The authors are grateful to Miguel Ayala, Lindsey Kitchenham, Dr. Michelle Edwards, Sylvia Lam and Stephanie Dejardin for their contributions to the Reseasco et al.19 validation work which this protocol is based on. We would also like to thank the mice and our wonderful animal care technicians, Michaela Randall and Michelle Cieplak.

<ol><li>Mathews, A., MacLeod, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cognitive+vulnerability+to+emotional+disorders%5BTitle%5D)+AND+%22Annual+Review+of+Clinical+Psychology%22%5BJournal%5D)">Cognitive vulnerability to emotional disorders.</a> Annual Review of Clinical Psychology. 1, 167-195 (2005).</li>
<li>Mathews, A., MacLeod, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cognitive+approaches+to+emotions%5BTitle%5D)+AND+%22Anual+Review+of+Psychology%22%5BJournal%5D)">Cognitive approaches to emotions.</a> Anual Review of Psychology. 45 (1), 25-50 (1994).</li>
<li>Blanchette, I., Richards, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+influence+of+affect+on+higher+level+cognition%3A+A+review+of+research+on+interpretation%2C+judgement%2C+decision+making+and+reasoning%5BTitle%5D)+AND+%22Cognition+and+Emotion%22%5BJournal%5D)">The influence of affect on higher level cognition: A review of research on interpretation, judgement, decision making and reasoning.</a> Cognition and Emotion. 24 (4), 561-595 (2010).</li>
<li>MacLeod, C., Cohen, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anxiety+and+the+interpretation+of+ambiguity%3A+A+text+comprehension+study%5BTitle%5D)+AND+%22Journal+of+Abnormal+Psychology%22%5BJournal%5D)">Anxiety and the interpretation of ambiguity: A text comprehension study.</a> Journal of Abnormal Psychology. 102 (2), 238-247 (1993).</li>
<li>Everaert, J., Podina, I. R., Koster, E. H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+comprehensive+meta-analysis+of+interpretation+biases+in+depression%5BTitle%5D)+AND+%22Clinical+Psychology+Review%22%5BJournal%5D)">A comprehensive meta-analysis of interpretation biases in depression.</a> Clinical Psychology Review. 58, 33-48 (2017).</li>
<li>Haselton, M. G., Nettle, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+paranoid+optimist%3A+An+integrative+evolutionary+model+of+cognitive+biases%5BTitle%5D)+AND+%22Personality+and+Social+Psychology+Review%22%5BJournal%5D)">The paranoid optimist: An integrative evolutionary model of cognitive biases.</a> Personality and Social Psychology Review. 10 (1), 47-66 (2006).</li>
<li>Mendl, M., Paul, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Getting+to+the+heart+of+animal+welfare.+The+study+of+animal+emotion%5BTitle%5D)+AND+%22Stichting+Animales%22%5BJournal%5D)">Getting to the heart of animal welfare. The study of animal emotion.</a> Stichting Animales. , Netherlands. (2017).</li>
<li>Harding, E. J., Paul, E. S., Mendl, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cognitive+bias+and+affective+state%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Cognitive bias and affective state.</a> Nature. 427 (6972), 312(2004).</li>
<li>Douglas, C., Bateson, M., Walsh, C., Bédué, A., Edwards, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Environmental+enrichment+induces+optimistic+cognitive+biases+in+pigs%5BTitle%5D)+AND+%22Applied+Animal+Behaviour+Science%22%5BJournal%5D)">Environmental enrichment induces optimistic cognitive biases in pigs.</a> Applied Animal Behaviour Science. 139 (1-2), 65-73 (2012).</li>
<li>Paul, E. S., Harding, E. J., Mendl, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measuring+emotional+processes+in+animals%3A+The+utility+of+a+cognitive+approach%5BTitle%5D)+AND+%22Neuroscience+and+Biobehavioral+Reviews%22%5BJournal%5D)">Measuring emotional processes in animals: The utility of a cognitive approach.</a> Neuroscience and Biobehavioral Reviews. 29 (3), 469-491 (2005).</li>
<li>Mendl, M., Burman, O. H. P., Parker, R. M. A., Paul, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cognitive+bias+as+an+indicator+of+animal+emotion+and+welfare%3A+Emerging+evidence+and+underlying+mechanisms%5BTitle%5D)+AND+%22Applied+Animal+Behaviour+Science%22%5BJournal%5D)">Cognitive bias as an indicator of animal emotion and welfare: Emerging evidence and underlying mechanisms.</a> Applied Animal Behaviour Science. 118 (3-4), 161-181 (2009).</li>
<li>Rygula, R., Pluta, H., Popik, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Laughing+rats+are+optimistic%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">Laughing rats are optimistic.</a> PLoS ONE. 7 (12), 51959(2012).</li>
<li>Boissy, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assessment+of+positive+emotions+in+animals+to+improve+their+welfare%5BTitle%5D)+AND+%22Physiology+and+Behavior%22%5BJournal%5D)">Assessment of positive emotions in animals to improve their welfare.</a> Physiology and Behavior. 92 (3), 375-397 (2007).</li>
<li>Ross, M., Garland, A., Harlander-Matauschek, A., Kitchenham, L., Mason, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Welfare-improving+enrichments+greatly+reduce+hens%27+startle+responses%2C+despite+little+change+in+judgment+bias%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Welfare-improving enrichments greatly reduce hens' startle responses, despite little change in judgment bias.</a> Scientific Reports. 9 (1), 1-14 (2019).</li>
