Journal of the American Association for Laboratory Animal Science Copyright 2014 by the American Association for Laboratory Animal Science
Vol 53, No 2 March 2014 Pages 141–145
Lack of Fear Response in Mice (Mus musculus) Exposed to Human Urine Odor Germain F Rivard,1,* Emily G Moser,1 Steven P D’Ambrose,1 and David M Lin2 A goal of the Guide for the Care and Use of Laboratory Animals is to improve animal welfare by minimizing sources of fear, anxiety, and stress. As a result, it includes recommendations on overcrowding, frequency of cage changes, enrichment, and group housing. However, human odorants are a potential but unexplored source of fear, anxiety, and stress. Although mice have been maintained for decades for animal research, whether mice perceive humans as predators is unknown. If so, this would necessitate changes in animal care and use procedures to minimize this source of chronic fear, anxiety, and stress. Odorants from predator urine are well known to elicit strong fear responses in mice, leading to modification of animal behavior and elevated levels of stress. To begin asking whether human odors influence mouse behavior, we tested the effect of human urine odor on fear response in mice. We assessed mouse behavior by using a modified shuttle cage to record various parameters of mouse exposure to odorants. We found that mice displayed fear responses to 2,4,5-trimethylthiazoline, a synthetic analog of red fox feces, but no fear response to DMSO, the diluent for 2,4,5-trimethylthiazoline. In contrast, mice exposed to human urine samples showed no significant fear response. Abbreviation: TMT, 2,4,5-trimethylthiazoline.
The use of laboratory mice is an essential component of academic research. Although precise figures are not available, approximately 12 to 15 million laboratory rodents are used each year in the United States for biomedical research, testing, and education.11 Because of the importance of mice in research, standardized care and use guidelines have been developed for maintaining and monitoring mouse welfare.6 These guidelines include, for example, limits on the number of mice that can be housed together, appropriate environmental enrichment procedures, minimal frequency for cage changes, and recommendations for group housing. These standards are necessary for minimizing fear, anxiety, and stress and to promote general animal health, wellbeing, and welfare. A central component of care and use is providing appropriate training to caretakers and research members on the correct techniques for handling mice. This training emphasizes how best to handle mice in a manner that reduces potentially fearful and stressful interactions. Surprisingly, however, no studies have ever been performed to address whether humans themselves induce fear, anxiety, or stress in mice. Humans, like all mammals, produce secretions such as sweat, saliva, dander, feces, and urine. Such secretions are important olfactory cues for mice. For example, 2 volatile chemicals produced by foxes and omnivores (2,4,5-trimethylthiazoline [TMT] and 2-phenylethylamine) and 2 nonvolatile proteinaceous compounds produced by cats and rats (lipocalin Feld4 and Mup13, respectively) are known to elicit fear-like or aversive behavior in mice.3,7 Mice respond to these predator cues with a variety of behavioral changes, including risk assessment, freezing, attempts to escape, and avoidance.1,2,5,7,10 Chronic exposure to these stressful situations with no means of escape is likely to affect laboratory mouse behavior and welfare. Chronic fear, anxiety, and stress are well-known to have Received: 04 Jun 2013. Revision requested: 13 Aug 2013. Accepted: 10 Sep 2013. 1Department of Clinical Sciences, Veterinary Behavior Medicine and 2Department of Biomedical Sciences, Cornell University, Ithaca, New York. *Corresponding author. Email: [email protected]
multiple effects on animal health.6,8,9 In addition, these can alter the outcome of studies that depend on accurate cortisol levels, hormone release, metabolic measurements, and overall homeostasis. Determining causes of fear, anxiety, and stress that influence mouse behavior and welfare is therefore essential for establishing an appropriate baseline for many types of experiments. Here we ask whether exposure to human odor induces a fear response in mice. For these initial tests, we chose to study the effects of human urine on mouse behavior, because urine from predators has been studied extensively for its effects on mice.3,4,10 We predicted that, among all secretions, urine would generate the most extreme behavioral responses. Any observed effect would lay the groundwork for potential future studies looking at other human secretions, such as saliva, sweat, and dander.
