Genetic background differences and nonassociative effects in mouse trace fear conditioning.
Open accessAbstract
Fear conditioning, including variants such as delay and trace conditioning that depend on different neural systems, is widely used to behaviorally characterize genetically altered mice. We present data from three strains of mice, C57/BL6 (C57), 129/SvlmJ (129), and a hybrid strain of the two (F(1) hybrids), trained on various versions of a trace fear-conditioning protocol. The initial version was taken from the literature but included unpaired control groups to assess nonassociative effects on test performance. We observed high levels of nonassociative freezing in both contextual and cued test conditions. In particular, nonassociative freezing in unpaired control groups was equivalent to freezing shown by paired groups in the tests for trace conditioning. A number of pilot studies resulted in a new protocol that yielded strong context conditioning and low levels of nonassociative freezing in all mouse strains. During the trace-CS test in this protocol, freezing in unpaired controls remained low in all strains, and both the C57s and F(1) hybrids showed reliable associative trace fear conditioning. Trace conditioning, however, was not obtained in the 129 mice. Our findings indicate that caution is warranted in interpreting mouse fear-conditioning studies that lack control conditions to address nonassociative effects. They also reveal a final set of parameters that are important for minimizing such nonassociative effects and demonstrate strain differences across performance in mouse contextual and trace fear conditioning.
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Flight or fight is the sum total of distinct components activated concurrently: a somatomotor component, a visceromotor component, and a pain-modulation component.
“Studies suggest that flight or fight is the sum total of distinct components activated concurrently: a somatomotor (skeletal muscle) component, a visceromotor (autonomic) component, and a pain-modulation component (see Figures 1 and 2).24,45,46”
From Fear and the Defense Cascade (2015) · cites this paper as [45]
The paper’s introductory discussion (indexed under Abstract; page unavailable) describes conditioned fear as involving autonomic responses and cessation of movement or freezing, which loosely corresponds to the visceromotor and somatomotor components. It does not describe a concurrent three-component fight-or-flight response or a pain-modulation component; searches for the component terms and for pain modulation found no such passage. (LLM, conf=0.82)
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“…interval (ITI) length, and animal handling influence the prevalence of nonassociative plasticity in fear conditioning (Kamprath and Wotjak, 2004; Smith et al., 2007; Burman et al., 2014), we developed an alternate trace fear-conditioning procedure (Protocol 2) that minimized nonassociative…”
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“Ketamine- (n = 10) and saline-exposed (n = 9) mice were given six tone-foot shock pairings with a 18-s trace interval between them (Chowdhury et al., 2005; Misane et al., 2005; Smith et al., 2007).”
MethodInfluentialDeletion of Fmr1 results in sex‐specific changes in behavior
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“This is indeed the case, as the 129/Sv strain performs poorly relative to the C57Bl/6J strain across a number of hippocampus-dependent learning paradigms (Motkowski et al., 1997; Smith et al., 2007).”
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Schmidt, M. J., Horváth, S., Ebert, P. J., Norris, J. L., Seeley, E. H., Brown, J. A., Gellért, L., Everheart, M., Garbett, K. A., Grice, T. W., et al. (2013) · Molecular Psychiatry · doi:10.1038/mp.2013.167 · Cited by 35 · see citing works
Deficits in trace fear memory in a mouse model of the schizophrenia risk gene TCF4
Brzózka, M. M., & Rossner, M. J. (2012) · Behavioural Brain Research · doi:10.1016/j.bbr.2012.10.001 · Cited by 35 · see citing works
Trace Fear Conditioning in Mice
Lugo, J. N., Smith, G. D., & Holley, A. J. (2014) · Journal of Visualized Experiments · doi:10.3791/51180 · Cited by 34 · see citing works
Coutellier, L. D., & Usdin, T. B. (2011) · Behavioural Brain Research · doi:10.1016/j.bbr.2011.02.043 · Cited by 31 · see citing works
Postweaning, forebrain-specific perturbation of the oxytocin system impairs fear conditioning
Pagani, J. H., Lee, H.-J., & Young, W. S. (2011) · Genes, Brain and Behavior · doi:10.1111/j.1601-183x.2011.00709.x · Cited by 30 · see citing works
“This method of unpaired training results in very low levels of freezing to the tone during retention (Smith et al. 2007), although levels may be higher than during baseline for the reasons just mentioned.”