<li>Lagisz, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimism%2C+pessimism+and+judgement+bias+in+animals%3A+A+systematic+review+and+meta-analysis%5BTitle%5D)+AND+%22Neuroscience+and+Biobehavioral+Reviews%22%5BJournal%5D)">Optimism, pessimism and judgement bias in animals: A systematic review and meta-analysis.</a> Neuroscience and Biobehavioral Reviews. 118, 3-17 (2020).</li>
<li>Canadian Council on Animal Care CCAC Animal Data Report 2019. , Available from: 
 <a target="_blank" href="https://ccac.ca/Documents/AUD/2019-Animal-Data-Report.pdf">https://ccac.ca/Documents/AUD/2019-Animal-Data-Report.pdf</a> (2019).</li>
<li>Report From the Commission to the European Parlaiment and the Council. European Commission. , Available from: 
 <a target="_blank" href="https://eur-lex.europa.eu/legal-content/EN/TXT/HTML/?uri=CELEX:52020DC0016&from=EN">https://eur-lex.europa.eu/legal-content/EN/TXT/HTML/?uri=CELEX:52020DC0016&from=EN</a> (2020).</li>
<li>Cryan, J. F., Holmes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Model+organisms%3A+The+ascent+of+mouse%3A+Advances+in+modelling+human+depression+and+anxiety%5BTitle%5D)+AND+%22Nature+Reviews+Drug+Discovery%22%5BJournal%5D)">Model organisms: The ascent of mouse: Advances in modelling human depression and anxiety.</a> Nature Reviews Drug Discovery. 4 (9), 775-790 (2005).</li>
<li>Resasco, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cancer+blues%3F+A+promising+judgment+bias+task+indicates+pessimism+in+nude+mice+with+tumors%5BTitle%5D)+AND+%22Physiology+and+Behavior%22%5BJournal%5D)">Cancer blues? A promising judgment bias task indicates pessimism in nude mice with tumors.</a> Physiology and Behavior. 238, 113465(2021).</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+%22Journal+of+Cerebral+Blood+Flow+and+Metabolism%22%5BJournal%5D)">The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research.</a> Journal of Cerebral Blood Flow and Metabolism. 40 (9), 1769-1777 (2020).</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>Gygax, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+A+to+Z+of+statistics+for+testing+cognitive+judgement+bias%5BTitle%5D)+AND+%22Animal+Behaviour%22%5BJournal%5D)">The A to Z of statistics for testing cognitive judgement bias.</a> Animal Behaviour. 95, 59-69 (2014).</li>
<li>Gaskill, B. N., Garner, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Power+to+the+people%3A+Power%2C+negative+results+and+sample+size%5BTitle%5D)+AND+%22Journal+of+the+American+Association+for+Laboratory+Animal+Science%3A+JAALAS%22%5BJournal%5D)">Power to the people: Power, negative results and sample size.</a> Journal of the American Association for Laboratory Animal Science: JAALAS. 59 (1), 9-16 (2020).</li>
<li>MacLellan, A., Adcock, A., Mason, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Behavioral+biology+of+mice%5BTitle%5D)+AND+%22Behavioral+Biology+of+Lab+Animals%22%5BJournal%5D)">Behavioral biology of mice.</a> Behavioral Biology of Lab Animals. , 89-111 (2021).</li>
<li>Walker, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mixed-strain+housing+for+female+C57BL%2F6%2C+DBA%2F2%2C+and+BALB%2Fc+mice%3A+Validating+a+split-plot+design+that+promotes+refinement+and+reduction%5BTitle%5D)+AND+%22BMC+Medical+Research+Methodology%22%5BJournal%5D)">Mixed-strain housing for female C57BL/6, DBA/2, and BALB/c mice: Validating a split-plot design that promotes refinement and reduction.</a> BMC Medical Research Methodology. 16 (11), (2016).</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>Nip, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Why+are+enriched+mice+nice+Investigating+how+environmental+enrichment+reduces+agonism+in+female+C57BL+%2F+6%2C+DBA+%2F+2%2C+and+BALB+%2F+c+mice%5BTitle%5D)+AND+%22Applied+Animal+Behaviour+Science%22%5BJournal%5D)">Why are enriched mice nice Investigating how environmental enrichment reduces agonism in female C57BL / 6, DBA / 2, and BALB / c mice.</a> Applied Animal Behaviour Science. 217, 73-82 (2019).</li>
<li>Tilly, S. C., Dallaire, J., Mason, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Middle-aged+mice+with+enrichment-resistant+stereotypic+behaviour+show+reduced+motivation+for+enrichment%5BTitle%5D)+AND+%22Animal+Behaviour%22%5BJournal%5D)">Middle-aged mice with enrichment-resistant stereotypic behaviour show reduced motivation for enrichment.</a> Animal Behaviour. 80 (3), 363-373 (2010).</li>
<li>Fureix, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stereotypic+behaviour+in+standard+non-enriched+cages+is+an+alternative+to+depression-like+responses+in+C57BL%2F6+mice%5BTitle%5D)+AND+%22Behavioural+Brain+Research%22%5BJournal%5D)">Stereotypic behaviour in standard non-enriched cages is an alternative to depression-like responses in C57BL/6 mice.</a> Behavioural Brain Research. 305, 186-190 (2016).</li>
<li>Nip, E. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22The+long-term+effects+of+environmental+enrichment+on+agonism+in+female+C57BL%2F6%2C+BALB%2Fc%2C+and+DBA%2F2+mice.%22">The long-term effects of environmental enrichment on agonism in female C57BL/6, BALB/c, and DBA/2 mice. Thesis Dissertation</a>. , University of Guelph. (2018).</li>
<li>Wei, J., Carroll, R. J., Harden, K. K., Wu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparisons+of+treatment+means+when+factors+do+not+interact+in+two-factorial+studies%5BTitle%5D)+AND+%22Amino+Acids%22%5BJournal%5D)">Comparisons of treatment means when factors do not interact in two-factorial studies.</a> Amino Acids. 42 (5), 2031-2035 (2012).</li>
