Inactive BehaviorMouse Ethogram

Mouse Ethogram: Awake but Still Behaviors

By November 15, 2019 No Comments

Definition

Awake but still behaviors collectively refer to behaviors in which the mouse is conscious and processing its immediate environment but is physically still and immobile, not exhibiting any locomotion.

Description

Awake but still behaviors are described by the absence of movement and the presence of consciousness. In this set of behaviors, the mouse is awake or alert but is not physically moving, thus falling under the wide category of inactive behaviors.

Awake but still behaviors may also be referred to as “awake but motionless,” “still and alert,” or “awake and alert.”

Awake but Still Behaviors in an Experimental Context

In an experimental setting, the phrase “awake but still” (or some variation of the phrase) will be used to denote that a mouse is at the simplest state of consciousness. This is made clear and exemplified by the categories comprising the stereotypy behavior scale which is used to score a mouse’s level of stereotypy (repetitive motions):

  • 0= Asleep
  • 1= Awake but still
  • 2= Grooming or mild licking;
  • 3= Continuous locomotion for the duration of 30 seconds along the horizontal plane without instances of rearing
  • 4= Continuous locomotion for the duration of 30 seconds along the horizontal plane with instances of rearing
  • 5= Bouts of locomotion along the horizontal plane (i.e., darting) without instances of rearing or sniffing

Although this particular scale goes up to 10, but the first few points (0-5) are given for the sake of illustrating how ‘awake but still’ is used within a categorical format scale to describe or measure a mouse’s behavior. In such measures of activity, ‘awake but still’ is typically ranked just above sleep behaviors but is still considered to be more inactive than maintenance behaviors.

Another example of how awake but still is used in an experimental setting is demonstrated by the psychostimulant-induced behavior rating scale where the levels are:

  • 1= Asleep/Inactive (asleep, awake but still)
  • 2= Light in-place directed activity (normal grooming, light sniffing)
  • 3= Normal exploratory behavior (slow intermittent locomotion, occasional rearing, sniffing or gnawing bedding)
  • 4=Fast exploratory behavior (fast exploratory locomotion or sniffing, increased rearing)
  • 5=In-place stereotyped behaviors (continuous sniffing, head bobbing, circling, intense self-grooming)
  • 6=Patterned locomotion (running in a pattern such as around the cage’s periphery)

The psychostimulant-induced behavior rating scale ranges from 1-9 but the first few points are listed here in order to demonstrate that ‘awake but still’ is on the inactive pole of the activity spectrum and may even be categorized in the same group as sleeping.

Types of Awake but Still Behaviors

  • Immobile: Immobility is defined as the complete absence of movement except for those necessary to keep breathing. A mouse is immobile yet awake and still, and aware of its immediate surroundings. In some cases, immobility may also be interpreted as a depressive behavior (a form of learned helplessness) wherein a mouse is doing the bare minimum in order to survive.
  • Freeze: Freeze differs from immobility in that it is observed in an agonistic encounter. When two mice face each other, a freeze behavior is considered as a retreat behavior likely to be exhibited by the less dominant mouse. Also, a mouse may freeze in reaction to aversive stimuli such as a predator in hopes of minimizing the potential threat of their encounter. Since predators can detect motion, freezing eliminates any motion and increases a mouse’s chances of survival by lowering the likelihood of their being detected.
  • Social Inactivity: Social inactivity consists of two or more mice being inactive at the same point in time. Mice, in close proximity to one another, may sit or lie together.

Related Behaviors

Several behaviors can be considered as awake but still since they fulfill the two basic criteria of absence of movement combined with wakefulness. However, these behaviors can be distinguished based on the context in which they occur. The following behaviors can be classified as awake but still:

  • Alert: An alert mouse is awake and paying attention to its environment. Alertness is associated with consciousness and awareness of surroundings. A mouse is sometimes alert when it is awake but not always. Thus, alertness is a cognitive process that is closely related to being awake but still.
  • Attend: A mouse is attending to a stimulus when it is paying attention to it and is oriented towards it. Attend can be an active behavior if it is happening in conjunction with locomotion. But, it can also be an awake but still behavior if a mouse is attending to stimuli without displaying any motion. Attend and alert are also similar to each other.
  • Hide: A mouse will retreat to an area that is safe, thus shielding and keeping itself out of view from dangerous predators or more dominant mice. When hiding, a mouse is likely to be awake but still in order to detect nearby stimuli but not be detected.

