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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Housing,+husbandry+and+handling+of+rodents+for+behavioral+experiments.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Mighty+Mouse:+the+impact+of+rodents+on+advances+in+biomedical+research.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+inherent+variability+and+experimental+parameters+on+the+reliability+of+small+animal+PET+data.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+Variability.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+the+cage+environment+on+rodent+physiology+and+behavior:+Implications+for+reproducibility+of+pre-clinical+rodent+research.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Non-invasive+Digital+Biomarker+for+the+Detection+of+Rest+Disturbances+in+the+SOD1G93A+Mouse+Model+of+ALS.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-cost+solution+for+rodent+home-cage+behaviour+monitoring.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+Handling+and+Restraint+Techniques.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taming+anxiety+in+laboratory+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+the+practicality+of+using+non-aversive+handling+methods+to+reduce+background+stress+and+anxiety+in+laboratory+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimising+reliability+of+mouse+performance+in+behavioural+testing:+the+major+role+of+non-aversive+handling.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+handling+limits+the+impact+of+stress+on+metabolic+endpoints.">Mouse handling limits the impact of stress on metabolic endpoints.</a> Physiology &amp; Behavior. 150, 31-37 (2015).</li><li>Wahlsten, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+data+from+different+labs:+lessons+from+studies+of+gene-environment+interaction.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Troublesome+variability+in+mouse+studies.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+handling+technique+and+handling+frequency+on+laboratory+mouse+welfare+is+sex-specific.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+handling+limits+the+impact+of+stress+on+metabolic+endpoints.">Mouse handling limits the impact of stress on metabolic endpoints.</a> Physiology &amp; Behavior. 150, 31-37 (2015).</li><li>Novak, J., Bailoo, J. D., Melotti, L., Rommen, J., W&#252;rbel, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Exploration+Based+Cognitive+Bias+Test+for+Mice:+Effects+of+Handling+Method+and+Stereotypic+Behaviour.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Handling+Tutorial.">Mouse Handling Tutorial.</a> NC3Rs. , (2016).</li><li>Gouveia, K., Hurst, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reducing+Mouse+Anxiety+during+Handling:+Effect+of+Experience+with+Handling+Tunnels.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+obstacles+preventing+the+uptake+of+tunnel+handling+methods+for+laboratory+mice:+An+international+thematic+survey.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ARRIVE+guidelines+2.0:+Updated+guidelines+for+reporting+animal+research.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid,+simple,+and+humane+method+for+submandibular+bleeding+of+mice+using+a+lancet.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+behavioral+z-scoring+increases+the+sensitivity+and+reliability+of+behavioral+phenotyping+in+mice:+relevance+to+emotionality+and+sex.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+Animal+Welfare+Meets+Human+Welfare:+A+Cross-Sectional+Study+of+Professional+Quality+of+Life,+Including+Compassion+Fatigue+in+Laboratory+Animal+Personnel.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+exposure+to+males,+including+men,+causes+stress+and+related+analgesia+in+rodents.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Cage+Enrichment+on+Behavior,+Welfare+and+Outcome+Variability+in+Female+Mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+the+welfare+of+laboratory+mice+in+their+home+environment+using+animal-based+measures+-+a+benchmarking+tool.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epidemiology+of+fighting+in+group-housed+laboratory+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aggression+in+group-housed+laboratory+mice:+why+can't+we+solve+the+problem.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Playful+handling+as+social+enrichment+for+individually-+and+group-housed+laboratory+rats.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=50-kHz+chirping+(laughter?)+in+response+to+conditioned+and+unconditioned+tickle-induced+reward+in+rats:+effects+of+social+housing+and+genetic+variables.">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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#174; Venous Blood Collection EDTA Tubes with Lavender BD Hemogard&#8482; 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&#8217;o cobs &#188;&#8221; 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&#8217;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/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/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/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/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/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

36.1K Views.  Campbell Family Mental Health Research Institute of CAMH. 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).