<li>Ruxton, G. D., Neuhäuser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((When+should+we+use+one-tailed+hypothesis+testing%5BTitle%5D)+AND+%22Methods+in+Ecology+and+Evolution%22%5BJournal%5D)">When should we use one-tailed hypothesis testing.</a> Methods in Ecology and Evolution. 1 (2), 114-117 (2010).</li>
<li>Young, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+odour+span+task%3A+A+novel+paradigm+for+assessing+working+memory+in+mice%5BTitle%5D)+AND+%22Neuropharmacology%22%5BJournal%5D)">The odour span task: A novel paradigm for assessing working memory in mice.</a> Neuropharmacology. 52 (2), 634-645 (2007).</li>
<li>Latham, N., Mason, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((From+house+mouse+to+mouse+house%3A+The+behavioural+biology+of+free-living+Mus+musculus+and+its+implications+in+the+laboratory%5BTitle%5D)+AND+%22Applied+Animal+Behaviour+Science%22%5BJournal%5D)">From house mouse to mouse house: The behavioural biology of free-living Mus musculus and its implications in the laboratory.</a> Applied Animal Behaviour Science. 86 (3-4), 261-289 (2004).</li>
<li>Jones, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assessing+animal+affect%3A+an+automated+and+self-initiated+judgement+bias+task+based+on+natural+investigative+behaviour%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Assessing animal affect: an automated and self-initiated judgement bias task based on natural investigative behaviour.</a> Scientific Reports. 8 (1), 12400(2018).</li>
<li>Novak, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+stereotypic+behaviour+and+chronic+mild+stress+on+judgement+bias+in+laboratory+mice%5BTitle%5D)+AND+%22Applied+Animal+Behaviour+Science%22%5BJournal%5D)">Effects of stereotypic behaviour and chronic mild stress on judgement bias in laboratory mice.</a> Applied Animal Behaviour Science. 174, 162-172 (2016).</li>
<li>Krakenberg, V., von Kortzfleisch, V. T., Kaiser, S., Sachser, N., Richter, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+effects+of+serotonin+transporter+genotype+on+anxiety-like+behavior+and+cognitive+judgment+bias+in+mice%5BTitle%5D)+AND+%22Frontiers+in+Behavioral+Neuroscience%22%5BJournal%5D)">Differential effects of serotonin transporter genotype on anxiety-like behavior and cognitive judgment bias in mice.</a> Frontiers in Behavioral Neuroscience. 13, 263(2019).</li>
<li>Krakenberg, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Technology+or+ecology%3F+New+tools+to+assess+cognitive+judgement+bias+in+mice%5BTitle%5D)+AND+%22Behavioural+Brain+Research%22%5BJournal%5D)">Technology or ecology? New tools to assess cognitive judgement bias in mice.</a> Behavioural Brain Research. 362, 279-287 (2019).</li>
<li>Krakenberg, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+different+social+experiences+on+emotional+state+in+mice%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Effects of different social experiences on emotional state in mice.</a> Scientific Reports. 10, 15255(2020).</li>
<li>Bračić, M., Bohn, L., Krakenberg, V., Schielzeth, H., Kaiser, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Once+an+optimist%2C+always+an+optimist%3F+Studying+cognitive+judgment+bias+in+mice%5BTitle%5D)+AND+%22EcoEvoRxiv%22%5BJournal%5D)">Once an optimist, always an optimist? Studying cognitive judgment bias in mice.</a> EcoEvoRxiv. , (2021).</li>
<li>Jones, S., Paul, E. S., Dayan, P., Robinson, E. S. J., Mendl, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pavlovian+influences+on+learning+differ+between+rats+and+mice+in+a+counter-balanced+Go%2FNoGo+judgement+bias+task%5BTitle%5D)+AND+%22Behavioural+Brain+Research%22%5BJournal%5D)">Pavlovian influences on learning differ between rats and mice in a counter-balanced Go/NoGo judgement bias task.</a> Behavioural Brain Research. 331, 214-224 (2017).</li>
<li>Roelofs, S., Boleij, H., Nordquist, R. E., vander Staay, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Making+decisions+under+ambiguity%3A+Judgment+bias+tasks+for+assessing+emotional+state+in+animals%5BTitle%5D)+AND+%22Frontiers+in+Behavioral+Neuroscience%22%5BJournal%5D)">Making decisions under ambiguity: Judgment bias tasks for assessing emotional state in animals.</a> Frontiers in Behavioral Neuroscience. 10 (119), 1-16 (2016).</li>
<li>Sherwin, C. M., Haug, E., Terkelsen, N., Vadgama, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Studies+on+the+motivation+for+burrowing+by+laboratory+mice%5BTitle%5D)+AND+%22Applied+Animal+Behaviour+Science%22%5BJournal%5D)">Studies on the motivation for burrowing by laboratory mice.</a> Applied Animal Behaviour Science. 88 (3-4), 343-358 (2004).</li>
<li>Deacon, R. M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Burrowing%3A+A+sensitive+behavioural+assay%2C+tested+in+five+species+of+laboratory+rodents%5BTitle%5D)+AND+%22Behavioural+Brain+Research%22%5BJournal%5D)">Burrowing: A sensitive behavioural assay, tested in five species of laboratory rodents.</a> Behavioural Brain Research. 200 (1), 128-133 (2009).</li>
<li>MacDougall-Shackleton, S. A., Bonier, F., Romero, L. M., Moore, I. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glucocorticoids+and+%26%2334%3Bstress%26%2334%3B+are+not+synonymous%5BTitle%5D)+AND+%22Integrative+Organismal+Biology%22%5BJournal%5D)">Glucocorticoids and &#34;stress&#34; are not synonymous.</a> Integrative Organismal Biology. 1 (1), 1-8 (2019).</li>
</ol>