Materials and Methods
Animals. The study was performed by using C57Bl/6 female mice (n = 18; age, 3 wk) donated by Harlan Laboratories (Indianapolis, IN). Mice arrived in 3 separate shipments (that is, lots). Mice were housed individually in static filter-top polycarbonate cages with stainless steel wire lid but without the filter on the top of the cage (Allentown Caging Equipment, Allentown, NJ). Bedding was composed of aspen shavings. Enrichment included the presence of PVC tubes and empty culture dishes in each cage. Animals were fed conventional mouse chow ad libitum with unlimited access to water from water bottles. Mice were housed individually for 7 to 10 d during the testing phase, to minimize human handling of mice, given that group-housed mice require individual identification (for example, ear punching or toe clipping) and more frequent cage changes, resulting in increased stress and handling. Mice were maintained under a reversed 12:12-h light:dark cycle. Testing occurred during the dark phase under red lights and by using an infrared camera. Testing was performed dur141
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ing the active phase to reduce stress caused by disturbing animals during their sleep–wake cycle. All mice were given 72 h to acclimate to their environment after delivery. Although all human contact could not be avoided, every effort was made to eliminate protein-binding odorants by using an enzymatic cleaner (Anti-Icky-Poo, Santa Clarita, CA) for hand washing and 70% alcohol for material preparation. Animal technicians were asked to wear personal protective equipment including Tyvek suits, facemasks, bonnets, booties, and gloves, and to use forceps to transfer mice. At time of testing, their cage was moved from the housing room into the antechamber. All experiments were approved by Cornell’s IACUC. Cornell University is AAALAC-accredited. Odorants and presentation. TMT (PheroTech, Delta, Canada) was diluted 1:10 with DMSO (Sigma Chemicals, St Louis, MO). Human urine was provided immediately prior to testing by the person conducting the test. The urine was collected in a sanitized glass jar, which was then placed in a 37 °C water bath. Samples were obtained within an hour of the actual test time. Urine was obtained from 2 college-age women, one of whom was an omnivore and the other a vegetarian. Controls (2 μL TMT or DMSO) and human urine samples (3 mL) were applied to a piece of filter paper (diameter, 35 mm) by using a micropipette; treated filter papers were placed in a culture dish and covered with a perforated lid (hole size, 1/16 to 3/32 in.) immediately before placing the culture dish in the test chamber. The culture dish was placed on the floor of the shuttle cage in the test chamber. The mouse could touch the culture plate, but the contents could not be accessed. Shuttle cage. We exposed mice to test and control odors by placing a mouse in a shuttle cage (Coulbourn Instruments, Whitehall, PA), which was divided into 2 chambers (that is, the test and control chambers). We modified the shuttle cage as follows. Black acrylic glass was placed on 3 sides of the shuttle cage, leaving one wall transparent for video recording. To prevent the introduced odorant from diffusing into the control chamber, vents on the tops of both chambers were opened to allow the odorant to escape from the test chamber. The wire floor was covered with a piece of acrylic glass. The shuttle cage was equipped with a computer program to control the opening and closing of the guillotine door and infrared sensors to detect movement between the 2 identical chambers. To reduce visual stimulation and arousal, tests were done during the dark phase of the reversed light cycle by using red light to allow for human but not mouse visibility. Red plastic covers were placed over the computer monitors, and an infrared camera was used to capture video. Experimental paradigm. Once all materials were in place, the mouse was transferred from its home cage to the control chamber by grasping the base of the mouse’s tail gently with forceps. The door remained closed for 2 min and then opened automatically and remained open for 3 min. During this final 3 min, the mouse had access to both chambers (Figure 1). Identical culture plates were present in both chambers, but odorant was present only in the plate inside the test chamber. After 3 min, the door automatically closed, and the mouse was immediately transferred from the shuttle cage back to its home cage. The culture dish was removed from the shuttle cage and discarded, and the shuttle cage, floor, forceps, and perforated lids were cleaned with isopropyl alcohol. The tester washed his or her hands with the enzymatic cleaner in preparation for subsequent tests. The perforated lids were reused, but with the same odorant.
Figure 1. Diagram of experimental paradigm. Mice are introduced into the control chamber, with the door of the shuttle cage closed. After 2 min, the door is opened, and the mouse is free to explore the test chamber containing the odorant for 3 min. The petri plate in the control chamber had no odorant.
Each mouse was tested, in order, with DMSO, human urine, and TMT. Mice were tested once each day with a single odorant, with a 24-h gap between tests to minimize stress. Eighteen mice were tested in this manner for 9 d, exposing each animal to each odorant 3 times. Fear assessment. Quantitative measures of mouse behavior were obtained by taking advantage of the shuttle cage’s ability to collect automated data. These include measurements of latency, number of entries, and cumulative time spent in the test chamber. These measures were interpreted as indicators of risk assessment and avoidance. In addition, qualitative measures of fear response were obtained by recording mouse behavior both prior to opening of the door and after odorant exposure. These recordings were randomized and scored by a blinded observer, a veterinarian who was completing a residency in Veterinary Behavioral Medicine at Cornell University at the time of this study. Criteria for observer-based assessment of fear response included risk assessment (slow approach, low head position, lifted tail, flat back, stretch–attend posture [that is, nose pointed toward stimulus while nearing the stimulus and resulting in an extended posture with a raised tail], and retraction), freezing (crouching, reduced forepaw–hindpaw distance, increased sniffing), attempts to escape and avoidance (characterized by hypervigilance with ears and nose constantly toward stimulus, rapid pacing, rapid retreat, seeking an escape route, frenzied wall migration, and digging) behaviors as compared with exploratory (increased exploration of the 2-chamber apparatus, standing, calm wall migration, normal forepaw–hindpaw distance, and nose pointed to different parts of chamber) nonfearful behaviors. To generate a fear severity score, the observer first underwent a training period during which all videos were reviewed blindly but not scored. The training period was done to establish the range of behavior observed in response to these odorants. Based on this initial calibration of behaviors, videos then were scored for fear response on a scale of 0 to 3: 0, mouse displayed only exploratory or nonfearful behavior; 1, observed fear behavior was mild in intensity and relatively brief (approximately 10% of the observed time); 2, fear behavior was moderately intense and lasted approximately 10% to 50% of the observed time; and 3, fear behavior was considered severe and accounted for more than 50% of the observation period. The observer viewed all videos of mice during the first 2 min (that is, no access to test chamber) twice and all videos of mice during the last 3 min (that is, door to test chamber open) 3 times. Scores of the same video were averaged to reduce bias associated with different viewings of the same video by a single observer.