Video: Handling Techniques to Reduce Stress in Mice

Using Chronic Social Stress to Model Postpartum Depression in Lactating Rodents

Exposure to chronic stress is a reliable predictor of depressive disorders, and social stress is a common ethologically relevant stressor in both animals and humans. However, many animal models of depression were developed in males and are not applicable or effective in studies of postpartum females. Recent studies have reported significant effects of chronic social stress during lactation, an ethologically relevant and effective stressor, on maternal behavior, growth, and behavioral neuroendocrinology. This manuscript will describe this chronic social stress paradigm using repeated exposure of a lactating dam to a novel male intruder, and the assessment of the behavioral, physiological, and neuroendocrine effects of this model. Chronic social stress (CSS) is a valuable model for studying the effects of stress on the behavior and physiology of the dam as well as her offspring and future generations. The exposure of pups to CSS can also be used as an early life stress that has long term effects on behavior, physiology, and neuroendocrinology.

This article describes the use of chronic resident intruder social stress as an ethologically relevant paradigm to model postpartum depression and anxiety in lactating rodents.

Exposure to chronic stress is a reliable predictor of depressive disorders, and social stress is a common ethologically relevant stressor in both animals ...

Uncovering Beat Deafness: Detecting Rhythm Disorders with Synchronized Finger Tapping and Perceptual Timing Tasks

A set of behavioral tasks for assessing perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) is presented here with the goal of uncovering rhythm disorders, such as beat deafness. Beat deafness is characterized by poor performance in perceiving durations in auditory rhythmic patterns or poor synchronization of movement with auditory rhythms (e.g., with musical beats). These tasks include the synchronization of finger tapping to the beat of simple and complex auditory stimuli and the detection of rhythmic irregularities (anisochrony detection task) embedded in the same stimuli. These tests, which are easy to administer, include an assessment of both perceptual and sensorimotor timing abilities under different conditions (e.g., beat rates and types of auditory material) and are based on the same auditory stimuli, ranging from a simple metronome to a complex musical excerpt. The analysis of synchronized tapping data is performed with circular statistics, which provide reliable measures of synchronization accuracy (e.g., the difference between the timing of the taps and the timing of the pacing stimuli) and consistency. Circular statistics on tapping data are particularly well-suited for detecting individual differences in the general population. Synchronized tapping and anisochrony detection are sensitive measures for identifying profiles of rhythm disorders and have been used with success to uncover cases of poor synchronization with spared perceptual timing. This systematic assessment of perceptual and sensorimotor timing can be extended to populations of patients with brain damage, neurodegenerative diseases (e.g., Parkinson&#8217;s disease), and developmental disorders (e.g., Attention Deficit Hyperactivity Disorder).

Behavioral tasks that allow for the assessment of perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) are presented. Synchronization of finger tapping to the beat of an auditory stimuli and detecting rhythmic irregularities provides a means of uncovering rhythm disorders.

A set of behavioral tasks for assessing perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) is presented here ...

Mindfulness in Motion (MIM): An Onsite Mindfulness Based Intervention (MBI) for Chronically High Stress Work Environments to Increase Resiliency and Work Engagement

A pragmatic mindfulness intervention to benefit personnel working in chronically high-stress environments, delivered onsite during the workday, is timely and valuable to employee and employer alike. Mindfulness in Motion (MIM) is a Mindfulness Based Intervention (MBI) offered as a modified, less time intensive method (compared to Mindfulness-Based Stress Reduction), delivered onsite, during work, and intends to enable busy working adults to experience the benefits of mindfulness. It teaches mindful awareness principles, rehearses mindfulness as a group, emphasizes the use of gentle yoga stretches, and utilizes relaxing music in the background of both the group sessions and individual mindfulness practice. MIM is delivered in a group format, for 1 hr/week/8 weeks. CDs and a DVD are provided to facilitate individual practice. The yoga movement is emphasized in the protocol to facilitate a quieting of the mind. The music is included for participants to associate the relaxed state experienced in the group session with their individual practice. To determine the intervention feasibility/efficacy we conducted a randomized wait-list control group in Intensive Care Units (ICUs). ICUs represent a high-stress work environment where personnel experience chronic exposure to catastrophic situations as they care for seriously injured/ill patients. Despite high levels of work-related stress, few interventions have been developed and delivered onsite for such environments. The intervention is delivered on site in the ICU, during work hours, with participants receiving time release to attend sessions. The intervention is well received with 97% retention rate. Work engagement and resiliency increase significantly in the intervention group, compared to the wait-list control group, while participant respiration rates decrease significantly pre-post in 6/8 of the weekly sessions. Participants value institutional support, relaxing music, and the instructor as pivotal to program success. This provides evidence that MIM is feasible, well accepted, and can be effectively implemented in a chronically high-stress work environment.