The authors have no conflicts of interest to disclose.

The scent-based digging protocol and results outlined here demonstrate the ability of this novel JB task to detect changes in mouse affective state. The task thus presents a valuable tool for diverse fields of research. Similar to any JB task, to assess animal affect it is critical that animals first learn to discriminate between cues (step 4.7.3) and that the ambiguous stimulus is interpreted as intermediate (step 5.3). Though simple, meeting these requirements can be challenging, particularly in laboratory mice for whi...

Measuring affectively modulated judgment bias (henceforth JB) has proven to be a useful tool for studying the emotional states of animals. This innovative approach borrows from human psychology since humans experiencing positive or negative affective states (emotions and longer-term moods) reliably demonstrate differences in the way they process information1,2,3. For example, humans experiencing anxiety or depression might interpret neutral facial expressions as negative, or neutral sentences as threatening4,5. It is likely that these biases have an adaptive value and are therefore conserved across species6,7. Researchers aiming to assess animal affect have cleverly taken advantage of this phenomenon, operationalizing optimism as the increased expectation of reward in response to neutral or ambiguous cues, and pessimism as the increased expectation of punishment or reward absence8,9. Thus, in an experimental setting, optimistic and pessimistic responses to ambiguous stimuli can be interpreted as indicators of positive and negative affect, respectively10,11.
Compared to other indicators of animal affect, JB tasks have the potential to be particularly valuable tools since they are capable of detecting both the valence and intensity of affective states10,11. The ability of JB tasks to detect positive states (e.g., Rygula et al.12) is especially useful since most indicators of animal affect are limited to the detection of negative states13. During JB tasks, animals are typically trained to respond to a positive discriminative cue predicting reward (e.g., high-frequency tone) and a negative discriminative cue predicting punishment (e.g.,&#160;low-frequency tone), before being presented with an ambiguous cue (e.g., intermediate tone)8. If in response to ambiguous cues an animal &#39;optimistically&#39; performs the trained response for the positive cue (as if expecting reward), this indicates a positive judgment bias. Alternatively, if animals demonstrate the negative trained response to avoid punishment, this is indicative of &#39;pessimism&#39; or negative judgment bias.
Since the development of the first successful JB task for animals by Harding and colleagues8, several JB tasks have been developed for a wide range of species across diverse research fields7. But despite their increasing popularity, animal JB tasks are often labor-intensive. Moreover, perhaps because they are methodologically different from the human tasks that inspired them, they sometimes produce null or counterintuitive results14 and commonly yield only small treatment effect sizes15. As a result, JB tasks can be challenging to develop and implement. In fact, for laboratory mice, the most widely used vertebrates in research16,17 and a species heavily relied upon to model affective disorders18, only one JB task has been successfully validated as sensitive to changes in affective state19 despite many attempts over the past decade (see supplementary material of Resasco et al.19&#160;for a summary). This article describes the recently validated murine JB task, detailing its biologically relevant design, and highlighting the ways that this humane task can be applied to test important hypotheses relevant to mouse affect. Overall, the protocol can be implemented to investigate the affective effects of any variable of interest on JB in mice. This would include categorical treatment variables as described here (drug or disease effects, environmental conditions, genetic background, etc.), or relationships with continuous variables (physiological changes, home cage behaviors, etc.).

