Anxiety Models in Rats and Mice

Anxiety Models in Rats and Mice

Anxiety is an adaptive response to stress. It is characterized by increased arousal, expectancy, autonomic and neuroendocrine activation, and specific behavior patterns. At low levels, anxiety can enhance performance and enable escape from danger. However, excessive or inappropriate anxiety may result in pathological impairment of normal everyday tasks. Anxiety is frequently distinguished from fear by its lack of specificity and its spontaneity in the absence of an imminent threat [1]. Pathological anxiety is among the most prevalent comorbid conditions in psychiatric disorders. The behavioral responses and brain mechanisms associated with an “anxious state” are so essential for survival that they must have evolved very early in the development of mammalian species and are probably highly conserved [2]. There is a wide variety of anxiety disorders, differing by the objects or situations that induce them. Anxiety disorders include panic disorder, generalized anxiety disorder, specific phobia, post-traumatic stress disorder, obsessive-compulsive disorder, etc. An estimated 18.1 % of U.S. adults had any anxiety disorder in the past year, and an estimated 31.1% of U.S. adults will experience any anxiety disorder at some time in their lives. Furthermore, anxiety can be co-morbid with many different disorders, such as depression, eating disorders, or irritable bowel syndrome.

Induction:

There are multiple approaches to assessing anxiety, the easiest of which is evaluating naïve rats or mice.  Enhanced induction is achieved through maternal separation (MS), MS + mild chronic stress (CS), MS + early weaning, single housing or chronic stress exposures.  Single housing and chronic stress exposures are simple and most effective in mice, while the MS paradigms are preferred for rats.

An example MS approach is as follows: Seven timed-pregnant dams will arrive in the facility at embryonic day 4 and will be housed under standard laboratory conditions. After the dams give birth, pups will be separated daily from postnatal day 2 until postnatal day 14 for 3 hours/day in the morning. As part of this procedure, the mother is removed from the home cage, and the home cage with the pups will be placed on a heating pad to prevent hypothermia. Alternatively, infrared lights can be used to keep the pups warm (30-33 ℃). The control pups are left undisturbed with their mothers. All offspring will then be weaned at postnatal day 21 and housed as singletons. The pups will be weighed twice per week to monitor the effect of stress on body weight.  A stronger anxiety phenotype can also be induced by adding a mild chronic stress (CS) paradigm for 5 consecutive days (postnatal day 85-89) to the maternal separation.  This addition involves a 4-hour restraint in a plastic tube during the extra induction days (MS + CS) [3]. Behavioral assays measuring depression and/or anxiety phenotypes will be performed at the age of 10-12 weeks old. Depending on the hypothesis, therapeutic interventions can start at gestation or just before behavioral analysis.

Disease Parameters & clinical assessment:

Anxiety-like behaviors in rodents should be assessed through a progressive test battery with each successive test being more stressful than the last.  Not all of the following tests need to be used in a given battery, but they should always move from least stressful to more stressful.

• Nestlet shredding/nest building- homecage behavior primarily for mice; anxiolytic agents will reduce this behavior
• Marble burying- homecage behavior primarily for mice; anxiolytic agents will reduce this behavior [4]
• Light-Dark box- anhedonia and photophobia; anxiolytics will increase the time spent on the light side of this apparatus [5]
• 3-chamber social interaction test- anxiolytics will increase interaction time with a conspecific [6]
• Open Field arena (OFA)- locomotor activity and anxiety. Rodents display a natural aversion to brightly lit open spaces, but they also have a drive to explore potentially threatening stimuli. Therefore, decreased levels of anxiety are correlated with increased exploratory behavior, especially the center of the arena.
• Elevated Plus Maze (EPM)- the gold standard for measuring anxiety-like behaviors. As with the OFA, the EPM is based on the animal’s natural aversion to open spaces and has been validated to assess anti-anxiety effects of pharmacological agents (e.g. benzodiazepines) and the genes involved in anxiety-related behaviors [7,8].

Optional Endpoints:

• Activation of microglia in brain
• IL-1β, TNF-a and IL-6 [9] in brain extracts
• IL-6 [10] and corticosterone [11] in plasma
• Cell death measured by labelling the 3’end of DNA fragments using terminal transferase (ApopTag) [12]
• Other blood and tissue collections
• Brain area dissections
• Other cytokine/chemokine analyses of blood and brain via Luminex®
• Other sandwich ELISAs
• CBC/clinical chemistry analysis
• Histopathology
• Other immunohistochemistry analyses

References

1. Duman, C.H. and R.S. Duman, Neurobiology and treatment of anxiety: signal transduction and neural plasticity.Handb Exp Pharmacol, 2005(169): p. 305-34.
2. Stein, D.J. and C. Bouwer, A neuro-evolutionary approach to the anxiety disorders. J Anxiety Disord, 1997. 11(4): p. 409-29.
3. Marais, L., et al., Maternal separation of rat pups increases the risk of developing depressive-like behavior after subsequent chronic stress by altering corticosterone and neurotrophin levels in the hippocampus. Neurosci Res, 2008. 61(1): p. 106-12.
4. Witkin, J.M. Animal models of obsessive-compulsive disorder. Curr Protoc Neurosci, 2008. 9: Unit 9.30. doi: 10.1002/0471142301.ns0930s45.
5. Lamont, M.G. et al., Binge drinking in male adolescent rats and its relationship to persistent behavioral impairment and elevated proinflammatory/proapoptotic proteins in the cerebellum. Psychopharmacology (Berl), 2020. doi: 10.1007/s00213-020-05458-3.
6. Choi, G.B. et al., The maternal interleukin-17a pathway in mice promotes autism-like phenotypes in offspring.Science, 2016. 351(6276): p.933-939.
7. Walf, A.A. and C.A. Frye, The use of the elevated plus maze as an assay of anxiety-related behavior in rodents.Nat Protoc, 2007. 2(2): p. 322-8.
8. Hoeffer, C.A., et al., Regulator of calcineurin 1 modulates expression of innate anxiety and anxiogenic responses to selective serotonin reuptake inhibitor treatment. J Neurosci, 2013. 33(43): p. 16930-44.
9. Roque, A., A. Ochoa-Zarzosa, and L. Torner, Maternal separation activates microglial cells and induces an inflammatory response in the hippocampus of male rat pups, independently of hypothalamic and peripheral cytokine levels. Brain Behav Immun, 2016. 55: p. 39-48.
10. Kim, K., et al., Pre-treatment peripheral biomarkers associated with treatment response in panic symptoms in patients with major depressive disorder and panic disorder: A 12-week follow-up study Compr Psychiatry, 2019. 95:152140.doi: 10.1016/j.comppsych.2019.152140.
11. Stenman, L.K., et al., Strain specific stress-modulating effects of candidate probiotics: A systematic screening in a mouse model of chronic restraint stress. Behav Brain Res, 2020. 379:112376. doi: 10.1016/j.bbr.2019.112376
12. Zhang, L.X., et al., Maternal deprivation increases cell death in the infant rat brain. Brain Res Dev Brain Res, 2002. 133(1): p. 1-11.

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