Mindfulness in Motion MIM: An Onsite Mindfulness Based Intervention MBI for Chronically High Stress Work Environments to Increase Resiliency and Work Engagement

The Mindfulness in Motion (MIM) protocol offers a pragmatic Mindfulness Based Intervention (MBI) on-site, for persons working in chronically high-stress work environments that significantly increases resiliency and work engagement. The protocol has proven feasible, beneficial, and is easily adaptable to other high-stress workplaces.

A pragmatic mindfulness intervention to benefit personnel working in chronically high-stress environments, delivered onsite during the workday, is timely ...

The Social Dimension of Stress: Experimental Manipulations of Social Support and Social Identity in the Trier Social Stress Test

In many situations humans are influenced by the behavior of other people and their relationships with them. For example, in stressful situations supportive behavior of other people as well as positive social relationships can act as powerful resources to cope with stress. In order to study the interplay between these variables, this protocol describes two effective experimental manipulations of social relationships and supportive behavior in the laboratory. In the present article, these two manipulations are implemented in the Trier Social Stress Test (TSST)&#8212;a standard stress induction paradigm in which participants are subjected to a simulated job interview. More precisely, we propose (a) a manipulation of the relationship between different protagonists in the TSST by making a shared social identity salient and (b) a manipulation of the behavior of the TSST-selection committee, which acts either supportively or unsupportively. These two experimental manipulations are designed in a modular fashion and can be applied independently of each other but can also be combined. Moreover, these two manipulations can also be integrated into other stress protocols and into other standardized social interactions such as trust games, negotiation tasks, or other group tasks.

Previous research on the social dimension of stress has focused on two important variables: social identity and social support. This protocol introduces an effective experimental manipulation of these two social variables and describes their implementation in a standard stress induction paradigm (Trier Social Stress Test).

In many situations humans are influenced by the behavior of other people and their relationships with them. For example, in stressful situations ...

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 ...

Visualization of Intensity Levels to Reduce the Gap Between Self-Reported and Directly Measured Physical Activity

Physical activity (PA) assessment needs tools that are inexpensive and easy to administer. Common questionnaires inquire time spent in light, moderate, and vigorous PA. However, inaccuracies may occur due to individually different understanding of PA intensity levels. Alternatively used direct measures (e.g., accelerometers) are susceptible to reactivity bias and may lack the ability to capture certain activities. Compared to accelerometer measurement, respondents report more time spent in higher-intensity PA. A video that visualizes PA intensity levels might help to overcome this problem. This report describes the design of a randomized controlled trial as a methodology to investigate the effect of a video on the difference between self-reported and directly measured PA. It is hypothesized that the video reduces the mean difference between the two measures. Individuals from the general population are recruited. Hip-worn accelerometers are used to collect directly measured PA data on seven consecutive days. Afterwards, participants are randomly allocated to the experimental and the control group. The experimental group receives a video demonstration on PA intensity levels and subsequent PA assessment via self-administered computer-assisted questionnaire. The control group receives PA assessment only. Thereafter, the data are processed to compare the difference between self-reported and accelerometer-based moderate-to-vigorous physical activity (MVPA) between the study groups using a two-sample t-test. This methodology is appropriate for investigating the effect of any existing or self-produced video on the difference between the two measurement methods. It can be used not only for persons from the general population, but for a variety of other populations and contexts as accurate measures are needed to evaluate PA levels.

This protocol describes a randomized controlled trial as a method to test the effect of a video demonstration on the intra-individual difference between self-reported and accelerometer-based moderate-to-vigorous physical activity.

Physical activity (PA) assessment needs tools that are inexpensive and easy to administer. Common questionnaires inquire time spent in light, moderate, ...

