The authors thank the Animal Care Committee of CAMH for supporting this work, as well as the animal caregivers of CAMH who provided extensive feedback on the usefulness of the procedure, motivating the execution of the described experiments and submission of the detailed protocol for other users. This work was in part funded by CAMH BreakThrough Challenge, awarded to TP, and by internal funds from CAMH.

<ol><li>Deacon, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Housing%2C+husbandry+and+handling+of+rodents+for+behavioral+experiments%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Housing, husbandry and handling of rodents for behavioral experiments.</a> Nature Protocols. 1 (2), 936(2006).</li>
<li>Bryda, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Mighty+Mouse%3A+the+impact+of+rodents+on+advances+in+biomedical+research%5BTitle%5D)+AND+%22Missouri+Medicine%22%5BJournal%5D)">The Mighty Mouse: the impact of rodents on advances in biomedical research.</a> Missouri Medicine. 110 (3), 207-211 (2013).</li>
<li>Martic-Kehl, M., Ametamey, S., Alf, M., Schubiger, P., Honer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impact+of+inherent+variability+and+experimental+parameters+on+the+reliability+of+small+animal+PET+data%5BTitle%5D)+AND+%22EJNMMI+Research%22%5BJournal%5D)">Impact of inherent variability and experimental parameters on the reliability of small animal PET data.</a> EJNMMI Research. 2 (1), 26(2012).</li>
<li>Howard, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Control+of+Variability%5BTitle%5D)+AND+%22ILAR+Journal%22%5BJournal%5D)">Control of Variability.</a> ILAR Journal. 43 (4), 194-201 (2002).</li>
<li>Toth, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+influence+of+the+cage+environment+on+rodent+physiology+and+behavior%3A+Implications+for+reproducibility+of+pre-clinical+rodent+research%5BTitle%5D)+AND+%22Experimental+Neurology%22%5BJournal%5D)">The influence of the cage environment on rodent physiology and behavior: Implications for reproducibility of pre-clinical rodent research.</a> Experimental Neurology. 270, 72-77 (2015).</li>
<li>Golini, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Non-invasive+Digital+Biomarker+for+the+Detection+of+Rest+Disturbances+in+the+SOD1G93A+Mouse+Model+of+ALS%5BTitle%5D)+AND+%22Frontiers+in+Neuroscience%22%5BJournal%5D)">A Non-invasive Digital Biomarker for the Detection of Rest Disturbances in the SOD1G93A Mouse Model of ALS.</a> Frontiers in Neuroscience. 14 (896), (2020).</li>
<li>Singh, S., Bermudez-Contreras, E., Nazari, M., Sutherland, R. J., Mohajerani, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Low-cost+solution+for+rodent+home-cage+behaviour+monitoring%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Low-cost solution for rodent home-cage behaviour monitoring.</a> PLoS One. 14 (8), 0220751(2019).</li>
<li>Stewart, K., Schroeder, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rodent+Handling+and+Restraint+Techniques%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Rodent Handling and Restraint Techniques.</a> Journal of Visualized Experiments. , (2021).</li>
<li>Hurst, J. L., West, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Taming+anxiety+in+laboratory+mice%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Taming anxiety in laboratory mice.</a> Nature Methods. 7 (10), 825-826 (2010).</li>
<li>Gouveia, K., Hurst, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improving+the+practicality+of+using+non-aversive+handling+methods+to+reduce+background+stress+and+anxiety+in+laboratory+mice%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Improving the practicality of using non-aversive handling methods to reduce background stress and anxiety in laboratory mice.</a> Scientific Reports. 9 (1), 20305(2019).</li>
<li>Gouveia, K., Hurst, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimising+reliability+of+mouse+performance+in+behavioural+testing%3A+the+major+role+of+non-aversive+handling%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Optimising reliability of mouse performance in behavioural testing: the major role of non-aversive handling.</a> Scientific Reports. 7, 44999(2017).</li>
<li>Ghosal, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+handling+limits+the+impact+of+stress+on+metabolic+endpoints%5BTitle%5D)+AND+%22Physiology+%26+Behavior%22%5BJournal%5D)">Mouse handling limits the impact of stress on metabolic endpoints.</a> Physiology & Behavior. 150, 31-37 (2015).</li>
<li>Wahlsten, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Different+data+from+different+labs%3A+lessons+from+studies+of+gene-environment+interaction%5BTitle%5D)+AND+%22Journal+of+Neurobiology%22%5BJournal%5D)">Different data from different labs: lessons from studies of gene-environment interaction.</a> Journal of Neurobiology. 54 (1), 283-311 (2003).</li>
<li>Nature Neuroscience. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Troublesome+variability+in+mouse+studies%5BTitle%5D)+AND+%22Nature+Neuroscience%22%5BJournal%5D)">Troublesome variability in mouse studies.</a> Nature Neuroscience. 12 (9), 1075(2009).</li>
<li>Sensini, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+impact+of+handling+technique+and+handling+frequency+on+laboratory+mouse+welfare+is+sex-specific%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">The impact of handling technique and handling frequency on laboratory mouse welfare is sex-specific.</a> Scientific Reports. 10 (1), 17281(2020).</li>
<li>Ghosal, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+handling+limits+the+impact+of+stress+on+metabolic+endpoints%5BTitle%5D)+AND+%22Physiology+%26+Behavior%22%5BJournal%5D)">Mouse handling limits the impact of stress on metabolic endpoints.</a> Physiology & Behavior. 150, 31-37 (2015).</li>
