some learning tasks, but intense or chronic stress exposure impairs performance on learning and memory tasks57. These shifts in cognitive performance are thought to be regulated by circuit dysregulation between the prefrontal cortex, amygdala, and hippocampus. However, the effects of restraint stress on cognitive flexibility are more mixed with inconsistent findings hindering a clear picture of how varied parameters of restraint alter reversal learning or set shifting58. With regards to behavioral effects of drugs, restraint stress has been shown, like other stressors, to increase behaviors related to substance use disorder across a number of drug classes59. However, these effects are somewhat selective in that restraint stress is not reported to drive drug seeking in operant-conditioning models of relapse but does increase associative learning of drugs effects as measured by conditioned place preference tasks, including reinstatement60. This discrepancy is thought to occur because restraint devices creates a separate context than operant conditioning chambers, whereas footshock stress can occur in the operant environment. To overcome this limitation, pairing stress exposure with cues that can be presented during operant self-administration sessions has demonstrated stressed-induced increases in drug taking and drug seeking61. The effects of chronic restraint on drug-related behaviors are less well characterized. Considering models of affective disorders like anxiety and depression, in both humans and animal models, stress exposure increases anxiety measures. In rodents, restraint stress assessments of anxiety include thigmotaxis responses in the open field apparatus measured by tracking the animal's locomotor response along the walls in the arena compared to the center. Animals exhibiting anxiety-like behaviors following restraint will spend more time near the sides and have fewer center crossings. Another approach to measure anxiety is the elevated plus maze. This task uses an apparatus in the shape of a cross elevated approximately 3 feet above the floor. Two arms of the apparatus include wall barriers; two arms have open sides. Animals exhibiting anxiety-like behaviors will spend more time in the enclosed arms than the open arms62,63. Depression-like behaviors are assessed through the forced swim task in which rodents are placed in a cylinder of water and measured for struggling responses compared to floating responses64. Whether the cessation of struggling to escape truly represents depression-like behavior has been called into question65, yet the task has shown utility as a screen for antidepressant medications. Depression-like behaviors can also be assessed through stress-induced reductions in sucrose preference66. These changes in consumption of sweet liquid are taken as an assessment of the anhedonia aspects of clinical depression.

In summary, restraint in mice and rats is a validated procedure to produce stress and anxiety responses in studies of physiology, genetics, immunology, and neuroscience. Compared to other stress-inducing approaches, restraint is inexpensive, easy to perform, useful to model aspects of human disorders including anxiety, depression, substance use disorder, and post-traumatic stress disorder, and can be adapted with a wide range of parameters. For these reasons, restraint stress continues to be a useful method to determine the impact of stress in rodents.

Disclosures

No conflicts of interest to report.

Acknowledgements

I appreciate the editorial feedback provided by Holly Rahurahu and Sam Shaffer.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
50 mL General Purpose Screwcap Conical TubeGlobe Scientific6288
Disposable Pipette BasinFisher Scientific13-681-500
ELISA Kit for CorticosteroneArbor AssaysK014-H
Tailveiner Restrainer TubeBraintree ScientificRTV

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Restraint StressRodent Stress ModelsAcute Restraint StressChronic Restraint StressPlasma CorticosteroneNorepinephrine ReleaseOptogenetic StimulationStress ResponseBehavioral TestingNeurobiological Mechanisms
Restraint to Induce Stress in Mice and Rats
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