Journal of Modern Foreign Psychology
2024. Vol. 13, no. 1, 78–91
doi:10.17759/jmfp.2024130107
ISSN: 2304-4977 (online)
Experimental Design and Behavioral Testing Protocol for the Evaluation of Cognitive Abilities and Social Behavior in Mice Following Early Life Stress
Abstract
This manuscript presents a protocol designed for the comprehensive investigation of early life stress (ELS) outcomes and a feasibility study conducted with this protocol. ELS alters normal development by interfering at various levels: hormonal changes, brain cellular architecture, epigenome, and chromosomal structural elements. The protocol combines classic behavioral tests with advanced molecular techniques to obtain comprehensive data and thus uncover the underlying mechanisms of ELS. In this protocol, the main source of stress is maternal separation. Briefly, a group of C57Bl/6 mice undergoes maternal separation; then, mice perform the radial maze test and the resident-intruder test. As a control, another group of mice stays undisturbed and performs the same behavioral tests in the same timeframe. After the behavioral tests, biosamples are collected, including urine for corticosterone measurements, peripheral blood, hippocampus, amygdala, and prefrontal cortex tissues for DNA isolation and its downstream analyses (DNA methylation profiling and telomere length measuring), and whole brains for immunohistochemistry analysis of the glucocorticoid receptor density. This protocol was successfully tested as a feasibility study for a large-scale investigation that addresses potential flaws to establish a robust methodology. This paper reports on a comprehensive approach to examining multiple aspects of development that interrogates a holistic analysis of multilayer and multidimensional data and may contribute valuable insights for both animal and human studies.
General Information
Keywords: early life stress, working memory, animal models, HPA axis
Journal rubric: Neurosciences and Cognitive Studies
Article type: scientific article
DOI: https://doi.org/10.17759/jmfp.2024130107
Funding. This research was supported by the award from the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) to the University of Houston and the Texas Center for Learning Disabilities (P50HD052117, PI: Jack Fletcher).
Acknowledgements. The authors are grateful to Drs. Olga Burenkova (Mason Laboratory, Department of Integrative Biology, University of Guelph), Fatin Atrooz, and Samina Salim (College of Pharmacy, University of Houston) for their assistance in designing, preparing, and executing the described experiments, and Aidan Nichols, Noelle Nguyen, Harini Kanamarlapudi, and Wu Wen-Wen (undergraduate students, University of Houston) for their assistance in performing the maternal separation procedure.
Received: 31.01.2024
Accepted:
For citation: Khafizova G.V., Naumova O.Y., Lopez A.L. III, Grigorenko E.L. Experimental Design and Behavioral Testing Protocol for the Evaluation of Cognitive Abilities and Social Behavior in Mice Following Early Life Stress [Elektronnyi resurs]. Sovremennaia zarubezhnaia psikhologiia = Journal of Modern Foreign Psychology, 2024. Vol. 13, no. 1, pp. 78–91. DOI: 10.17759/jmfp.2024130107.
Full text
Introduction
Materials and Methods
Animals
Maternal separation
Behavioral assessments
Radial arm maze test
Social defeat paradigm
Urine collecting for corticosterone measurement
Blood collecting for methylation profiling
Brain sectioning for methylation profiling and telomere length analysis
Brain dissection for immunohistochemistry (IHC)
Results
PND |
Females (grams; ± st.dev) |
Males (grams; ± st.dev) |
||
|
Control nest |
Jackson Lab |
MS nest |
Jackson Lab |
31 |
12.5 ± 1.29 |
14.7 ± 1.8 |
11.7 ± 3.5 |
16.5 ± 2.6 |
35 |
13.5 ± 2.1 |
17.8 ± 1.1 |
13.3 ± 2.5 |
20.7 ± 1.8 |
47 |
17 ± 0.58 |
18.75 ± 0.95 |
17.7 ± 2.1 |
22.75 ± 1.65 |
Fig. 2. The experiment roadmap. A) Dam with litter. At this stage, the number of pups in a litter is evaluated; litters with less than 3 pups are excluded from the protocol; B) During the MS, developmental milestones (eyes opening, ear positioning, and hair growth) are tracked; C) In radial maze test the number of re-entries in the baited arm for each animal is counted; D) In SD test the number and duration of attacks is noted, as well as specific submissive poses. Urine samples are collected and processed for the enzyme-linked immunosorbent assay (ELISA) to measure the amount of corticosterone in a sample; the ELISA is performed according to the protocol described in [4]; E) Blood sample is collected using cardiac puncture for subsequent DNA extraction followed by telomere length analysis using a quantitative real-time PCR (the protocol is described in [16]), and epigenotyping via genome-wide DNA methylation array. Prior to the brain tissue collection, the MS and control animals are randomly assigned to two groups for 2 different downstream analyses of the target brain regions — amygdala, hippocampus, and prefrontal cortex: (1) immunohistochemical analysis (see Methods, [29]) of GR density according to the manual described in [14], and (2) DNA extraction followed by the telomere length measurement and DNA methylation profiling using the above molecular genetic techniques.
Discussion
- While the first set of 4 litters undergoes MS, an order for 4 pregnant dams should be placed so they are delivered near the end of MS.
- When the first set is weaned and left to grow (for approximately 2 weeks), the second set of 4 litters is ready for MS.
- When the second set of litters is weaned and left to grow, the first set can perform behavioral experiments. Thus, the researchers have two weeks to conduct the maze test and the SD test and to sacrifice animals. Having completed this first round, they can engage the second set of mice. As a result, nine weeks are needed to process approximately 50 mice. This number can be increased if there are multiple rooms for MS, behavior rooms, and enough personnel.
Conclusion
References
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