<li>Novak, J., Bailoo, J. D., Melotti, L., Rommen, J., Würbel, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+Exploration+Based+Cognitive+Bias+Test+for+Mice%3A+Effects+of+Handling+Method+and+Stereotypic+Behaviour%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">An Exploration Based Cognitive Bias Test for Mice: Effects of Handling Method and Stereotypic Behaviour.</a> PLoS One. 10 (7), 0130718(2015).</li>
<li>Gouveia, K., Waters, J., Hurst, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+Handling+Tutorial%5BTitle%5D)+AND+%22NC3Rs%22%5BJournal%5D)">Mouse Handling Tutorial.</a> NC3Rs. , (2016).</li>
<li>Gouveia, K., Hurst, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reducing+Mouse+Anxiety+during+Handling%3A+Effect+of+Experience+with+Handling+Tunnels%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Reducing Mouse Anxiety during Handling: Effect of Experience with Handling Tunnels.</a> PLoS One. 8 (6), 66401(2013).</li>
<li>Henderson, L. J., Smulders, T. V., Roughan, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identifying+obstacles+preventing+the+uptake+of+tunnel+handling+methods+for+laboratory+mice%3A+An+international+thematic+survey%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Identifying obstacles preventing the uptake of tunnel handling methods for laboratory mice: An international thematic survey.</a> PLoS One. 15 (4), 0231454(2020).</li>
<li>Percie Du Sert, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+ARRIVE+guidelines+2.0%3A+Updated+guidelines+for+reporting+animal+research%5BTitle%5D)+AND+%22PLOS+Biology%22%5BJournal%5D)">The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research.</a> PLOS Biology. 18 (7), 3000410(2020).</li>
<li>Golde, W. T., Gollobin, P., Rodriguez, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+rapid%2C+simple%2C+and+humane+method+for+submandibular+bleeding+of+mice+using+a+lancet%5BTitle%5D)+AND+%22Lab+Animal%22%5BJournal%5D)">A rapid, simple, and humane method for submandibular bleeding of mice using a lancet.</a> Lab Animal. 34 (9), 39-43 (2005).</li>
<li>Guilloux, J. P., Seney, M., Edgar, N., Sibille, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integrated+behavioral+z-scoring+increases+the+sensitivity+and+reliability+of+behavioral+phenotyping+in+mice%3A+relevance+to+emotionality+and+sex%5BTitle%5D)+AND+%22Journal+of+Neuroscience+Methods%22%5BJournal%5D)">Integrated behavioral z-scoring increases the sensitivity and reliability of behavioral phenotyping in mice: relevance to emotionality and sex.</a> Journal of Neuroscience Methods. 197 (1), 21-31 (2011).</li>
<li>LaFollette, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Laboratory+Animal+Welfare+Meets+Human+Welfare%3A+A+Cross-Sectional+Study+of+Professional+Quality+of+Life%2C+Including+Compassion+Fatigue+in+Laboratory+Animal+Personnel%5BTitle%5D)+AND+%22Frontiers+in+Veterinary+Science%22%5BJournal%5D)">Laboratory Animal Welfare Meets Human Welfare: A Cross-Sectional Study of Professional Quality of Life, Including Compassion Fatigue in Laboratory Animal Personnel.</a> Frontiers in Veterinary Science. 7 (114), (2020).</li>
<li>Sorge, R. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Olfactory+exposure+to+males%2C+including+men%2C+causes+stress+and+related+analgesia+in+rodents%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Olfactory exposure to males, including men, causes stress and related analgesia in rodents.</a> Nature Methods. 11 (6), 629-632 (2014).</li>
<li>Bailoo, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+Cage+Enrichment+on+Behavior%2C+Welfare+and+Outcome+Variability+in+Female+Mice%5BTitle%5D)+AND+%22Frontiers+in+Behavioral+Neuroscience%22%5BJournal%5D)">Effects of Cage Enrichment on Behavior, Welfare and Outcome Variability in Female Mice.</a> Frontiers in Behavioral Neuroscience. 12, (2018).</li>
<li>Spangenberg, E. M., Keeling, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assessing+the+welfare+of+laboratory+mice+in+their+home+environment+using+animal-based+measures+-+a+benchmarking+tool%5BTitle%5D)+AND+%22Laboratory+Animals%22%5BJournal%5D)">Assessing the welfare of laboratory mice in their home environment using animal-based measures - a benchmarking tool.</a> Laboratory Animals. 50 (1), 30-38 (2016).</li>
<li>Theil, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+epidemiology+of+fighting+in+group-housed+laboratory+mice%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">The epidemiology of fighting in group-housed laboratory mice.</a> Scientific Reports. 10 (1), 16649(2020).</li>
<li>Weber, E. M., Dallaire, J. A., Gaskill, B. N., Pritchett-Corning, K. R., Garner, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aggression+in+group-housed+laboratory+mice%3A+why+can%27t+we+solve+the+problem%5BTitle%5D)+AND+%22Lab+Animal%22%5BJournal%5D)">Aggression in group-housed laboratory mice: why can't we solve the problem.</a> Lab Animal. 46 (4), 157-161 (2017).</li>
<li>Cloutier, S., Baker, C., Wahl, K., Panksepp, J., Newberry, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Playful+handling+as+social+enrichment+for+individually-+and+group-housed+laboratory+rats%5BTitle%5D)+AND+%22Applied+Animal+Behaviour+Science%22%5BJournal%5D)">Playful handling as social enrichment for individually- and group-housed laboratory rats.</a> Applied Animal Behaviour Science. 143 (2), 85-95 (2013).</li>
<li>Panksepp, J., Burgdorf, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((50-kHz+chirping+%28laughter%3F%29+in+response+to+conditioned+and+unconditioned+tickle-induced+reward+in+rats%3A+effects+of+social+housing+and+genetic+variables%5BTitle%5D)+AND+%22Behavioural+Brain+Research%22%5BJournal%5D)">50-kHz chirping (laughter?) in response to conditioned and unconditioned tickle-induced reward in rats: effects of social housing and genetic variables.</a> Behavioural Brain Research. 115 (1), 25-38 (2000).</li>
</ol>