<table><tbody><tr><td>Absolute ethanol</td><td>Commercial alcohol</td><td>P016EAAN</td><td>Dilute to 70% with distilled water, for cleaning</td></tr><tr><td>Centrifuge tubes</td><td>Fischer</td><td>55395</td><td>15 mL tubes used to dilute essences here. However, size may be selected based on total volume required for sample size</td></tr><tr><td>Cheerios (original)</td><td>Cheerios</td><td>N/A</td><td>Commercially available. Used as reward to train animals to enter annex cage for handling</td></tr><tr><td>Corncob bedding</td><td>Envigo</td><td>7092</td><td>Teklad 1/8 corncob bedding used in digging pots and animal cages</td></tr><tr><td>Cotton pads</td><td>Equate</td><td>N/A</td><td>Commercially available. Modified in lab to fit within tissue cassettes for scent dispensing</td></tr><tr><td>Digging pots</td><td>Rubbermaid</td><td>N/A</td><td>Commercially available. Containers were modified to fit the apparatus in the lab</td></tr><tr><td>Dried, sweetened banana chips</td><td>Stock and Barrel</td><td>N/A</td><td>Commercially available. High value reward in JB task</td></tr><tr><td>JB apparatus</td><td></td><td>N/A</td><td>The apparatus was made in the lab</td></tr><tr><td>JWatcher event recording software</td><td>Animal Behavior Laboratory, Macquarie University</td><td>Version 1.0</td><td>Freely available for download online. Used to score digging behavior during JB task</td></tr><tr><td>Mint extract</td><td>Fleibor</td><td>N/A</td><td>Commercially &quot;Menta (Soluci&amp;oacute;n)&quot;. Discriminative stimulus odor</td></tr><tr><td>Rodent Diet</td><td>Envigo</td><td>2914</td><td>Teklad global 14% protein rodent maintenance diets. Low value reward in JB task and regular diet in home cage</td></tr><tr><td>SAS statistical software</td><td>SAS</td><td>Version 9.4</td><td>Other comparable software programs (e.g. R) are also appropriate</td></tr><tr><td>Vanilla extract</td><td>Fleibor</td><td></td><td>Commercially available &quot;Vainilla (Soluci&amp;oacute;n)&quot;. Discriminative stimulus odor</td></tr><tr><td>Video camera</td><td>Sony</td><td>DCR-SX22</td><td>Sony handycam.</td></tr></tbody></table>

assessment of mouse judgment bias through an olfactory digging task

Judgment biases (JB) are differences in the way that individuals in positive and negative affective/emotional states interpret ambiguous information. This phenomenon has long been observed in humans, with individuals in positive states responding to ambiguity 'optimistically' and those in negative states instead showing 'pessimism'. Researchers aiming to assess animal affect have taken advantage of these differential responses, developing tasks to assess judgment bias as an indicator of affective state. These tasks are becoming increasingly popular across diverse species and fields of research. However, for laboratory mice, the most widely used vertebrates in research and a species heavily relied upon to model affective disorders, only one JB task has been successfully validated as sensitive to changes in affective state. Here, we provide a detailed description of this novel murine JB task, and evidence of its sensitivity to mouse affect. Though refinements are still necessary, assessment of mouse JB opens the door for answering both practical questions regarding mouse welfare, and fundamental questions about the impact of affective state in translational research.

Results presented here reflect relevant findings from Experiment 1 of Resasco et al.19. Subjects in this experiment were 18 female C57BL/6NCrl (&#39;C57&#39;) and 18 Balb/cAnNCrl (&#39;Balb&#39;) mice. Animals arrived at the facility at 3-4 weeks of age and were randomly assigned to environmentally enriched or conventional housing treatments (EH or CH, respectively) in mixed strain quartets25. Each cage contained one C57 and one Balb, in addition to two DBA/2NCrl mice being...

Watch this Scientific Journal Video about Assessment of Mouse Judgment Bias through an Olfactory Digging Task at JoVE.com

Assessment of Mouse Judgment Bias through an Olfactory Digging Task

This article provides a detailed description of a novel mouse judgment bias protocol. Evidence of this olfactory digging task's sensitivity to affective state is also demonstrated and its utility across diverse research fields is discussed.