Function of Behavior

Awake but still behaviors, depending on the context, have many functions, including:

  • To elude predators: In a dangerous situation, such as when crossing paths with a predator, the safest decision may be to just freeze or hide nearby. Therefore, in the proper context, the inactive behaviors benefit the mouse by ensuring its survival.
  • To defend itself in agonistic encounters: Some awake but still behaviors, like freeze or hide, may be observed in the context of an agonistic situation with another mouse where dominance behaviors are elicited. Thus, freezing and hiding are useful awake but still behaviors that serve as a defense tactic to avoid further threats or to minimize the damage that has been done (such as if the behavior ultimately leads to submission as the resolution of the agonistic encounter).
  • To conserve or regain energy: When a mouse enters into hiding, a type of awake but still behavior, it is essentially regaining the energy that it spent while escaping or fighting. Therefore, being awake but still is a way to regain energy, which can be used up later, while being aware of the immediate surroundings.
  • To build social ties: In the case of social inactivity, social bonding occurs while requiring minimal energy resources from the mouse. Therefore, it is beneficial to the mouse to be able to be a part of its group while exerting a minimal amount of energy.

Application of the Behavior

Still but awake behaviors may be observed in many contexts, including:

  • An agonistic setting: In an agonistic setting, awake but still behaviors can be exhibited when a mouse is trying to hide or freeze.
  • A social setting: When mice are together, they may exhibit awake but still behaviors collectively through social inactivity.
  • The presence of a predator: When in the presence of a predator, a mouse may react by hiding. Thus, a mouse is able to stay aware of its surroundings but reduce the chances of being detected by staying still.
  • The context of learned helplessness: When a mouse becomes helpless, it may be awake but still as a form of despair, demonstrating longer bouts of immobility. In fact, learned helplessness is a disease model that is used to induce depression and is characterized by high instances of immobility. More on this in the Disease Models section.

Research Techniques

Depending on the research question and experiment, a multitude of research techniques can be utilized in order to study a mouse that is awake but still, including:

  • Pharmaceutical studies: Studies which involve giving mice drugs and studying their behavioral profile will usually take note if a mouse is awake but still, since this is the most basic level of consciousness. Anesthetic studies in which mice are given sedatives will take note of whether a mouse is awake but still in order to denote that the mice are awake and conscious but not active.
  • Electroencephalogram studies: Electroencephalogram (EEG) studies involve taking recordings of the brain which measure brain activity by capturing the rhythm of the brain’s oscillatory patterns which occur due to neuronal firing. Awake but still is relevant to EEG studies because it takes a mouse’s baseline recording, thus enabling scientists to compare activity levels and determine how brainwave patterns vary from one state to the next.
  • Behavioral studies: Behavioral studies directly measure the various awake but still behaviors. Mice are subjected to different behavioral tests, thus exposing them to different contexts that are likely to elicit relevant awake but still behaviors. More on this below.