MethodDeveloping and validating trace fear conditioning protocols in C57BL/6 mice
Burman, M. A., Simmons, C. A., Hughes, M., & Lei, L. (2013) · Journal of Neuroscience Methods · doi:10.1016/j.jneumeth.2013.11.005 · Cited by 29 · see citing works
“Previous work conducting similar protocol development in mice demonstrated that nonassociative freezing levels were high in a variety of mouse strains (Hwang et al., 2010; Smith et al., 2007).”
BackgroundLearning and memory is modulated by cannabidiol when administered during trace fear-conditioning
Uhernik, A. L., Montoya, Z. T., Balkissoon, C. D., & Smith, J. P. (2018) · Neurobiology of Learning and Memory · doi:10.1016/j.nlm.2018.02.009 · Cited by 28 · see citing works
Gileadi, T. E., Swamy, A. K., Hore, Z. L., Horswell, S. D., Ellegood, J., Mohan, C., Mizuno, K., Lundebye, A., Giese, K.-P., Stockinger, B., et al. (2021) · Environmental Health Perspectives · doi:10.1289/ehp7352 · Cited by 26 · see citing works
“However, this difference was not significant [tð14Þ=2:131, p=0:051; Figure 4J] and freezing behavior was generally low, as previously reported for C57BL/6J mice (Smith et al. 2007; Vigil et al. 2017).”
ResultThe role of cannabinoid 1 receptor expressing interneurons in behavior
Brown, J. A., Horváth, S., Garbett, K. A., Schmidt, M. J., Everheart, M., Gellért, L., Ebert, P. J., & Mirnics, K. (2013) · Neurobiology of Disease · doi:10.1016/j.nbd.2013.11.001 · Cited by 25 · see citing works
Buhusi, M. C., Scripa, I., Williams, C. L., & Buhusi, C. V. (2013) · Timing & Time Perception · doi:10.1163/22134468-00002003 · Cited by 24 · see citing works
Disruption of Shmt1 Impairs Hippocampal Neurogenesis and Mnemonic Function in Mice1–3
Abarinov, E. V., Beaudin, A. E., Field, M. S., Perry, C. A., Allen, R. H., Stabler, S. P., & Stover, P. J. (2013) · Journal of Nutrition · doi:10.3945/jn.113.174417 · Cited by 21 · see citing works
Comparison of inbred mouse substrains reveals segregation of maladaptive fear phenotypes
Temme, S. J., Bell, R. Z., Pahumi, R., & Murphy, G. G. (2014) · Frontiers in Behavioral Neuroscience · doi:10.3389/fnbeh.2014.00282 · Cited by 21 · see citing works
“Based on previous literature (Smith et al., 2007; Matynia et al., 2008) and our prior work, strain specific shock intensities were used (0.5 mA for 129S1 and 129S6 mice and 0.75 mA for C57B6 mice) to elicit similar levels of freezing while preventing over/undertraining.”
InfluentialKim, D.-H., Jang, Y.-S., Jeon, W. K., & Han, J. (2019) · Experimental Neurobiology · doi:10.5607/en.2019.28.2.146 · Cited by 18 · see citing works
Ghoweri, A. O., Gagolewicz, P. J., Frazier, H. N., Gant, J. C., Andrew, R. D., Bennett, B. M., & Thibault, O. (2020) · Journal of Alzheimer s Disease · doi:10.3233/jad-200617 · Cited by 16 · see citing works
An assessment of olfaction and responses to novelty in three strains of mice
Smith, D. R., Burruss, D. R., & Johnson, A. W. (2009) · Behavioural Brain Research · doi:10.1016/j.bbr.2009.01.024 · Cited by 16 · see citing works
Sex differences in interval timing and attention to time in C57Bl/6J mice
Buhusi, M. C., Bartlett, M. J., & Buhusi, C. V. (2017) · Behavioural Brain Research · doi:10.1016/j.bbr.2017.02.023 · Cited by 15 · see citing works
“This raises questions in interpretation, since differences in performance are reported in different strains, genders, and paradigms: fear conditioning [6], spatial memory, and social behavior [7].”