The authors have no conflict of interest to disclose.

This study and method development are based on the observation that handling techniques in mice are still overlooked by the scientific community, and that some labs are still reluctant to implement habituation or handling techniques to reduce stress and reactivity of their animals prior to experiments. While representing a time commitment, animal handling provides beneficial effects to the animals that may contribute to the success of the experiments to be performed and prevents experiments from having to be performed multiple times due to data variability or animal over-reactivity. The use of the 3D-handling technique decreased escape attempts in mice. It also increased interaction with the experimenter and decreased anxiety-like phenotypes in our 6-month-old male mice. Further, 3D-handling decreased data variability and decreased corticosterone levels in 2.5 month-old female mice after only 3 initial days of handling. This approach relies on gentle manipulations to habituate the mouse to handling by the experimenter facilitating smoother transport and easier intervention.
Something worth emphasizing from the 3D-handling technique is that the progression of handling methods occurs in response to the reactivity of the mouse, depending on the achievement of the milestones described above and in Table 1. Animals should have reduced reactivity to one handling step before progressing to the next steps. Attempting to progress too quickly to the &#34;shelter&#34; or &#34;nose-poke&#34; steps on animals that are not sufficiently habituated would likely result in increased stress and potentially reduce the effectiveness of the procedure. Similarly, the reactivity of the animal on each day of handling should be monitored and should be considered when deciding if additional handling days are required. If animals do not respond well to the shelter test on the first day, not meeting criteria for achieving the first milestone, the first day of handling could be repeated until completion of the milestone. Similarly, if animals fail to respond to the nose poke test on the second day, the second day may also be repeated. Another caveat to note with this approach is that the risk of mice jumping away is greater on the first day of handling, in particular in jumpy strains like C57BL6. Following the guidelines described above should reduce the risk of jumping, and provide ways to limit such behaviors. Duration of the handling and progression through the steps may vary depending on the strains, particularly if working with transgenic models known to exhibit anxious phenotypes.
Several factors can contribute to reducing the effectiveness of the presented 3D-handling technique. One such factor is the potential fear or hesitancy from the experimenter, in the event of the experimenter being not familiar with mouse handling, or being scared of mice. Therefore, the effect on the handler is also something to consider. However, the gradual increase in the level of interaction with the mice allows novice experimenters to develop confidence and greater skills at performing the handling technique as they proceed through the handling steps. The proposed steps/milestones (the shelter and nose poke tests) can help counter potential human variability in novice handlers, ensuring that animals reach similar levels of habituation. It has been reported that fostering positive human-animal interactions with animals had resulted in greater quality of life and compassion satisfaction in animal care staff24. As such, the gentling from handling presents benefits to both the handler and the animal during any general interaction or intervention.
With its impact on decreasing the number of attempts to pick-up the mouse, in both 6-month-old males and 2.5 month-old females, the 3D-handling provides an alternative to tunnel handling or other techniques, facilitating easier transfer of animals from their cage to experimental apparatus. The 3D-handling technique also increased the interaction of 6-month-old male mice with the experimenter. This was not observed in 2.5 month-old female mice, but female mice remained easier to pick up, compared to tail-handled mice. This suggests that the 3D-handling technique may be more suitable for experiments requiring direct interactions between the animal and the experimenter, such as the Morris water maze (despite potential sex/age confounding factors discussed later). Others have developed and used manual handling techniques, consisting of picking up the animals with cupped hands, without additional manipulation10. While these techniques showed beneficial effects, data in the literature often present handling protocols with habituation periods exceeding 10 days9,16. Additionally, cupped-handling without the refined interaction provided by 3D-handling may not be suitable for jumpy strains that continue to jump out and away from the hands. While we did not do a direct comparison in this study to the cup method, the 3D-handling addresses this and relies on refined moves to foster interaction between the mouse and the handler. The study by Ghosal et al.16 used a cup-handling technique combined with massage for 5 days, and showed this technique limits the impact of stress on metabolic endpoints, highlighting the need for refined moves and interaction during handling for better efficacy. Based on this cup-massage technique, the 3D-handling uses additional interaction to habituate mice. Using the 3D-handling approach, handlers ensure that all mice reach a similar level of habituation by performing standardized moves and by adapting the duration of the procedure to each animal depending on its need (in the present study, all mice passed the milestones and finished the 3D-handling protocol in 3 days). This approach can be considered &#34;personalized&#34; to each mouse, so all animals reach the desired level of habituation on each day of handling. As mentioned earlier, if animals do not reach the milestones described in the protocol, this technique can be adjusted by increasing the number of days. This technique showed beneficial effects for reducing variability between animals in behavioral studies and physiological measurement (CORT levels), suggesting that this approach could contribute to the reduction of intra-study variability and reduce the impact of experimental error potentially driving biased results in preclinical studies.
Supporting results suggested that mice subjected to 3D- and tunnel handling exhibit reduced anxiety in the novelty suppressed feeding test, compared to tail-handled mice. Considering combined data from the NSF and the EPM, both approaches showed significant effects at reducing anxiety in 6-month-old male mice. This replicates the findings that animals habituated to tunnel handling had improved performance in tests for anxiety9,15 after 10+ days of handling, and further demonstrate the potential of the 3D-handling to exhibit similar effects. This also showed that 3D-handled 6-month-old male mice approach and voluntarily interact more with their experimenter than 6-month-old male mice subjected to tunnel and tail handling. Importantly, 2.5-month-old female mice subjected to 3D-handling had reduced levels of CORT, which is in agreement with previously published results9. The two studies (Study #1 in 6 month old males and Study #2 in 2.5 month old females) confirmed, in two different ways that the handling has beneficial impact on anxiety-like phenotypes (either on behavioral outcomes in Study #1, or on CORT levels in Study #2).
A possible contributing factor to the effect is the sex of the experimenter, in this case male. It has been shown by Sorge et al.25&#160; that the presence of male experimenters can lead to an increase in CORT and anxiety like behaviors in male but not female mice. This is in contrast with results from the present study. The major difference between this study and the study from Sorge et al.25 is that the approach described here consists on habituating the mice to handling, by fostering positive (non-reinforced) interaction with the experimenter, while Sorge et al.25 used na&#239;ve rodents that never interacted with human beings. One can expect that na&#239;ve mice could have a strong reaction against human experimenters if they do not learn that the experimenter does not represent a threat. However, the present study was only performed with a male experimenter, and future studies should investigate if such effects are reproducible with a female experimenter. Though isolating these factors is outside the scope of this paper, it is worth highlighting the importance of identifying such sources of variability when implementing handling habituation, or in experimental design more generally.
The present study also confirmed efficacy of the tunnel-handling technique at reducing anxiety-like behaviors and CORT levels in mice9,10,11. An additional benefit of this approach is that the tunnel can be left in the cage as enrichment26, which may also contribute to a reduced stress/anxiety response, altogether contributing to improved welfare10,11. In this case, the role of the experimenter is to manipulate the tunnel only, with each animal, for one minute. However, as described by Gouveia et al19, the tunnel does not necessarily need to remain in the home cage and instead can be presented to the animals only when required to transfer the animal, without causing additional stress. Both approaches, the tunnel and 3D-handling techniques, offer benefits that should be assessed by the lab and the experimenters in order to determine which approach is the most appropriate for their needs. In the present study, the tunnel was left in the cage, and the effects we observed on anxiety-like behaviors may be due to a combination of tunnel handling and enrichment.
While both provide beneficial effects, the 3D- and tunnel-handling techniques are not without limitations. A shared limitation is that it can be time consuming and potentially discouraging for animal facilities to implement such procedures. However, the added benefits are invaluable, improving animal welfare by reducing stress and improving interaction with experimenter and animal care providers (as described in Spangenberg and Kelling27), and research reliability and reproducibility. Evidence from our facility suggests that this technique improves interactions between animals and husbandry staff, facilitating cage change and health monitoring. From other users in our facility, contention and overall manipulation are reported as being significantly easier with handled mice, consistent with our findings that mice handled with the 3D-technique are less likely to flee when being picked up and in our example, 6-month-old males are more prone to interact with their experimenter. Follow up studies could quantify such effects to demonstrate further the usefulness of the technique. Altogether, this 3D-handling approach, as well as the tunnel-approach, contributes to the rule of the 3Rs, particularly by refining routine animal interactions to minimize the stress in response to handling. Given the observed reduction in variability of data, this also has the potential to reduce the number of animals needed to obtain consistent results and refining the approach used to limit variability.
Another point of discussion based on the data presented is that this study was performed with animals being single-housed. Single housing was preferred as it limits the potential agonistic behaviors (particularly in male mice), that can contribute to inter-individual variability28,29. For consistency between groups, all animal were single housed. It is also interesting to note that positive experimenter-animal interactions in rats in the form of rat tickling, was able to mitigate some of the effects of social isolation in single housed rats30,31. It is possible that handling techniques involving direct contact between animal and experimenter, such as the 3D-handling technique or the cup-massage technique described by Ghosal et al.11 could have a similar effect. Future studies could explore this question by comparing the effects of handling techniques in single and group housed animals. Past studies investigated the impact of cup and tunnel handling approaches with mice in a group-housed setting, and obtained similar results7,8. This confirms that it is possible to use the handling protocols described herein with animals kept in single-house or group-house conditions, keeping in mind the possibility of agonistic behavior when taking one animal out of the cage and placing it back in (particularly in male mice, or in aggressive mouse lines). In such cases, it is recommended to use a temporary cage before regrouping all the animals together.
To conclude, the proposed 3D-handling approach contributes to reducing reactivity and stress in mice. It also increases data reliability by reducing variability after 3 days of handling. Similar results are observable with the tunnel handling, in our case after 10 days of tunnel handling. In comparison with the tunnel handling technique, the 3D-handling technique provided the benefit of increasing interaction with an experimenter in our 6-month-old male mice, which can be critical in some cases. If the 3D- or tunnel handling technique were to be implemented in all animal facilities that would represent a major improvement for data generation and would greatly contribute to the reduction of animal use in research.