Behavioral Tests for Assessing Awake but Still Behaviors

Awake but still behavior can be observed at any point during a mouse’s awake-phase. The following behavioral tests and techniques can be used to study awake but still behaviors:

  • Video analysis: Video recordings which capture the daily activity of mice are useful for quantifying and measuring active and inactive behaviors because they enable scientists to measure the amount of time that a mouse spends in the awake but still state.
  • Photobeam integration and analysis: Movement tracking can be sensed through photobeam integration. If a mouse is moving, photobeams will be blocked or interrupted, thus detecting movement. This is useful for measuring awake but still behavior since a photobeam will remain interrupted for the duration in which a mouse is still.
  • Forced Swim Test: Immobility may be observed in the context of learned helplessness in this experimental setting. A mouse is placed in a cylinder containing water and is expected to swim non-stop in an energetic effort to escape and maintain its ability to breathe. This set-up essentially mimics a stressful environment. In such a situation, a mouse is induced to model depression or despair and immobility serves as an index of depression. Instances of immobility, the awake but still behavior that is typically studied using this experimental paradigm, are measured and compared across experimental groups.
  • Tail Suspension Test: In the tail suspension test, a mouse will be hung upside down on a hook by its tail, thereby creating a stressful situation from which it will try to escape. This test lasts for about 6 minutes and a mouse will progressively become more immobile and helpless. When immobile, a mouse is awake but still, a behavioral response that occurs due to helplessness from the inability to escape.
  • Social Interaction Test: In the Social Interaction Test, mice are placed collectively in an experimental cage and are expected to interact with one another. At the beginning of the test, mice will display many bouts of investigative behaviors. Across time, this activity and investigative period will decrease as more bouts of social inactivity (a form of awake but still where mice are collectively at rest while maintaining alertness) increase.

Pharmaceutical Studies on the Behavior

Using pharmaceutical studies, certain substances, chemical, or drugs, are administered to mice, enabling researchers to closely study how the various awake but still behaviors are subsequently affected.

Psychostimulants Decrease Awake but Still Behaviors

Normal healthy mice will be awake but still at various time points throughout their awake-phase. Mice which are given psychostimulants like amphetamine or cocaine will display fewer instances of awake but still behaviors and more instances of fast exploratory behaviors and stereotypy. For example, when C57BL/6 mice are given low doses of amphetamine or cocaine, they exhibit in-place stereotypy. When given higher doses, they display repetitive behaviors characterized by rearing and patterned locomotion throughout the cage. By contrast, mice administered with saline will display low instances of these behaviors and, by comparison, spend more time in the awake but still state.

Imipramine Injections Reduce Depression-related Immobility

Learned helplessness, a condition of becoming helpless due to constant exposure to stressful stimuli combined with the inability to escape said stimuli, is associated with higher levels of immobility. Intraperitoneal injections of imipramine, a tricyclic antidepressant, are able to decrease depression-related immobility.

Abnormalities

Stereotypy

Stereotypy is the repetition of movements performed excessively and without a clear goal or function. There are many different types of stereotypies. A mouse can repetitively jump, route-track, bar-mouth, and chew. Stereotypy is a common side-effect of drugs, thus it is a behavior that is commonly observed in pharmaceutical studies studying behavior in mice. Stereotypy is inversely associated with awake but still behaviors. As a mouse demonstrates more stereotypical behaviors, they spend less time being awake but still. Thus, a major goal of behavioral researchers is to find ways to reduce stereotypy and increase awake but still behaviors.

Depression

Depression is a psychological syndrome that refers to low affect and dropped energy levels. In humans, it is characterized by changed thoughts, feels, and an altered state of well-being. Mice are used as animal models in behavioral research, especially as subjects to test pharmacological interventions for managing depression. Mice which model depression spend more time simply being awake but still or immobile. As a result, they show low activity levels. Thus, the goal of pharmaceutical studies is to reverse this and decrease the amount of time depressed mice spend being awake but still.

Disease Models of Awake but Still

Awake but still behaviors are essential for optimal functioning and everyday survival. Yet, these behaviors are sometimes thrown off or put off balance by abnormalities. Disease models can be used in a research setting in order to find solutions as to how to restore awake but still behaviors to their normal profile.

Home-Cage Induced Stereotypy

Stereotypy can be induced through home cage housing. Mice that are housed in low environmentally enriched cages demonstrate higher levels of stereotypy than mice that are in environmentally enriched housing. Nesting material is the key factor here. Mice that are not provided with nesting material will develop stereotypical behavior, thus reducing the instances of awake but still behaviors they exhibit. Awake but still behaviors are considered to be observed in inverse frequency when compared to stereotypy. Thus, stereotypy as induced through home-cage housing, is a disease model that has reduced instances of awake but still behaviors.