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Statistical analysis. To assess the dependent variables (that is, latency to enter test chamber, cumulative time spent in the test chamber, and number of entries into the test chamber), a mixed model was run by using experimenter–urine provider, number of times exposed to sample, mouse lot, and treatment as fixed effects and by controlling for individual mice as a random effect. Variables with P values greater than 0.05 were not considered statistically significant, and removed from the model. Values were corrected for multiple comparisons by using Bonferroni correction. A similar mixed model was run to assess the averaged severity fear scores for the dependent variables, first 2 min before the shuttle door opened, and last 3 min after the door opened, with the same fixed and random variables as listed earlier and the same criteria for removal from the model. Output included mean values, standard errors, significance, and pairwise comparisons of significant treatment (TMT compared with DMSO compared with urine) and lot effects. Some dependent variables were transformed to meet the assumption of the model. Statistics were determined by using Statistical Product and Service Solution software (IBM, Armonk, NY). A P value of less than 0.05 was considered statistically significant.
Results
Preliminary studies using commercial preparations of human dander, saliva, and urine suggested that urine was likely to have the greatest effect on mouse behavior (data not shown). Furthermore, a comparison of frozen, commercial urine and freshly obtained urine indicated fresh urine was more likely to elicit a fear response in mice. Mice were exposed, in order, to DMSO, freshly provided human urine, and TMT. Eighteen mice were tested, producing a total of 162 recorded behavioral responses. Of these, 20 videos were removed because of technical errors in the presentation of the odor or in the recording. These included trials in which the urine had been collected more than 1 d previously or was diluted and in which the control was not presented correctly according to the protocol. Of the remaining 142 videos, 47 recordings comprised mouse response to DMSO, 41 to human urine, and 54 to TMT. Statistical analysis of data that included the 20 removed trials still produced the same results (data not shown). We scored mouse behavior for fear response during the first 2 min prior to opening the automatic door. Observer assessment of fear response showed no obvious differences in mouse behavior on placement in the shuttle cage regardless of the odorant being tested (Figure 2; mixed model; P = 0.96). This finding is consistent with the interpretation that odors do not diffuse from the test chamber into the control chamber during the first 2 min prior to opening of the door. Mouse fear response during exposure to an odorant. We next asked whether mice displayed a fear response when exposed to the odorant during their final 3 min in the shuttle cage. We first looked at the quantitative information recorded by the shuttle cage. We could detect no differences for any of these measures among the 3 odorants (Figure 3; mixed-model analysis; latency, P = 0.31; cumulative time spent in test chamber, P = 0.21; number of entries into test chamber, P = 0.75). We then asked whether we could detect a fear response through double-blind scoring of mouse behavior during exposure to an odorant. Exposure of mice to TMT resulted in a significant fear response relative to exposure to DMSO (Figure 4; mixed model, P < 0.03). Mice responded to TMT whether they had been exposed once or multiple times, indicating a consistent fear response. Prior studies2,4,10 generally exposed mice once to TMT. We found that mice appear to show consistent responses
Figure 2. No significant differences in average fear response based on behavioral analysis were seen during the first 2 min prior to exposure to an odorant, regardless of the odorant that was to be tested.
to TMT over time and over multiple exposures. We found an additional significant factor in our tests of TMT. Mice were received in 3 separate lots from the vendor. Mice from one lot were less fearful when exposed to TMT than were those from the other lots (mixed-model analysis, P = 0.045). In addition, we asked whether human urine resulted in fear responses among mice. Video assessment of mouse response to urine was performed in a manner identical to that for TMT. However, we were unable to detect any difference in fear response among mice to human urine compared with DMSO (Figure 4; mixed model; P = 0.83). Our studies of human urine used samples obtained from 2 college-age women, one of whom was an omnivore and the other a vegetarian. We asked whether mice responded differentially to human urine as a consequence of diet. No significant difference (P = 0.45) was observed between urine from the omnivore compared with that of the vegetarian.