Mice and rats are essential assets to preclinical studies1,2 for multiple purposes, including endocrinal, physiological, pharmacological or behavioral studies2. From the increasing number of studies involving animals, it arose that uncontrolled environmental variables including human interaction influence various outcomes in biomedical research3,4,5. This is responsible for significant variability observed across experiments and research laboratories4,5, posing a major caveat in animal research.
Various approaches have been implemented with the goal of limiting the impact of environmental stressors and reducing reactivity to human interaction. For example, to limit the impact of environmental stressors, standardization of housing conditions and automated housing systems6,7 have been implemented across laboratories. Regarding interaction with human beings, commonly used approaches for handling and transporting animals had little regard for animal discomfort and stress. For instance, picking up animals by their tail or using forceps8 increases baseline anxiety9,10,11, reduces exploration9,12 and contributes greatly to inter-individual variability within and across studies13,14. As a result, other approaches were developed, such as the cup handling technique, which is applicable to mice and rats. In this approach, the animals are &#34;cupped&#34; out of their cage, and held by the experimenters with their hands forming a cup9,10,11. Another useful alternative to tail handling involves the use of a polycarbonate tunnel to transfer mice9,10,15. This approach eliminates direct interaction between the mouse and the experimenter. Both the cup and tunnel approaches showed efficacy in reducing anxiety-like behaviors and fear of the experimenter that can be exaggerated by aversive handling techniques, such as tail pick up/tail handling9,10.
Therefore, increasing evidence demonstrates the usefulness of proper mouse handling for reducing variability between individuals9,11, and improving animal welfare10. However, the techniques mentioned above are still faced with limitations. The cup handling technique has been implemented with schedules ranging from 10 days (10 sessions over 2 weeks16) up to 15 weeks17, which is a considerable amount of time for facility staff and experimenters. Additionally, the effectiveness of cup handling varies by strain9 and conventional cup handling in open hands may lead to na&#239;ve mice or particularly jumpy strains to jump from the hand9,18. Tunnel handling results in more consistent and generally quicker results in gentling19. Tunnels are also used as home cage enrichment. They help animals habituate to handling quickly and provide the added benefits of enrichment. Tunnel handling, however, has limitations when transferring animals between apparatuses. Interestingly, Hurst and West9, and Henderson et al.20 demonstrated that using gentle and brief manual handling to transfer animals from the tunnel to the apparatus does not affect their phenotype.
To provide an alternative to existing methods, with achievable habituation in a short period of time, this article describes a novel technique that expands on the cup handling technique, therefore requiring no particular equipment. This approach uses milestones to gauge the level of comfort mice have with the handling process. It shows efficacy at decreasing mouse reactivity and stress (at the behavioral and hormonal levels), facilitates routine handling and contributes to reducing variability between animals. Details of this technique are provided here, and its efficacy at reducing anxiety-like behaviors, improving interaction with experimenters, and limiting peripheral stress-hormone (corticosterone) release are demonstrated in two separate studies (male and female mice), in comparison with tunnel handling (positive control) and tail handling techniques (negative control).

<table><tbody><tr><td>23 G x 1 in. BD PrecisionGlide general use sterile hypodermic needle. Regular wall type and regular bevel.</td><td>BD</td><td>2546-CABD305145</td><td>Needles for Blood collection</td></tr><tr><td>BD Vacutainer&amp;reg; Venous Blood Collection EDTA Tubes with Lavender BD Hemogard&amp;trade; closure, 2.0ml (13x75mm), 100/pk</td><td>BD</td><td>367841</td><td>EDTA Coated tubes for blood collection</td></tr><tr><td>Bed&amp;rsquo;o cobs &amp;frac14;&amp;rdquo; Corn cob laboratory animal bedding</td><td>Bed-O-Cobs</td><td>BEDO1/4</td><td>Novel bedding for novelty suppressed feeding</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>Centrifuge 5424 R</td><td>For centrifugation of blood.</td></tr><tr><td>Corticosterone ELISA Kit</td><td>Arbor Assays</td><td>K003-H1W</td><td></td></tr><tr><td>Digital Camera</td><td>Panasonic</td><td>HC-V770</td><td>Camera to record EPM/Experimenter interactions</td></tr><tr><td>Elevated Plus Maze</td><td>Home Made</td><td>n/a</td><td>Custom Maze made of four black Plexiglas arms (two open arms (29cm long by 7 cm wide) and two enclosed arms (29 cm long x7 cm wide with 16 cm tall walls)) that form a cross shape with the two open arms opposite to each other held 55 cm above the floor</td></tr><tr><td>Ethanol</td><td>Medstore House Brand</td><td>39753-P016-EA95</td><td>Dilute to 70% with Distilled water, for cleaning</td></tr><tr><td>Ethovision XT 15</td><td>Noldus</td><td>n/a</td><td>Automated animal tracking software</td></tr><tr><td>Laboratory Rodent Diet</td><td>LabDiet</td><td>Rodent Diet 5001</td><td>Standard Rodent diet</td></tr><tr><td>Memory Card</td><td>Kingstone Technology</td><td>SDA3/64GB</td><td>For video recording and file transfer</td></tr><tr><td>Novelty Suppressed Feeding Chamber</td><td>Home Made</td><td>n/a</td><td>Custom test plexiglass test chamber with clear floors and walls 62cm long, by 31cm wide by 40cm tall .</td></tr><tr><td>Parlycarbonate tubes</td><td>Home Made</td><td>n/a</td><td>13 cm in length and 5cm in diameter</td></tr><tr><td>Purina Yesterday&amp;rsquo;s news recycled newspaper bedding</td><td>Purina</td><td>n/a</td><td>Standard Bedding</td></tr><tr><td>Spectrophotometer</td><td>Biotek</td><td>Epoch Microplate Reader</td><td></td></tr></tbody></table>

handling techniques to reduce stress in mice

Laboratory animals are subjected to multiple manipulations by scientists or animal care providers. The stress this causes can have profound effects on&#160;animal well-being and can also be a confounding factor for experimental variables such as anxiety measures. Over the years, handling techniques that minimize handling-related stress have been developed with a particular focus on rats, and little attention to mice. However, it has been shown that mice can be habituated to manipulations using handling techniques. Habituating mice to handling reduces stress, facilitates routine handling, improves animal wellbeing, decreases data variability, and improves experimental reliability. Despite beneficial effects of handling, the tail-pick up approach, which is particularly stressful, is still widely used. This paper provides a detailed description and demonstration of a newly developed mouse-handling technique intended to minimize the stress experienced by the animal during human interaction. This manual technique is performed over 3 days (3D-handling technique) and focuses on the animal&#39;s capacity to habituate to the experimenter. This study also shows the effect of previously established tunnel handling techniques (using a polycarbonate tunnel) and the tail-pick up technique. Specifically studied are their effects on anxiety-like behaviors, using behavioral tests (Elevated-Plus Maze and Novelty Suppressed Feeding), voluntary interaction with experimenters and physiological measurement (corticosterone levels). The 3D-handling technique and the tunnel handling technique reduced anxiety-like phenotypes. In the first experiment, using 6-month-old male mice, the 3D-handling technique significantly improved experimenter interaction. In the second experiment, using 2.5-month-old female, it reduced corticosterone levels. As such, the 3D-handling is a useful approach in scenarios where interaction with the experimenter is required or preferred, or where tunnel handling may not be possible during the experiment.