Judgment biases (JB) are differences in the way that individuals in positive and negative affective/emotional states interpret ambiguous information. This ...

assessment-of-mouse-judgment-bias-through-an-olfactory-digging-task

Research

JoVE Journal

Behavior

2.5K Views.  University of Guelph. Dit is de allereerste muisbeoordelingsbiastaak die wordt gevalideerd als gevoelig voor veranderingen in affectieve toestand, met andere woorden, stemming of emotie. Het kan worden gebruikt om de effecten van elke variabele op het effect van de muis te onderzoeken. Dit protocol maakt gebruik van een ethologisch ontwerp, waardoor het voor muizen gemakkelijk is om te leren, en het is ook welzijnsvriendelijk, omdat de taak het gebruik van straf of negatieve versterking vermijdt.Deze taak k...

Video: Assessment of Mouse Judgment Bias through an Olfactory Digging Task

Assessment of Sensorimotor Function in Mouse Models of Parkinson's Disease

Sensitive and reliable behavioral outcome measures are essential to the evaluation of potential therapeutic treatments in preclinical trials for many neurodegenerative diseases. In Parkinson's disease, sensorimotor tests sensitive to varying degrees of nigrostriatal dysfunction are fundamental for testing the efficacy of potential therapeutics. Reliable and quite elegant sensorimotor measures exist for rats, however many of these tests measure sensorimotor asymmetry within the rat and are not entirely suitable for the newer genetic mouse models of PD. We have put together a battery of sensorimotor tests inspired by the sensitive tests in rats and adapted for mice. The test battery highlighted in this study is chosen for a) its sensitivity in a wide variety of mouse models of PD, b) its ease in implementing into a study, and c) its low expense. These tests have proven useful in characterizing novel genetic mouse models of PD as well as in testing potential disease-modifying therapies.

In Parkinson's disease and movement disorders in general, sensitive and reliable behavioral assays are essential for testing novel potential therapeutics. Here, we describe a manageable battery of sensorimotor tests for mice that are sensitive to varying degrees of injury to the nigrostriatal system and useful for preclinical studies.

Sensitive and  reliable behavioral outcome measures are essential to the evaluation of  potential therapeutic treatments in preclinical trials for many  ...

The Attentional Set Shifting Task: A Measure of Cognitive Flexibility in Mice

Cognitive impairment, particularly involving dysfunction of circuitry within the prefrontal cortex (PFC), represents a core feature of many neuropsychiatric and neurodevelopmental disorders, including depression, post-traumatic stress disorder, schizophrenia and autism spectrum disorder. Deficits in cognitive function also represent the most difficult symptom domain&#160;to successfully treat, as serotonin reuptake inhibitors and tricyclic antidepressants have only modest effects. Functional neuroimaging studies and postmortem analysis of human brain tissue implicate the PFC as being a primary region of dysregulation in patients with these disorders. However, preclinical behavioral assays used to assess these deficits in mouse models which can be readily manipulated genetically and could provide the basis for studies of new treatment avenues have been underutilized. Here we describe the adaptation of a behavioral assay, the attentional set shifting task (AST), to be performed in mice to assess prefrontal cortex mediated cognitive deficits. The neural circuits underlying behavior during the AST are highly conserved across humans, nonhuman primates and rodents, providing excellent face, construct and predictive validity.

The goal of this protocol is to perform a behavioral assay such as the attentional set shifting task (AST) to assess prefrontal cortex-mediated cognitive flexibility in mice.

Cognitive impairment, particularly involving dysfunction of circuitry within the prefrontal cortex (PFC), represents a core feature of many ...

Testing for Odor Discrimination and Habituation in Mice

This video demonstrates a technique to establish the presence of a normally functioning olfactory system in a mouse. The test helps determine whether the mouse can discriminate between non-social odors and social odors, whether the mouse habituates to a repeatedly presented odor, and whether the mouse demonstrates dishabituation when presented with a novel odor. Since many social behavior tests measure the experimental animal&#8217;s response to a familiar or novel mouse, false positives can be avoided by establishing that the animals can detect and discriminate between social odors. There are similar considerations in learning tests such as fear conditioning that use odor to create a novel environment or olfactory cues as an associative stimulus. Deficits in the olfactory system would impair the ability to distinguish between contexts and to form an association with an olfactory cue during fear conditioning. 
In the odor habitation/dishabituation test, the mouse is repeatedly presented with several odors. Each odor is presented three times for two minutes. The investigator records the sniffing time directed towards the odor as the measurement of olfactory responsiveness. A typical mouse shows a decrease in response to the odor over repeated presentations (habituation). The experimenter then presents a novel odor that elicits increased sniffing towards the new odor (dishabituation). After repeated presentation of the novel odor the animal again shows habituation. This protocol involves the presentation of water, two or more non-social odors, and two social odors. In addition to reducing experimental confounds, this test can provide information on the function of the olfactory systems of new knockout, knock-in, and conditional knockout mouse lines.