Behavioral Approaches to Studying Innate Stress in Zebrafish

Responding appropriately to stressful stimuli is essential for survival of an organism. Extensive research has been done on a wide spectrum of stress-related diseases and psychiatric disorders, yet further studies into the genetic and neuronal regulation of stress are still required to develop better therapeutics. The zebrafish provides a powerful genetic model to investigate the neural underpinnings of stress, as there exists a large collection of mutant and transgenic lines. Moreover, pharmacology can easily be applied to zebrafish, as most drugs can be added directly to water. We describe here the use of the 'novel tank test' as a method to study innate stress responses in zebrafish, and demonstrate how potential anxiolytic drugs can be validated using the assay. The method can easily be coupled with zebrafish lines harboring genetic mutations, or those in which transgenic approaches for manipulating precise neural circuits are used. The assay can also be used in other fish models. Together, the described protocol should facilitate the adoption of this simple assay to other laboratories.

This manuscript describes a simple method to measure stress behaviorally in adult zebrafish. The approach takes advantage of the innate tendency that zebrafish prefer the bottom half of a tank when in a stressful state. We also describe methods for coupling the assay with pharmacology.

Responding appropriately to stressful stimuli is essential for survival of an organism. Extensive research has been done on a wide spectrum of ...

Modified Blood Collection from Tail Veins of Non-anesthetized Mice with a Vacuum Blood Collection System and Eyeglass Magnifier

Blood sample collection is the basis of experimental animal research. It is of importance to obtain adequate blood samples for various research purposes. The tail veins of mice are small, and it is sometimes difficult to obtain the required blood volume by conventional puncture methods. This study investigates the superiority of repeated blood sample collection from tail veins of mice through use of a vacuum blood collection system and eyeglass magnifier (experimental group) compared to conventional blood sampling methods (conventional group), performed by beginners and experts, respectively. With the help of an eyeglass magnifier, a butterfly needle tip is inserted into the tail vein of each mouse in the experimental group. When the vein is penetrated successfully, a blood sample is collected in the vacuum collection tube by insertion of the rubber end of a butterfly needle into the vacuum blood collection tube. The plunger is then used to collect blood without the help of the eyeglass magnifier in the conventional group. Success rates of blood sample collection by the beginners and experts were shown to be 70% vs. 100% (p &#60; 0.01) in the experimental group and 35% vs. 85% (p &#60; 0.01) in the conventional group. For both beginners and experts, puncture times required for obtaining required blood sample were significantly lower in the experimental group compared to the conventional group (2.40 &#177; 0.75 vs. 2.90 &#177; 0.31, p &#60; 0.05; 1.15 &#177; 0.37 vs. 1.55 &#177; 0.76, p &#60; 0.05). In conclusion, the presented blood sampling technique is feasible and easy to practice and enables frequent sampling of adequate blood volumes from non-anesthetized mice.&#160;

This study reports blood sampling from tail vein in mice using a vacuum extraction tube system with eyeglass magnifier. Our method is easy to practice and could be used for repeat blood sampling in mice.

Blood sample collection is the basis of experimental animal research. It is of importance to obtain adequate blood samples for various research purposes. ...

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, ...

Longitudinal Two-Photon Imaging of Dorsal Hippocampal CA1 in Live Mice

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from subcellular to network levels and at temporal scales from milliseconds to weeks. In addition, two-photon imaging can be combined with a variety of behavioral tasks to explore the causal relationships between brain function and behavior. However, in mammals, limited penetration and scattering of light have limited two-photon intravital imaging mostly to superficial brain regions, thus precluding longitudinal investigation of deep-brain areas such as the hippocampus. The hippocampus is involved in spatial navigation and episodic memory and is a long-standing model used to study cellular as well as cognitive processes important for learning and recall, both in health and disease. Here, a preparation that enables chronic optical access to the dorsal hippocampus in living mice is detailed. This preparation can be combined with two-photon optical imaging at cellular and subcellular resolution in head fixed, anesthetized live mice over several weeks. These techniques enable repeated imaging of neuronal structure or activity-evoked plasticity in tens to hundreds of neurons in the dorsal hippocampal CA1. Furthermore, this chronic preparation can be used in combination with other techniques such as micro-endoscopy, head-mounted wide field microscopy or three-photon microscopy, thus greatly expanding the toolbox to study cellular and network processes involved in learning and memory.

This method describes a chronic preparation that allows optical access to the hippocampus of living mice. This preparation can be used to perform longitudinal optical imaging of neuronal structural plasticity and activity-evoked cellular plasticity over a period of several weeks.

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from ...