Two separate studies were performed with C57BL/6 mice. Study #1 included 6-month-old males and Study #2 included 2.5-month-old females (N=36/study) from Jackson Laboratories (Cat #000664). Mice arrived in the facility at the age of 2 months. While Study #2 females were handled and tested two weeks after arrival, Study #1 males were only handled and tested at the age of 6 months (delay due to global pandemic shutdown). During this time, one mouse from Study #2 died, prior to starting handling experiments. The Study #1 male mice were cared for by animal facility staff. All mice were maintained on a 12 hour light/dark cycle (7:00 ON, 19:00 OFF), given access to food and water ad libitum. Their home cage was filled with recycled newspaper as bedding material, as well as nesting material. Mice were housed individually, in order to limit potential agonistic behavior in group-housed males during handling session or after procedures such as blood collection or behavioral testing. Mice were randomized into three groups: tail handling, tunnel handling and 3D-handling, and handled in the open-room according&#160;to the design of their respective group (Figure 2). The tunnel-handled group received the tunnel as an enrichment for 1 week prior to handling session. They were then handled for ten (10) consecutive days, prior to behavioral testing. One week after completion of the different handling sessions, behavioral testing commenced. On day 16, mice were tested in the EPM, and then in the experimenter interaction test. Two days later, mice were tested in the NSF. Finally, on day 24, blood was drawn 15 min after a one-minute handling session of the same type as the initial handling.
For behavioral testing, tunnel-handled animals were transferred from their cage to the apparatus using the tunnel as much as possible. However, for the Elevated-Plus Maze experiment, the dimensions of the maze made it difficult to remove or place animals in the maze using the tunnel. In this case, animals were transferred from tunnels to cupped hands, and transported to the maze. 3D-handled mice were handled over the three days, concurrent with days 8-10 of tunnel handling (Figure 2). Tail handled mice were not habituated to handling but were tail handled during interactions with experimenters. During the time of the study, cage change was performed by the experimenter to ensure the use of the appropriate handling technique used for each group.
In the experimenter interaction test, animals were tested for their willingness to voluntarily interact with the experimenter and the ease of handling in an experimental context (Figure 3). ANOVA performed on the number of attempts to pick up the mouse from the cage showed a significant effect of the handling approach in Study #1 males (F(2,31)=6.36, p=0.004), and in Study #2 females (F(2,33)=12.21, p=0.0001). Scheffe&#39;s post hoc analyses revealed that the number of attempts required to pick up the mice was significantly reduced by both 3D (p=0.0061 in Study #1 males, and p=0.0002 in Study #2 females) and tunnel handling (p=0.04 in Study #1 males, and p=0.003 in Study #2 females), in comparison to the tail handled group (Figure 3A). ANOVA performed on the time spent in the same quadrant as the hand showed significant effect of handling in Study #1 males (F(2,31)=5.38, p=0.009), and in Study #2 females (F(2,33)=3.5, p=0.04; Figure 3B). Scheffe&#39;s post hoc analyses showed that Study #1 male mice handled with the 3D-handling technique spent significantly more time in the same quadrant than the experimenter&#39;s hand, compared to tail-handled mice (p=0.012). There were no significant differences between handling groups in Study #2, 2.5 month-old females. Degree of interaction with the experimenter is further demonstrated by the combined heat-maps of the center points of the mice (Figure 3C-E). These illustrate how the 3D-handled male mice from Study #1 spent more time proximal to the hand, including areas near the hand, while tail handled mice had the least overall interaction with the hand.
The effects of the 3D- and tunnel handling were compared to tail handling in two tests of anxiety-like behaviors, the novelty suppressed feeding (NSF) test and the elevated plus maze (EPM). In the NSF test, ANOVA performed on the latency to approach showed an effect of handling technique used in Study #1 males (F(2,31)=3.5, p=0.04). Scheffe&#39;s post hoc analyses in Study #1 males showed trends from 3D-handled mice (p=0.08), and from the tunnel-handled mice (p=0.08), with reduced latency to approach compared to tail handled mice (Figure 4A). No effects were observed in Study #2. ANOVA performed on the latency to approach in the mouse home cage (data not shown) showed no effect of handling (p=0.88 in Study #1 males, and p=0.16 in Study #2 females). ANOVA performed on the percent time in the open arms in the EPM revealed a significant effect of handling in Study #2 females (F(2,33)=3.5, p=0.04). No effects were observed in Study #1 males (F(2,31)=2.1, p=0.1; Figure 4B). Scheffe&#39;s post hoc analyses only revealed a trend towards increased time spent in the open arms in tunnel handled mice from Study #2, compared to tail handled mice (p=0.07). Regarding the percent entries in the open arms (Figure 4C), ANOVA revealed no effect of handling, neither in Study #1 males nor in Study #2 females (F(2,31)=1.12, p=0.33; and F(2,33)=1.3, p=0.26, respectively). Behavioral scores were summarized in a z-score, as in Guilloux et al.23, informing on potential reduction of anxiety-like behaviors compared to tail handled mice (Figure 4D). ANOVA on the z-scores showed a significant effect of handling in Study #1 males (F(2,31)=5.6, p=0.008) but not in Study #2 females (F(2,33)=1.07, p=0.35). Scheffe&#39;s post hoc analyses showed that 3D-handling and tunnel handling significantly decreased z-score (p=0.04 and 0.01, respectively), compared to tail handling, suggesting that both approaches reduces anxiety-like behaviors in Study #1 males.
Corticosterone levels after handling were also assessed 15 minutes after a brief handling session (Figure 5). ANOVA found a significant effect of handling in Study #2 females (F(2,33)=4.44, p=0.01), but not in Study #1 males (F(1,31)=0.53, p=0.59). In Study #2 females, post hoc analyses revealed a significant decrease in corticosterone levels in mice from the 3D-handling group compared to the tail handling group (p=0.02).