This manuscript describes a protocol to examine the olfactory system of rodents. The olfactory habituation/dishabituation test will allow investigators to determine whether a mouse habituates to a repeatedly presented odor and whether the mouse demonstrates dishabituation when presented a novel odor.

This video demonstrates a technique to establish the presence of a normally functioning olfactory system in a mouse. The test helps determine whether the ...

Simple and Computer-assisted Olfactory Testing for Mice

Olfaction is highly conserved among species and is required for reproduction and survival.
In humans, olfaction is also one of the senses that is affected with aging and is a strong predictor of neurodegenerative diseases. Thus, olfaction testing is used as a non-invasive diagnostic method to detect neurological deficits early on. In order to understand the mechanisms underlying olfactory network susceptibility, olfactory research in rodents has gained momentum in the past decade.
Here, we present a very simple, time efficient and reproducible olfactory testing method of innate odor perception and sensitivity in mice without the need of any prior food or water restriction. The tests are performed in a familiar environment to the mice, require only the scents and a 2 min session of odorant exposure. The analysis is performed, post-hoc, using computer-assisted commands on ImageJ and can be, therefore, carried out from start to end by one researcher.
This protocol does not require any special hardware or setup and is indicated for any laboratory interested in testing olfactory perception and sensitivity.

We present a simple and unbiased olfactory test in mice. With this protocol olfactory discrimination, preference, avoidance and sensitivity to a novel odor as compared to water can be assessed in single behavioral sessions. This method is indicated for a single experimenter and analysis is based on computer-assisted video processing.

Olfaction is highly conserved among species and is required for reproduction and survival.
In humans, olfaction is also one of the senses that is affected ...

An Operant Intra-/Extra-dimensional Set-shift Task for Mice

Alterations in executive control and cognitive flexibility, such as attentional set-shifting abilities, are core features of several neuropsychiatric diseases. The most widely used neuropsychological tests for the evaluation of attentional set-shifting in human subjects are the Wisconsin Card Sorting Test (WCST) and the CANTAB Intra-/Extra-dimensional set shift task (ID/ED). These tasks have proven clinical relevance and have been modified and successfully adapted for research in animal models. However, currently available tasks for rodents present several limitations, mainly due to their manual-based testing procedures, which are hampering translational advances in psychiatric medicine. To overcome these limitations and to better mimic the original version in primates, we present the development of a novel operant-based two-chamber ID/ED "Operon" task for rodents. We demonstrated the effectiveness of this novel task to measure different facets of cognitive flexibility in mice including attentional set formation and shifting, and reversal learning. Moreover, we show the high flexibility of this task in which three different perceptual dimensions can be manipulated with a high number of stimuli cues for each dimension. This novel ID/ED Operon task can be an effective preclinical tool for drug testing and/or large genetic screening relevant to the study of executive dysfunction and cognitive symptoms found in psychiatric disorders.

Attentional set-shifting cognitive abnormalities are a core feature of many psychiatric diseases. Here, we present the development of a novel automatic system for the effective study of attentional set-shifting abilities, executive functions and other cognitive abilities in mice with high translational validity to human studies.

Alterations in executive control and cognitive flexibility, such as attentional set-shifting abilities, are core features of several neuropsychiatric ...

Olfactory Behavioral Testing in the Adult Mouse

The rodent olfactory system is of increasing interest to scientists, studied, in part, in systems biology because of its stereotyped, yet accessible circuitry. In addition, this area's unique ability to generate new neurons throughout an organism's lifetime makes it an attractive system for developmental and regenerative biologists alike. Such interest necessitates a means for a quick, yet reliable assessment of olfactory function. Many tests of olfactory ability are complex, variable or not specifically designed for mice. Also, some tests are sensitive to memory deficits as well as defects in olfactory abilities, confounding obtained results.
Here, we describe a simple battery of tests designed to identify defects in olfactory sensitivity and preference. First, an initial general health assessment allows for the identification of animals suitable for further testing. Second, mice are exposed to various dilutions of scents to ascertain whether there is a threshold difference. Third, mice are presented with various scents, both attractive and aversive, that allow for the assessment of olfactory preference. These simple studies should make the initial characterization of olfactory behavior accessible for labs of varied resources and expertise.

Fundamental, yet unique properties of the rodent olfactory system have led to its increasing study among biologists. A relatively simple assessment of its function is then also needed. Here we describe sensitive tests for the characterization of mouse olfactory sensitivity and preference.

The rodent olfactory system is of increasing interest to scientists, studied, in part, in systems biology because of its stereotyped, yet accessible ...

Biology

Assessing Olfactory Impairment in Mice Due to Inhaled Environmental Pollutants

Studying the Effects of Inhaled Environmental Pollutants on Olfactory Function in Mice

Olfactory impairment is a significant public health problem and independently predicts the risk of neurodegenerative diseases. Inhaled environmental pollutants exposure may impair olfaction; thereby, there is an urgent need for methods to evaluate the effects of inhaled environmental pollutants exposure on olfaction. Mice are ideal models for olfactory experiments because of their highly developed olfactory system and behavioral characteristics. To assess the effects of inhaled environmental pollutants exposure on olfactory function in mice, a detailed buried food test and social odor discrimination experiment is provided, including the experiment preparation, the selection and construction of experimental facilities, the testing process, and indexes of time. Meanwhile, timekeeping equipment, operational details, and the experimental environment are discussed to ensure the success of the assay. Zinc sulfate is used as the treatment to demonstrate the feasibility of the experimental approach. The protocol provides a simple and clear operational process for assessing the effects of inhaled environmental pollutants on olfactory function in mice.