BackgroundZhang, L., Chen, X., Sindreu, C. B., Song, L., Storm, D. R., Zweifel, L. S., & Xia, Z. (2019) · PLoS ONE · doi:10.1371/journal.pone.0219152 · Cited by 12 · see citing works
Herrmann, T., Gerth, M., Dittmann, R., Pensold, D., Ungelenk, M., Liebmann, L., & Hübner, C. A. (2022) · Frontiers in Molecular Neuroscience · doi:10.3389/fnmol.2021.807090 · Cited by 12 · see citing works
Hwang, Y. K., Song, J.-C., Han, S., Cho, J., Smith, D. R., Gallagher, M., & Han, J. (2010) · Brain Research · doi:10.1016/j.brainres.2010.05.048 · Cited by 11 · see citing works
“The considerable levels of freezing in the unpaired C57BL/6 mice would appear inconsistent with the finding of Smith et al. (2007) who reported much lower levels of freezing in unpaired C57BL/6 mice in a similar trace conditioning protocol.”
InfluentialMarch, A., Borchelt, D., Golde, T. E., & Janus, C. (2014) · PubMed Central · Cited by 9 · see citing works
Endres, K., Reinhardt, S., Geladaris, A., Knies, J., Grimm, M. O. W., Hartmann, T., & Schmitt, U. (2016) · BMC Neuroscience · doi:10.1186/s12868-016-0280-9 · Cited by 8 · see citing works
“Such peculiarities regarding behavioral tasks have already been reported in literature for different mouse strains [27].”
Roland, M., Berglas, E., Pines, R., Carata, I., Castillo, A., Nashed, M., Sclafani, A. J., & Bodnar, R. J. (2023) · Pharmacology Biochemistry and Behavior · doi:10.1016/j.pbb.2023.173514 · Cited by 3 · see citing works
Buhusi, C. V., Meyer, A. E., Oprisan, S. A., & Buhusi, M. C. (2022) · Timing & Time Perception · doi:10.1163/22134468-bja10052 · Cited by 1 · see citing works
Behavioral and Genetic Architecture of Fear Conditioning and Related PhenotypesPreprint
Zeid, D., Seemiller, L. R., Wagstaff, D. A., & Gould, T. J. (2021) · SSRN Electronic Journal · doi:10.1016/j.nlm.2023.107837 · Cited by 1 · see citing works
Eyeblink Conditioning – A Behavioral Model of Procedural and Declarative Learning
Disterhoft, J. F., & Weiss, C. (2017) · Elsevier eBooks · doi:10.1016/b978-0-12-809324-5.21087-0 · Cited by 1 · see citing works
Contribution of Opioid and Stress Systems to Impulsive Action in Rodent Models
Mahoney, M. K. (2015) · QSpace (Queen's University Library)
Lonsdorf, T. B. (2019) · Oxford University Press eBooks · doi:10.1093/oso/9780198793014.003.0009
Divergence in Neuronal Calcium Dysregulation in Brain Aging and Animal Models of AD
Ghoweri, A. O. (2020) · UKnowledge (University of Kentucky) · doi:10.13023/etd.2020.521
Vigil, J. J., Tiemeier, E., Chalmers, N. E., Herson, P. S., & Quillinan, N. (2025) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2025.01.28.635307
“The next day, animals were returned to context A for 2-minutes without presentation of CS or US to assess contextual fear and extinguish any non-associative cues(Poon & Schmid, 2012; Smith et al., 2007).”
- Authors
- Smith, D. R., Gallagher, M., & Stanton, M. E.
- Affiliations
- Johns Hopkins University (US)
- Venue
- Learning & Memory
- Publisher
- Cold Spring Harbor Laboratory
- Published
- 2007-09
- Volume
- 14
- Issue
- 9
- Pages
- 597-605
- ISSN
- 1072-0502 (ISSN-L)
- 1549-5485
- PMC
- PMC1994077
- OpenAlex
- W2119515211
- Cited by
- 47
- References
- 69
- Citations by year
- 2025: 1 · 2023: 2 · 2022: 2 · 2021: 2 · 2020: 2 · 2019: 4 · 2018: 1 · 2017: 4 · 2016: 2 · 2015: 4 · 2014: 4 · 2013: 6 · 2012: 2
- Type
- posted content
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Learning & Memory (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Neuroscience › Cognitive Neuroscience › Memory and Neural Mechanisms
- Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
- Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neurotransmitter Receptor Influence on Behavior
- Keywords
- trace fear conditioning, C57BL/6 mice, 129/Sv mice, F1 hybrids, contextual fear conditioning
- MeSH terms
- Acoustic Stimulation, Animals, Association Learning, Conditioning, Psychological, Fear, Hippocampus, Hybridization, Genetic, Male, Mice, Inbred C57BL, Species Specificity, Reflex, Startle, Mice
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