To determine if the handling techniques had a significant impact on the variability of data obtained, we applied Bartlett&#39;s test of homogeneity of variance. Our results found no significant difference in variability in the Study #2 female mice across measurements (% time EPM B(2,33)=4.95, p=0.087; % Entries EPM B(2, 33)=3.68, p=0.16; NSF B(2, 33)=0.20, p=0.91; CORT B(2, 33)=1.69, p=0.42). However, in Study #1 male mice, there was a significant heterogeneity of variance in the NSF test (B(2,31)=8.08, p=0.0175) and in measured CORT levels (B(2,32)=11.63, p=0.0029), but not in either of the measures for EPM (% time EPM B(2,32)=1.16, p=0.56; % Entries EPM B(2,32)=2.79, p=0.25). Using the F-test to compare two variances showed that in the NSF test variance was significantly reduced for Study #1 males by both the 3D (F(1,21)=4.22, p=0.04) and tunnel handling techniques (F(1,22)=4.01;p=0.03) in comparison to tail handling. For the concentration of CORT after handling, only 3D-handling significantly reduced variability (F(1,20)=9.65, p=0.0019) in comparison to tail handling.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62593/62593fig01.jpg" /> 
Figure 1. Representative images of the 3D-Handling Procedure.&#160;&#160;The images illustrate the 3D-handling procedure. A) Hand in cage: The experimenter&#39;s hand is placed in the cage and kept still, allowing the mouse to habituate to the presence of the hand in the cage. B) Flat hand: upon first removal from the cage, the mouse is placed on the flat palm of the hand. The mouse can freely walk around the palm and move between adjacent flat hands. C) Roll: Relax palm of the hand to form a loose &#34;cup&#34; around the mouse. Gently tilt the cup into the opposite hand the mouse should freely move to this hand, if not gently guide it into the other hand. D)&#160;Shelter: position the mouse at the edge of the hand then bring both hands together and very slowly form cup around the mouse. The mouse should not be restrained and an opening should be left so the mouse may escape. Hold for ~5-10 s and then open to flat hands. E) Head/Back Petting: While the mouse is exploring the flat palm of the hand, gently pet the mouse on the head and back. This habituates the mouse to the approach of the experimenter from above. F) Nose Poke: When the mouse appears to be habituated to handling, attempt to gently touch the mouse directly on the snout. If the mouse does not move its head away it is well habituated to handling. G) It is possible to perform a short (2-3 s) neck pinch on the last day, to measure the habituation of the animals in the event of future interventions requiring contention. When habituated to handling, mice remain immobile during the neck pinch, while non-habituated mice will try to escape by rotating their tail to get freed from the contention.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62593/62593fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62593/62593fig02.jpg" /> 
Figure 2. Experimental Design.&#160; After arrival in the facility, tail handled mice received no habituation. Tunnel-handled mice were habituated to the tunnels in their home cage for one week before the start of handling. Tunnel handled mice were handled with the tunnel handling technique for 10 days (First day of handling = Day 1), while 3D-handled mice were habituated for three days (Day 8-10). Mice were then subjected to the elevated plus maze (EPM) (Day 16), experimenter interaction test (Day 19), novelty suppressed feeding (Day 21), and a brief handling session followed by serum collection for CORT measurement (Day 24).&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62593/62593fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62593/62593fig03.jpg" /> 
Figure 3. Impact of the three handling techniques on ease of handling and willingness to interact with experimenter.&#160; A) Average number of pick up attempts required to remove a mouse from the cage. Study #1 Male (left panel, Tail Handling N= 12, Tunnel Handling N=12 and 3D handling N=11) and Study #2 female (right panel, N=12 per group) mice from both tunnel and 3D-handled groups displayed a significant reduction in the number of attempts required to remove them from the cage compared to tail handled mice. B) Average amount of time spent by an animal in the same quadrant of the cage as the experimenter&#39;s hand. Study #1 male mice handled with the 3D-technique showed a significant increase in time spent in the same quadrant as the experimenter&#39;s hand. C-E) Average heat-maps of mouse center-point by time rendered in Ethovision XT 14, visually demonstrated the increased exploration and interaction with experimenter of the Study #1 3D-handled male mice. Error bars indicate SEM. *p&#60;0.05, **p&#60;0.01 compared to Tail Handled group.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62593/62593fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62593/62593fig4v4.jpg" /> 
Figure 4. Impact of the three handling techniques on anxiety-like behaviors.&#160; A) Latency to approach and feed on the pellet in the novelty suppressed feeding chamber in Study #1 male mice (Tail Handling N= 12, Tunnel Handling N=12 and 3D handling N=11) and in Study #2 female mice (N=12/group). Data from the Study #1 male mice in the 3D-handling and tunnel-handling groups showed a trend towards significant reduction of latency to approach the pellet. B) Means of % of time spent in the open arms of the elevated plus maze. There were no significant differences between groups in Study #1 males, and a trend towards more time in open arms by Study #2 females in the tunnel-handling group. C) Entries in the open arms: There were no significant differences between groups in Study #1 males, nor in Study #2 females. D) Z-score summarizing the anxiety-like behaviors. Using the data presented in A, B and C, a z-score was calculated using the Tail-handled mice as reference. Decrease in the z-score suggests a decrease in anxiety-like behaviors measured by the NSF and EPM tests. Study #1 male mice handled using the 3D- or tunnel technique showed a reduced anxiety-like phenotype compared to Tail-handled mice. Error bars indicate SEM. *p&#60;0.05 comparison to tail-handled group. t depicts trending level of significance (p&#60;0.1) compared to tail handled group.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62593/62593fig4v3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62593/62593fig05.jpg" /> 
Figure 5. Levels of corticosterone after handling.&#160; Serum was collected 15 min&#160;after a brief handling session and then CORT levels were measured by ELISA in both Study #1 male (Tail Handling N= 12, Tunnel Handling N=12 and 3D handling N=11) and in Study #2 female mice (N=12/group). Study #2 female mice handled via the 3D-handling technique showed reduced corticosterone levels compared to mice handled by the tail. ANOVA in Study #1 male mice did not reach significance for differences between groups (p=0.5). Error bars indicate SEM. *p&#60;0.05 compared to Tail Handled group.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62593/62593fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/62593/62593tbl1.jpg" /> 
Table 1.