Discussion
To our knowledge, no studies have examined the effects of any human odor on mouse behavior. Our studies assessed, for the first time, whether human urine odor elicits an observable fear response in mice. We showed no detectable fear response in mice to human urine compared with DMSO odors. Although laboratory animal technicians handling mice are unlikely to routinely introduce mice to urine odors, we chose urine because we predicted that it would have the most dramatic effect on mouse behavior. We presented mice with a specific sequence of odorants. DMSO was presented first, followed by human urine and then TMT. This ordered presentation was performed to prevent ‘preconditioning’ the mice with a fear response to human urine because they had previously been exposed to TMT. We presented the same mouse with the same sequence of odorants 3 times. TMT is known to elicit an innate and consistent fear response in mice.2,4 Because whether human urine odor would induce a fear response was unknown, we were uncertain whether such a response would be innate or learned. We reasoned that if the response were learned, presenting the odorant multiple times would result in reduced fear response. However, if the response were innate, we expected that the severity of the response would remain high on all 3 exposures. We did not detect a quantitative or qualitative fear response among mice in response to human urine odor by using our paradigm. There could be multiple explanations for this lack of response, which was similar to that of mice to DMSO. First, human urine odor may not elicit any fear response in mice because humans are not recognized by mice as natural predators. Second, this lack of response might be due to the domestication of mice by humans. Selection aimed at adapting mice for study 143
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Figure 3. No significant differences in quantitative assessment (mean ± SEM) of (A) latency to enter the test chamber, (B) number of entries into the test chamber, or (C) cumulative time spent in the test chamber were observed in mice exposed to various odors.
Figure 4. Behavior-based assessment of fear response (mean ± SEM) showed significant differences in mouse behavior when presented with TMT as compared with DMSO. No significant differences were seen on exposure to human urine compared with DMSO odors.
as a model organism could, over time, result in the selective breeding of animals nonfearful to human exposure. Third, the lack of response may reflect adequate socialization of pups to humans during early development. Fourth, perhaps human urine odor does elicit a fear response in mice, but not one that is strong enough to be detected by using our paradigm. One final caveat is that these mice were handled by humans while at the vendor facility. This handling may have affected the fear response to human urine odor in our studies. One potential concern regarding our scoring system is that the degree of severity was determined according to the reviewer’s overall subjective assessment of fear. Nevertheless, we were able to clearly show differences in animal behavior upon exposure to TMT. Therefore, we anticipate that we could have identified differences upon exposure to human urine odor if the level of fear response among mice was similar to that of TMT. Although we were able to identify a fear response in mice exposed to TMT, we also discovered an unexpected effect. We obtained mice from the vendor on 3 separate occasions, and mice from each lot were tested in our shuttle cage. Mice from one particular lot showed less fear response than did those in the other 2. Why this occurred is unclear. All mice were from the
same vendor, the same age, and the same genetic background, and the same testers tested mice in all 3 lots. Therefore, we are unable to explain why this one lot of mice showed a different fear response. Perhaps these less-responsive mice were socialized more extensively or were from a different breeding room at the vendor facility. In summary, our study assessed the effect of human urine odor on fear response in mice. Because mice are an essential model for human disease, causes of variability that may bias potential results must be identified and eliminated. Our study is the first to assess whether exposure to human urine odor can induce a behavioral response in mice. The study found no overt response to fresh human urine. However, future studies may address other human secretions (for example, dander, saliva, sweat), perfumes, and masking agents, as well as additional strains of mice.
Acknowledgments
We thank Harlan Laboratories for donating the mice used in these experiments. We thank Thom Cleland for use of the shuttle cage and Francoise Vermeylen (Statistical Consulting Unit, Cornell University) for performing the statistical analysis. We thank Mary Martin (Center for Animal Research and Education, Cornell University) for helpful comments on the manuscript; Kelsey Piel, Katherine Walden, and Shanna Johnson for running shuttle-cage trials; and Laura Lin for assistance with illustrations. This work was funded by a grant from the Johns Hopkins Center for Alternatives to Animal Testing.
References
1. Berton F, Vogel E, Belzung C. 1998. Modulation of mice anxiety in response to cat odor as a consequence of predator diet. Physiol Behav 65:247–254. 2. Fendt M, Endres T, Lowry CA, Apfelbach R, McGregor IS. 2005. TMT-induced autonomic and behavioral changes and the neural basis of its processing. Neurosci Biobehav Rev 29:1145–1156. 3. Ferrero DM, Lemon JK, Fluegge D, Pashkovski SL, Korzan WJ, Datta SR, Spehr M, Fendt M, Liberles SD. 2011. Detection and avoidance of a carnivore odor by prey. Proc Natl Acad Sci USA 108:11235–11240.