Learned Helplessness Model of Depression

Immobility is one of the major characteristics of depression. When modeling depression, mice show prolonged instances of immobility.

Learned helplessness, a method for inducing depression, is achieved by exposing mice to inescapable stressful situations, such as repetitive foot shocks which last about 15 seconds. Thus, they learn to be helpless and put no effort into avoiding stress since they have learned that stress is inescapable. In fact, mice that have been induced to have behavioral learned helplessness will show longer duration of immobility in the Tail Suspension and Forced Swim Tests, thus demonstrating a depression-like phenotype.

Summary

  • Awake but still, as the name implies, refers to a state wherein the mouse is alert and conscious but not exhibiting any movement or locomotion.
  • Awake but still behaviors may also be referred to as “still and alert” or “awake and alert.”
  • In experimental settings, awake but still is used as a part of a categorical scale and is one step above being asleep. In some cases, it is even grouped together with sleep.
  • Behaviors in which the mouse is awake but still include alert, attend, immobile, freeze, hide, and social inactivity.
  • Awake but still is a useful state for the mouse to be in since it enables it to elude predators, defend itself in agonistic encounters, conserve or regain energy, and build social ties.
  • Awake but still behaviors can be exhibited in various situations, including an agonistic setting, a social setting, in the presence of a predator or aversive stimuli, and in the context of learned helplessness.
  • Commonly used research techniques for studying awake but still behaviors include pharmaceutical studies, EEG studies, and behavioral studies.
  • Awake but still behaviors can be measured through several behavioral study approaches and techniques, such as video analysis, photo beam integration, the Forced Swim Test, the Tail Suspension Test, and the Social Interaction Test.
  • As a general rule, psycho-stimulants decrease the number of times that a mouse is awake but still during its awake-phase since they affect the mouse in such a way that it exhibits more locomotion and stereotypy and less stillness.
  • Antidepressants are able to decrease immobility that is associated with learned helplessness and depression.
  • Depression and stereotypy are abnormalities that affect awake but still behaviors.
  • Stereotypy can be induced through housing mice in cages deprived of nesting materials, thus decreasing the amount of time they spend being awake and still.
  • Learned helplessness is a model of depression wherein mice show a high duration of immobility, a type of awake but still behavior, after being exposed to inescapable stressful situations.

References

  1. http://mousebehavior.org/still-and-alert/
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  3. Van Swearingen, Amanda ED, Q. David Walker, and Cynthia M. Kuhn. “Sex differences in novelty-and psychostimulant-induced behaviors of C57BL/6 mice.” Psychopharmacology225.3 (2013): 707-718.
  4. Jensen, Victoria N., et al. “Repeated measurement of respiratory muscle activity and ventilation in mouse models of neuromuscular disease.” JoVE (Journal of Visualized Experiments) 122 (2017): e55599.
  5. Su, Chun-Lin, et al. “Epigenetic regulation of BDNF in the learned helplessness-induced animal model of depression.” Journal of psychiatric research 76 (2016): 101-110.
  6. Fureix, Carole, et al. “Stereotypic behaviour in standard non-enriched cages is an alternative to depression-like responses in C57BL/6 mice.” Behavioural brain research 305 (2016): 186-190.
  7. Novak, Janja, et al. “Effect of cage-induced stereotypies on measures of affective state and recurrent perseveration in CD-1 and C57BL/6 mice.” PloS one 11.5 (2016): e0153203.
  8. Gross, Alexandra Nam-Mi, Anna Katarina Julia Engel, and Hanno Würbel. “Simply a nest? Effects of different enrichments on stereotypic and anxiety-related behaviour in mice.” Applied animal behaviour science 134.3-4 (2011): 239-245.

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