Watch this Scientific Journal Video about Handling Techniques to Reduce Stress in Mice at JoVE.com

Handling Techniques to Reduce Stress in Mice

This paper describes a handling technique in mice, the 3D-handling technique, which facilitates routine handling by reducing anxiety-like behaviors and presents details on two existing related techniques (tunnel and tail handling).

Laboratory animals are subjected to multiple manipulations by scientists or animal care providers. The stress this causes can have profound effects ...

handling-techniques-to-reduce-stress-in-mice

Nanyang Technological University

Research

JoVE Journal

Behavior

37.5K Views.  Campbell Family Mental Health Research Institute of CAMH. Mijn naam is Dr.Thomas Prevot. Ik ben wetenschapper in het Center for Addiction and Mental Health, of CAMH, en een universitair docent aan de Universiteit van Toronto in het departement Psychiatrie. Mijn fractie heeft een bijzondere interesse in het verminderen van angst en reactiviteit bij muizen bij de uitgangssituatie om de gegevensvariabiliteit te verminderen en het dierenwelzijn te verbeteren.Muizen hebben bewezen van onschatbare waarde te zijn in basiswetenschap en preklinisch on...

Video: Handling Techniques to Reduce Stress in Mice

A Mouse Model of Subchronic and Mild Social Defeat Stress for Understanding Stress-induced Behavioral and Physiological Deficits

Stressful life events often increase the incidence of depression in humans. To study the mechanisms of depression, the development of animal models of depression is essential. Because there are several types of depression, various animal models are needed for a deeper understanding of the disorder. Previously, a mouse model of subchronic and mild social defeat stress (sCSDS) using a modified chronic social defeat stress (CSDS) paradigm was established. In the paradigm, to reduce physical injuries from aggressors, the duration of physical contact between the aggressor and a subordinate was reduced compared to in the original CSDS paradigm. sCSDS mice showed increased body weight gain, food intake, and water intake during the stress period, and their social behaviors were suppressed after the stress period. In terms of the face validity of the stress-induced overeating and overdrinking following the increased body weight gain, the sCSDS mice may show some features related to atypical depression in humans. Thus, a mouse model of sCSDS may be useful for studying the pathogenic mechanisms underlying depression. This protocol will help establish the sCSDS mouse model, especially for studying the mechanisms underlying stress-induced weight gain and polydipsia- and hyperphagia-like symptoms.

Here, methods for developing a mouse model of subchronic and mild social defeat stress are described and used to investigate the pathogenic features of depression including hyperphagia- and polydipsia-like symptoms following increased body weight.

Stressful life events often increase the incidence of depression in humans. To study the mechanisms of depression, the development of animal models of ...

Systematic Assessment of Well-Being in Mice for Procedures Using General Anesthesia

In keeping with the 3R Principle (Replacement, Reduction, Refinement) developed by Russel and Burch, scientific research should use alternatives to animal experimentation whenever possible. When there is no alternative to animal experimentation, the total number of laboratory animals used should be the minimum needed to obtain valuable data. Moreover, appropriate refinement measures should be applied to minimize pain, suffering, and distress accompanying the experimental procedure. The categories used to classify the degree of pain, suffering, and distress are non-recovery, mild, moderate, or severe (EU Directive 2010/63). To determine which categories apply in individual cases, it is crucial to use scientifically sound tools.
The well-being-assessment protocol presented here is designed for procedures during which general anesthesia is used. The protocol focuses on home cage activity, the Mouse Grimace Scale, and luxury behaviors such as burrowing and nest building behavior as indicators of well-being. It also uses the free exploratory paradigm for trait anxiety-related behavior. Fecal corticosterone metabolites as indicators of acute stress are measured over the 24-h post-anesthetic period.
The protocol provides scientifically solid information on the well-being of mice following general anesthesia. Due to its simplicity, the protocol can easily be adapted and integrated in a planned study. Although it does not provide a scale to classify distress in categories according to the EU Directive 2010/63, it can help researchers estimate the degree of severity of a procedure using scientifically sound data. It provides a way to improve the assessment of well-being in a scientific, animal-centered manner.

We developed a protocol to assess well-being in mice during procedures using general anesthesia. A series of behavioral parameters indicating levels of well-being as well as glucocorticoid metabolites were analyzed. The protocol can serve as a general aid to estimate the degree of severity in a scientific, animal-centered manner.

In keeping with the 3R Principle (Replacement, Reduction, Refinement) developed by Russel and Burch, scientific research should use alternatives to animal ...

The Unpredictable Chronic Mild Stress Protocol for Inducing Anhedonia in Mice

Depression is a highly prevalent and debilitating condition, only partially addressed by current pharmacotherapies. The lack of response to treatment by many patients prompts the need to develop new therapeutic alternatives and to better understand the etiology of the disorder. Pre-clinical models with translational merits are rudimentary for this task. Here we present a protocol for the unpredictable chronic mild stress (UCMS) method in mice. In this protocol, adolescent mice are chronically exposed to interchanging unpredictable mild stressors. Resembling the pathogenesis of depression in humans, stress exposure during the sensitive period of mice adolescence instigates a depressive-like phenotype evident in adulthood. UCMS can be used for screenings of antidepressants on the variety of depressive-like behaviors and neuromolecular indices. Among the more prominent tests to assess depressive-like behavior in rodents is the sucrose preference test (SPT), which reflects anhedonia (core symptom of depression). The SPT will also be presented in this protocol. The ability of UCMS to induce anhedonia, instigate long-term behavioral deficits and enable reversal of these deficits via chronic (but not acute) treatment with antidepressants strengthens the protocol&#39;s validity compared to other animal protocols for inducing depressive-like behaviors.

Here we present the unpredictable chronic mild stress protocol in mice. This protocol induces a long-term depressive-like phenotype and enables to assess the efficacy of putative antidepressants in reversing the behavioral and neuromolecular depressive-like deficits.

Depression is a highly prevalent and debilitating condition, only partially addressed by current pharmacotherapies. The lack of response to treatment by ...

A Chronic Immobilization Stress Protocol for Inducing Depression-Like Behavior in Mice

Depression is not yet fully understood, but various causative factors have been reported. Recently, the prevalence of depression has increased. However, therapeutic treatments for depression or research on depression is scarce. Thus, in the present paper, we propose a mouse model of depression induced by movement restriction. Chronic mild stress (CMS) is a well-known technique to induce depressive-like behavior. However, it necessitates a complex procedure consisting of a combination of various mild stresses. In contrast, chronic immobilization stress (CIS) is a readily accessible chronic stress model, modified from a restraint model that induces depressive behavior by restricting movement using a restrainer for a certain period. To evaluate the depressive-like behaviors, the sucrose preference test (SPT), the tail suspension test (TST), and the ELISA assay to measure stress marker corticosterone levels are combined in the present experiment. The described protocols illustrate the induction of CIS and evaluation of the changes in behavior and physiological factors for the validation of depression.

This article provides a simplified and standardized protocol for induction of depressive-like behavior in chronically immobilized mice by using a restrainer. In addition, behavior and physiological techniques to verify induction of depression are explained.

Depression is not yet fully understood, but various causative factors have been reported. Recently, the prevalence of depression has increased. However, ...

Chronic Stress Shifts Effort-Related Choice Behavior in a Y-Maze Barrier Task in Mice

Mood disorders, including major depressive disorder, can be precipitated by chronic stress. The Y-maze barrier task is an effort-related choice test that measures motivation to expend effort and obtain reward. In mice, chronic stress exposure significantly impacts motivation to work for a higher value reward when a lesser value reward is freely available compared to unstressed mice. Here we describe the chronic corticosterone administration paradigm, which produces a shift in effortful responding in the Y-maze barrier task. In the Y-maze task, one arm contains 4 food pellets, while the other arm contains only 2 pellets. After mice learn to select the high reward arm, barriers with progressively increasing height are then introduced into the high reward arm over multiple test sessions. Unfortunately, most chronic stress paradigms (including corticosterone and social defeat) were developed in male mice and are less effective in female mice. Therefore, we also discuss chronic non-discriminatory social defeat stress (CNSDS), a stress paradigm we developed that is effective in both male and female mice. Repeating results with multiple distinct chronic stressors in male and female mice combined with increased usage of translationally relevant behavior tasks will help to advance the understanding of how chronic stress can precipitate mood disorders.