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4. Hacquemand R, Choffat N, Jacquot L, Brand G. 2013. Comparison between low doses of TMT and cat odor exposure in anxiety- and fear-related behaviors in mice. Behav Brain Res 238:227–231. 5. Hebb AL, Zacharko RM, Gauthier M, Trudel F, Laforest S, Drolet G. 2004. Brief exposure to predator odor and resultant anxiety enhances mesocorticolimbic activity and enkephalin expression in CD1 mice. Eur J Neurosci 20:2415–2429. 6. Institute for Laboratory Animal Research. 2011. Guide for the care and use of laboratory animals, 8th ed. Washington (DC): National Academies Press. 7. Papes F, Logan DW, Stowers L. 2010. The vomeronasal organ mediates interspecies defensive behaviors through detection of protein pheromone homologs. Cell 141:692–703.
8. Snyder JS, Soumier A, Brewer M, Pickel J, Cameron HA. 2011. Adult hippocampal neurogenesis buffers stress responses and depressive behaviour. Nature 476:458–461. 9. Strekalova T, Steinbusch HW. 2010. Measuring behavior in mice with chronic stress depression paradigm. Prog Neuropsychopharmacol Biol Psychiatry 34:348–361. 10. Takahashi LK, Nakashima BR, Hong H, Watanabe K. 2005. The smell of danger: a behavioral and neural analysis of predator odor-induced fear. Neurosci Biobehav Rev 29:1157–1167. 11. United States Congress, Office of Technology Assessment. 1986. Alternatives to animal use in research, testing, and education. Washington (DC): US Government Printing Office.
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Lack of Fear Response in Mice (Mus musculus) Exposed to Human Urine Odor Germain F Rivard,1,* Emily G Moser,1 Steven P D’Ambrose,1 and David M Lin2 A goal of the Guide for the Care and Use of Laboratory Animals is to improve animal welfare by minimizing sources of fear, anxiety, and stress. As a result, it includes recommendations on overcrowding, frequency of cage changes, enrichment, and group housing. However, human odorants are a potential but unexplored source of fear, anxiety, and stress. Although mice have been maintained for decades for animal research, whether mice perceive humans as predators is unknown. If so, this would necessitate changes in animal care and use procedures to minimize this source of chronic fear, anxiety, and stress. Odorants from predator urine are well known to elicit strong fear responses in mice, leading to modification of animal behavior and elevated levels of stress. To begin asking whether human odors influence mouse behavior, we tested the effect of human urine odor on fear response in mice. We assessed mouse behavior by using a modified shuttle cage to record various parameters of mouse exposure to odorants. We found that mice displayed fear responses to 2,4,5-trimethylthiazoline, a synthetic analog of red fox feces, but no fear response to DMSO, the diluent for 2,4,5-trimethylthiazoline. In contrast, mice exposed to human urine samples showed no significant fear response. Abbreviation: TMT, 2,4,5-trimethylthiazoline.
The use of laboratory mice is an essential component of academic research. Although precise figures are not available, approximately 12 to 15 million laboratory rodents are used each year in the United States for biomedical research, testing, and education.11 Because of the importance of mice in research, standardized care and use guidelines have been developed for maintaining and monitoring mouse welfare.6 These guidelines include, for example, limits on the number of mice that can be housed together, appropriate environmental enrichment procedures, minimal frequency for cage changes, and recommendations for group housing. These standards are necessary for minimizing fear, anxiety, and stress and to promote general animal health, wellbeing, and welfare. A central component of care and use is providing appropriate training to caretakers and research members on the correct techniques for handling mice. This training emphasizes how best to handle mice in a manner that reduces potentially fearful and stressful interactions. Surprisingly, however, no studies have ever been performed to address whether humans themselves induce fear, anxiety, or stress in mice. Humans, like all mammals, produce secretions such as sweat, saliva, dander, feces, and urine. Such secretions are important olfactory cues for mice. For example, 2 volatile chemicals produced by foxes and omnivores (2,4,5-trimethylthiazoline [TMT] and 2-phenylethylamine) and 2 nonvolatile proteinaceous compounds produced by cats and rats (lipocalin Feld4 and Mup13, respectively) are known to elicit fear-like or aversive behavior in mice.3,7 Mice respond to these predator cues with a variety of behavioral changes, including risk assessment, freezing, attempts to escape, and avoidance.1,2,5,7,10 Chronic exposure to these stressful situations with no means of escape is likely to affect laboratory mouse behavior and welfare. Chronic fear, anxiety, and stress are well-known to have Received: 04 Jun 2013. Revision requested: 13 Aug 2013. Accepted: 10 Sep 2013. 1Department of Clinical Sciences, Veterinary Behavior Medicine and 2Department of Biomedical Sciences, Cornell University, Ithaca, New York. *Corresponding author. Email: [email protected]
multiple effects on animal health.6,8,9 In addition, these can alter the outcome of studies that depend on accurate cortisol levels, hormone release, metabolic measurements, and overall homeostasis. Determining causes of fear, anxiety, and stress that influence mouse behavior and welfare is therefore essential for establishing an appropriate baseline for many types of experiments. Here we ask whether exposure to human odor induces a fear response in mice. For these initial tests, we chose to study the effects of human urine on mouse behavior, because urine from predators has been studied extensively for its effects on mice.3,4,10 We predicted that, among all secretions, urine would generate the most extreme behavioral responses. Any observed effect would lay the groundwork for potential future studies looking at other human secretions, such as saliva, sweat, and dander.