Author Spotlight: Assessing the Olfactory Effects of Airborne Pollutants &#8212; Buried Food and Social Odor Tests

This paper provides a detailed description of the buried food test and social odor discrimination experiment to assess the effects of inhaled environmental pollutants exposure on olfactory function in mice.

Olfactory impairment is a significant public health problem and independently predicts the risk of neurodegenerative diseases. Inhaled environmental ...

Environment

Olfactory Assays for Mouse Models of Neurodegenerative Disease

In many neurodegenerative diseases and particularly in Parkinson&#8217;s disease, deficits in olfaction are reported to occur early in the disease process and may be a useful behavioral marker for early detection. Earlier detection in neurodegenerative disease is a major goal in the field because this is when neuroprotective therapies have the best potential to be effective. Therefore, in preclinical studies testing novel neuroprotective strategies in rodent models of neurodegenerative disease, olfactory assessment could be highly useful in determining therapeutic potential of compounds and translation to the clinic. In the present study we describe a battery of olfactory assays that are useful in measuring olfactory function in mice. The tests presented in this study were chosen because they measure olfaction abilities in mice related to food odors, social odors, and non-social odors. These tests have proven useful in characterizing novel genetic mouse models of Parkinson&#8217;s disease as well as in testing potential disease-modifying therapies.

Impairment in olfactory function is a common feature in many neurodegenerative disorders including Parkinson, Alzheimer, and Huntington diseases. In the present article, we describe a set of tests for assessing olfaction discrimination and detection in mice that can be used to measure olfactory abilities in mouse models of neurodegenerative diseases.

In many neurodegenerative diseases and particularly in Parkinson&#8217;s disease, deficits in olfaction are reported to occur early in the disease process ...

Neuroscience

Designing a Flexible, Cost-Effective Olfactometer for Rodent Olfactory Experiments

Constructing an Olfactometer for Rodent Olfactory Behavior Studies

The use of olfactometers to study rodent behavior and brain activity during olfactory tasks is crucial for understanding brain circuits. These sophisticated devices allow researchers to precisely control and deliver odor stimuli, enabling the investigation of complex olfactory processes in rodents. Although commercially available olfactometers are convenient, they present challenges when technical issues arise, often requiring costly assistance and potentially disrupting research timelines. This article details the construction of a custom olfactometer specifically designed for mouse olfactory behavior experiments, providing a comprehensive list of parts and step-by-step instructions. The olfactometer is controlled through MATLAB, offering a user-friendly interface for researchers. Importantly, the open-source code allows users to modify and adapt the system, tailoring behavioral tasks to meet specific experimental needs. Building a customized olfactometer empowers users with the knowledge and capability to perform custom experimental design and troubleshooting independently, saving both time and resources. This approach not only enhances research flexibility but also fosters a deeper understanding of the equipment's functionality, ultimately leading to more robust and reliable olfactory studies in rodents.

Constructing an Olfactometer for Rodent Olfactory Behavior Studies Near-Infrared Spectroscopy Hyperscanning Study in Psychological Counseling

This protocol describes the construction of an olfactometer for go/no-go olfactory behavior experiments. Step-by-step instructions, along with images, are provided to ensure the successful construction of the olfactometer. Information for troubleshooting issues encountered during the process is also included.

The use of olfactometers to study rodent behavior and brain activity during olfactory tasks is crucial for understanding brain circuits. These ...

An Objective and Reproducible Test of Olfactory Learning and Discrimination in Mice

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and parenting. Importantly, mice can be trained to associate novel odors with specific behavioral responses to provide insight into olfactory circuit function. This protocol details the procedure for training mice on a Go/No-Go operant learning task. In this approach, mice are trained on hundreds of automated trials daily for 2&#8211;4 weeks and can then be tested on novel Go/No-Go odor pairs to assess olfactory discrimination, or be used for studies on how odor learning alters the structure or function of the olfactory circuit. Additionally, the mouse olfactory bulb (OB) features ongoing integration of adult-born neurons. Interestingly, olfactory learning increases both the survival and synaptic connections of these adult-born neurons. Therefore, this protocol can be combined with other biochemical, electrophysiological, and imaging techniques to study learning and activity-dependent factors that mediate neuronal survival and plasticity.

Here, we train mice on an associative learning task to test odor discrimination. This protocol also allows for studies on learning-induced structural changes in the brain.

Assessment of Mouse Judgment Bias through an Olfactory Digging Task

Samenvatting

Meer video's verkennen