The Y-maze barrier task is a behavior test that examines motivation to expend effort for reward. Here, we discuss testing multiple well-validated chronic stressors including chronic corticosterone and social defeat stress with this behavior, as well as the novel chronic non-discriminatory social defeat stress (CNSDS), which is effective in females.

Mood disorders, including major depressive disorder, can be precipitated by chronic stress. The Y-maze barrier task is an effort-related choice test that ...

Using a Murine Model of Psychosocial Stress in Pregnancy as a Translationally Relevant Paradigm for Psychiatric Disorders in Mothers and Infants

The peripartum period is considered a sensitive period where adverse maternal exposures can result in long-term negative consequences for both mother and offspring, including the development of neuropsychiatric disorders. Risk factors linked to the emergence of affective dysregulation in the maternal-infant dyad have been extensively studied. Exposure to psychosocial stress during pregnancy has consistently emerged as one of the strongest predictors. Several rodent models have been created to explore this association; however, these models rely on the use of physical stressors or a limited number of psychosocial stressors presented in a repetitive fashion, which do not accurately capture the type, intensity, and frequency of stressors experienced by women. To overcome these limitations, a chronic psychosocial stress (CGS) paradigm was generated that employs various psychosocial insults of different intensity presented in an unpredictable fashion. The manuscript describes this novel CGS paradigm where pregnant female mice, from gestational day 6.5 to 17.5, are exposed to various stressors during the day and overnight. Day stressors, two per day separated by a 2 h break, range from exposure to foreign objects or predator odor to frequent changes in bedding, removal of bedding, and cage tilting. Overnight stressors include continuous light exposure, changing cage mates, or wetting bedding. We have previously shown that exposure to CGS results in the development of maternal neuroendocrine and behavioral abnormalities, including increased stress reactivity, the emergence of fragmented maternal care patterns, anhedonia, and anxiety-related behaviors, core features of women suffering from perinatal mood and anxiety disorders. This CGS model, therefore, becomes a unique tool that can be used to elucidate molecular defects underlying maternal affective dysregulation, as well as trans-placental mechanisms that impact fetal neurodevelopment and result in negative long-term behavioral consequences in the offspring.

The chronic psychosocial stress (CGS) paradigm employs clinically relevant stressors during pregnancy in mice to model psychiatric disorders of mothers and infants. Here, we provide a step-by-step procedure of applying the CGS paradigm and downstream assessments to validate this model.

The peripartum period is considered a sensitive period where adverse maternal exposures can result in long-term negative consequences for both mother and ...

Social Isolation Model: A Noninvasive Rodent Model of Stress and Anxiety

Anxiety disorders are one of the leading causes of disability in the United States (US). Current treatments are not always effective and less than 50% of patients achieve full remission. A critical step in developing a novel anxiolytic is to develop and utilize an animal model, such as mice, to study pathological changes and test drug target(s), efficacy, and safety. Current approaches include genetic manipulation, chronic administration of anxiety-inducing molecules, or the administration of environmental stress. These methods, however, may not realistically reflect anxiety induced throughout daily life. This protocol describes a novel anxiety model, which mimics the intentional or unintentional patterns of social isolation in modern life. The social isolation-induced anxiety model minimizes perceived distractions and invasiveness and utilizes wild type C57BL/6 mice. In this protocol, 6- to 8-week-old mice (male and female) are singly housed in opaque cages to visually block the external environment, such as neighboring mice, for 4 weeks. No environmental enrichments (such as toys) are provided, bedding material is reduced by 50%, any treatment of drug is administered as an agar form, and the exposure/handling of the mice is minimized. Socially isolated mice generated using this protocol exhibit greater anxiety-like behavior, aggression,&#160;as well as decreased cognition.

Presented here is a social isolation (SI)-induced anxiety mouse model that utilizes wild type C56BL/6J mice to induce stress and anxiety-like behavior with minimal handling and no invasive procedures. This model reflects modern life patterns of social isolation and is ideal for studying anxiety and related disorders.

Anxiety disorders are one of the leading causes of disability in the United States (US). Current treatments are not always effective and less than 50% of ...

Neuroscience

Adolescent Social Stress Paradigm in C57BL&#47;6 Mice for Behavioral Research

Social Defeat Stress Model for Adolescent C57BL&#47;6 Male and Female Mice

Social adversity in adolescence is prevalent and can negatively impact mental health trajectories. Modeling social stress in adolescent male and female rodents is needed to understand its effects on ongoing brain development and behavioral outcomes. The chronic social defeat stress paradigm (CSDS) has been widely used to model social stress in adult C57BL/6 male mice by leveraging on the aggressive behavior displayed by an adult male rodent to an intruder invading its territory. An advantage of this paradigm is that it allows to categorize defeated mice into resilient and susceptible groups based on their individual differences in social behavior 24 h after the last defeat session. Implementing this model in adolescent C57BL/6 mice has been challenging because adult or adolescent mice do not typically attack early adolescent male or female mice and because adolescence is a short period of life, encompassing discreet temporal windows of vulnerability. This limitation was overcome by adapting an accelerated version of the CSDS to be used for adolescent male and female mice. This 4-day stress paradigm with 2 physical attack sessions per day uses a C57BL/6 male adult to prime the CD-1 mouse for aggressiveness such that it readily attacks the male or female adolescent mouse. This model was termed accelerated social defeat stress (AcSD) for adolescent mice. Adolescent exposure to AcSD induces social avoidance 24 h later in both males and females, but only in a subset of defeated mice. This vulnerability occurs despite the number of attacks being consistent across sessions between resilient and susceptible groups. The AcSD model is short enough to allow exposure during discrete periods within adolescence, allows the segregation of mice according to the presence or absence of social avoidance behavior, and is the first model available to study social defeat stress in adolescent C57BL/6 female mice.

Author Spotlight: Understanding Adolescent Social Adversity Effects on Neurodevelopment in Mice

We developed an accelerated social defeat stress model for adolescent C57BL/6 mice, which works in both males and females and allows exposure during discrete adolescent periods. Exposure to this model induces social avoidance, but only in a subset of defeated male and female mice.

Social adversity in adolescence is prevalent and can negatively impact mental health trajectories. Modeling social stress in adolescent male and female ...

Impact of Restraint Stress on Physiology and Neurobiology in Rodent Models

Restraint to Induce Stress in Mice and Rats

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.

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.

Across all animal species, exposure to stressful conditions induces stress responses. One method to study the effects of stress using rodent models is the ...

Inducing Depression-like Behavior in a Mouse via Immobilization Stress

Source: Son, H., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/59546">A Chronic Immobilization Stress Protocol for Inducing Depression-Like Behavior in Mice.</a> J. Vis. Exp. (2019).
This video demonstrates the method of inducing chronic stress in a mouse through repeated immobilization, leading to corticosterone release and reduced glutamate receptor activity in neurons, which results in depression-like behavior.

Handling Techniques to Reduce Stress in Mice

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