Materials and Methods
Animals. The study was performed by using C57Bl/6 female mice (n = 18; age, 3 wk) donated by Harlan Laboratories (Indianapolis, IN). Mice arrived in 3 separate shipments (that is, lots). Mice were housed individually in static filter-top polycarbonate cages with stainless steel wire lid but without the filter on the top of the cage (Allentown Caging Equipment, Allentown, NJ). Bedding was composed of aspen shavings. Enrichment included the presence of PVC tubes and empty culture dishes in each cage. Animals were fed conventional mouse chow ad libitum with unlimited access to water from water bottles. Mice were housed individually for 7 to 10 d during the testing phase, to minimize human handling of mice, given that group-housed mice require individual identification (for example, ear punching or toe clipping) and more frequent cage changes, resulting in increased stress and handling. Mice were maintained under a reversed 12:12-h light:dark cycle. Testing occurred during the dark phase under red lights and by using an infrared camera. Testing was performed dur141
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ing the active phase to reduce stress caused by disturbing animals during their sleep–wake cycle. All mice were given 72 h to acclimate to their environment after delivery. Although all human contact could not be avoided, every effort was made to eliminate protein-binding odorants by using an enzymatic cleaner (Anti-Icky-Poo, Santa Clarita, CA) for hand washing and 70% alcohol for material preparation. Animal technicians were asked to wear personal protective equipment including Tyvek suits, facemasks, bonnets, booties, and gloves, and to use forceps to transfer mice. At time of testing, their cage was moved from the housing room into the antechamber. All experiments were approved by Cornell’s IACUC. Cornell University is AAALAC-accredited. Odorants and presentation. TMT (PheroTech, Delta, Canada) was diluted 1:10 with DMSO (Sigma Chemicals, St Louis, MO). Human urine was provided immediately prior to testing by the person conducting the test. The urine was collected in a sanitized glass jar, which was then placed in a 37 °C water bath. Samples were obtained within an hour of the actual test time. Urine was obtained from 2 college-age women, one of whom was an omnivore and the other a vegetarian. Controls (2 μL TMT or DMSO) and human urine samples (3 mL) were applied to a piece of filter paper (diameter, 35 mm) by using a micropipette; treated filter papers were placed in a culture dish and covered with a perforated lid (hole size, 1/16 to 3/32 in.) immediately before placing the culture dish in the test chamber. The culture dish was placed on the floor of the shuttle cage in the test chamber. The mouse could touch the culture plate, but the contents could not be accessed. Shuttle cage. We exposed mice to test and control odors by placing a mouse in a shuttle cage (Coulbourn Instruments, Whitehall, PA), which was divided into 2 chambers (that is, the test and control chambers). We modified the shuttle cage as follows. Black acrylic glass was placed on 3 sides of the shuttle cage, leaving one wall transparent for video recording. To prevent the introduced odorant from diffusing into the control chamber, vents on the tops of both chambers were opened to allow the odorant to escape from the test chamber. The wire floor was covered with a piece of acrylic glass. The shuttle cage was equipped with a computer program to control the opening and closing of the guillotine door and infrared sensors to detect movement between the 2 identical chambers. To reduce visual stimulation and arousal, tests were done during the dark phase of the reversed light cycle by using red light to allow for human but not mouse visibility. Red plastic covers were placed over the computer monitors, and an infrared camera was used to capture video. Experimental paradigm. Once all materials were in place, the mouse was transferred from its home cage to the control chamber by grasping the base of the mouse’s tail gently with forceps. The door remained closed for 2 min and then opened automatically and remained open for 3 min. During this final 3 min, the mouse had access to both chambers (Figure 1). Identical culture plates were present in both chambers, but odorant was present only in the plate inside the test chamber. After 3 min, the door automatically closed, and the mouse was immediately transferred from the shuttle cage back to its home cage. The culture dish was removed from the shuttle cage and discarded, and the shuttle cage, floor, forceps, and perforated lids were cleaned with isopropyl alcohol. The tester washed his or her hands with the enzymatic cleaner in preparation for subsequent tests. The perforated lids were reused, but with the same odorant.
Figure 1. Diagram of experimental paradigm. Mice are introduced into the control chamber, with the door of the shuttle cage closed. After 2 min, the door is opened, and the mouse is free to explore the test chamber containing the odorant for 3 min. The petri plate in the control chamber had no odorant.
Each mouse was tested, in order, with DMSO, human urine, and TMT. Mice were tested once each day with a single odorant, with a 24-h gap between tests to minimize stress. Eighteen mice were tested in this manner for 9 d, exposing each animal to each odorant 3 times. Fear assessment. Quantitative measures of mouse behavior were obtained by taking advantage of the shuttle cage’s ability to collect automated data. These include measurements of latency, number of entries, and cumulative time spent in the test chamber. These measures were interpreted as indicators of risk assessment and avoidance. In addition, qualitative measures of fear response were obtained by recording mouse behavior both prior to opening of the door and after odorant exposure. These recordings were randomized and scored by a blinded observer, a veterinarian who was completing a residency in Veterinary Behavioral Medicine at Cornell University at the time of this study. Criteria for observer-based assessment of fear response included risk assessment (slow approach, low head position, lifted tail, flat back, stretch–attend posture [that is, nose pointed toward stimulus while nearing the stimulus and resulting in an extended posture with a raised tail], and retraction), freezing (crouching, reduced forepaw–hindpaw distance, increased sniffing), attempts to escape and avoidance (characterized by hypervigilance with ears and nose constantly toward stimulus, rapid pacing, rapid retreat, seeking an escape route, frenzied wall migration, and digging) behaviors as compared with exploratory (increased exploration of the 2-chamber apparatus, standing, calm wall migration, normal forepaw–hindpaw distance, and nose pointed to different parts of chamber) nonfearful behaviors. To generate a fear severity score, the observer first underwent a training period during which all videos were reviewed blindly but not scored. The training period was done to establish the range of behavior observed in response to these odorants. Based on this initial calibration of behaviors, videos then were scored for fear response on a scale of 0 to 3: 0, mouse displayed only exploratory or nonfearful behavior; 1, observed fear behavior was mild in intensity and relatively brief (approximately 10% of the observed time); 2, fear behavior was moderately intense and lasted approximately 10% to 50% of the observed time; and 3, fear behavior was considered severe and accounted for more than 50% of the observation period. The observer viewed all videos of mice during the first 2 min (that is, no access to test chamber) twice and all videos of mice during the last 3 min (that is, door to test chamber open) 3 times. Scores of the same video were averaged to reduce bias associated with different viewings of the same video by a single observer.
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Lack of fear in mice exposed to human urine odor
Statistical analysis. To assess the dependent variables (that is, latency to enter test chamber, cumulative time spent in the test chamber, and number of entries into the test chamber), a mixed model was run by using experimenter–urine provider, number of times exposed to sample, mouse lot, and treatment as fixed effects and by controlling for individual mice as a random effect. Variables with P values greater than 0.05 were not considered statistically significant, and removed from the model. Values were corrected for multiple comparisons by using Bonferroni correction. A similar mixed model was run to assess the averaged severity fear scores for the dependent variables, first 2 min before the shuttle door opened, and last 3 min after the door opened, with the same fixed and random variables as listed earlier and the same criteria for removal from the model. Output included mean values, standard errors, significance, and pairwise comparisons of significant treatment (TMT compared with DMSO compared with urine) and lot effects. Some dependent variables were transformed to meet the assumption of the model. Statistics were determined by using Statistical Product and Service Solution software (IBM, Armonk, NY). A P value of less than 0.05 was considered statistically significant.
Results
Preliminary studies using commercial preparations of human dander, saliva, and urine suggested that urine was likely to have the greatest effect on mouse behavior (data not shown). Furthermore, a comparison of frozen, commercial urine and freshly obtained urine indicated fresh urine was more likely to elicit a fear response in mice. Mice were exposed, in order, to DMSO, freshly provided human urine, and TMT. Eighteen mice were tested, producing a total of 162 recorded behavioral responses. Of these, 20 videos were removed because of technical errors in the presentation of the odor or in the recording. These included trials in which the urine had been collected more than 1 d previously or was diluted and in which the control was not presented correctly according to the protocol. Of the remaining 142 videos, 47 recordings comprised mouse response to DMSO, 41 to human urine, and 54 to TMT. Statistical analysis of data that included the 20 removed trials still produced the same results (data not shown). We scored mouse behavior for fear response during the first 2 min prior to opening the automatic door. Observer assessment of fear response showed no obvious differences in mouse behavior on placement in the shuttle cage regardless of the odorant being tested (Figure 2; mixed model; P = 0.96). This finding is consistent with the interpretation that odors do not diffuse from the test chamber into the control chamber during the first 2 min prior to opening of the door. Mouse fear response during exposure to an odorant. We next asked whether mice displayed a fear response when exposed to the odorant during their final 3 min in the shuttle cage. We first looked at the quantitative information recorded by the shuttle cage. We could detect no differences for any of these measures among the 3 odorants (Figure 3; mixed-model analysis; latency, P = 0.31; cumulative time spent in test chamber, P = 0.21; number of entries into test chamber, P = 0.75). We then asked whether we could detect a fear response through double-blind scoring of mouse behavior during exposure to an odorant. Exposure of mice to TMT resulted in a significant fear response relative to exposure to DMSO (Figure 4; mixed model, P < 0.03). Mice responded to TMT whether they had been exposed once or multiple times, indicating a consistent fear response. Prior studies2,4,10 generally exposed mice once to TMT. We found that mice appear to show consistent responses
Figure 2. No significant differences in average fear response based on behavioral analysis were seen during the first 2 min prior to exposure to an odorant, regardless of the odorant that was to be tested.
to TMT over time and over multiple exposures. We found an additional significant factor in our tests of TMT. Mice were received in 3 separate lots from the vendor. Mice from one lot were less fearful when exposed to TMT than were those from the other lots (mixed-model analysis, P = 0.045). In addition, we asked whether human urine resulted in fear responses among mice. Video assessment of mouse response to urine was performed in a manner identical to that for TMT. However, we were unable to detect any difference in fear response among mice to human urine compared with DMSO (Figure 4; mixed model; P = 0.83). Our studies of human urine used samples obtained from 2 college-age women, one of whom was an omnivore and the other a vegetarian. We asked whether mice responded differentially to human urine as a consequence of diet. No significant difference (P = 0.45) was observed between urine from the omnivore compared with that of the vegetarian.
Discussion
To our knowledge, no studies have examined the effects of any human odor on mouse behavior. Our studies assessed, for the first time, whether human urine odor elicits an observable fear response in mice. We showed no detectable fear response in mice to human urine compared with DMSO odors. Although laboratory animal technicians handling mice are unlikely to routinely introduce mice to urine odors, we chose urine because we predicted that it would have the most dramatic effect on mouse behavior. We presented mice with a specific sequence of odorants. DMSO was presented first, followed by human urine and then TMT. This ordered presentation was performed to prevent ‘preconditioning’ the mice with a fear response to human urine because they had previously been exposed to TMT. We presented the same mouse with the same sequence of odorants 3 times. TMT is known to elicit an innate and consistent fear response in mice.2,4 Because whether human urine odor would induce a fear response was unknown, we were uncertain whether such a response would be innate or learned. We reasoned that if the response were learned, presenting the odorant multiple times would result in reduced fear response. However, if the response were innate, we expected that the severity of the response would remain high on all 3 exposures. We did not detect a quantitative or qualitative fear response among mice in response to human urine odor by using our paradigm. There could be multiple explanations for this lack of response, which was similar to that of mice to DMSO. First, human urine odor may not elicit any fear response in mice because humans are not recognized by mice as natural predators. Second, this lack of response might be due to the domestication of mice by humans. Selection aimed at adapting mice for study 143
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Figure 3. No significant differences in quantitative assessment (mean ± SEM) of (A) latency to enter the test chamber, (B) number of entries into the test chamber, or (C) cumulative time spent in the test chamber were observed in mice exposed to various odors.
Figure 4. Behavior-based assessment of fear response (mean ± SEM) showed significant differences in mouse behavior when presented with TMT as compared with DMSO. No significant differences were seen on exposure to human urine compared with DMSO odors.
as a model organism could, over time, result in the selective breeding of animals nonfearful to human exposure. Third, the lack of response may reflect adequate socialization of pups to humans during early development. Fourth, perhaps human urine odor does elicit a fear response in mice, but not one that is strong enough to be detected by using our paradigm. One final caveat is that these mice were handled by humans while at the vendor facility. This handling may have affected the fear response to human urine odor in our studies. One potential concern regarding our scoring system is that the degree of severity was determined according to the reviewer’s overall subjective assessment of fear. Nevertheless, we were able to clearly show differences in animal behavior upon exposure to TMT. Therefore, we anticipate that we could have identified differences upon exposure to human urine odor if the level of fear response among mice was similar to that of TMT. Although we were able to identify a fear response in mice exposed to TMT, we also discovered an unexpected effect. We obtained mice from the vendor on 3 separate occasions, and mice from each lot were tested in our shuttle cage. Mice from one particular lot showed less fear response than did those in the other 2. Why this occurred is unclear. All mice were from the
same vendor, the same age, and the same genetic background, and the same testers tested mice in all 3 lots. Therefore, we are unable to explain why this one lot of mice showed a different fear response. Perhaps these less-responsive mice were socialized more extensively or were from a different breeding room at the vendor facility. In summary, our study assessed the effect of human urine odor on fear response in mice. Because mice are an essential model for human disease, causes of variability that may bias potential results must be identified and eliminated. Our study is the first to assess whether exposure to human urine odor can induce a behavioral response in mice. The study found no overt response to fresh human urine. However, future studies may address other human secretions (for example, dander, saliva, sweat), perfumes, and masking agents, as well as additional strains of mice.
Acknowledgments
We thank Harlan Laboratories for donating the mice used in these experiments. We thank Thom Cleland for use of the shuttle cage and Francoise Vermeylen (Statistical Consulting Unit, Cornell University) for performing the statistical analysis. We thank Mary Martin (Center for Animal Research and Education, Cornell University) for helpful comments on the manuscript; Kelsey Piel, Katherine Walden, and Shanna Johnson for running shuttle-cage trials; and Laura Lin for assistance with illustrations. This work was funded by a grant from the Johns Hopkins Center for Alternatives to Animal Testing.
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