Limited Bedding and Nesting as a Model for Early-Life Adversity in Mice
Summary
This protocol describes an animal model for studying how early-life adversity, provoked by an impoverished environment and unpredictable maternal care during the early postnatal period, affects brain development and the future risk of mental disorders.
Abstract
Early-life adversity (ELA), such as abuse, neglect, lack of resources, and an unpredictable home environment, is a known risk factor for developing neuropsychiatric disorders such as depression. Animal models for ELA have been used to study the impact of chronic stress on brain development, and typically rely on manipulating the quality and/or quantity of maternal care, as this is the major source of early-life experiences in mammals, including humans. Here, a detailed protocol for employing the Limited Bedding and Nesting (LBN) model in mice is provided. This model mimics a low-resource environment, which provokes fragmented and unpredictable patterns of maternal care during a critical developmental window (postnatal days 2-9) by limiting the amount of nesting materials given to the dam to build a nest for her pups and separating the mice from the bedding via a mesh platform in the cage. Representative data are provided to illustrate the changes in maternal behavior, as well as the diminished pup weights and long-term changes in basal corticosterone levels, that result from the LBN model. As adults, offspring reared in the LBN environment have been shown to exhibit an aberrant stress response, cognitive deficits, and anhedonia-like behavior. Therefore, this model is an important tool to define how the maturation of stress-sensitive brain circuits is altered by ELA and results in long-term behavioral changes that confer vulnerability to mental disorders.
Introduction
The early postnatal period is a critical developmental window in which environmental influences can shift the trajectory of development. For example, early-life adversity (ELA) can alter brain development to provoke long-term changes in cognitive and emotional function. Examples of ELA include physical or emotional abuse, neglect, inadequate resources, and an unpredictable home environment occurring during childhood or adolescence1. It is known that ELA is a risk factor for developing disorders such as depression, substance use disorder, post-traumatic stress disorder (PTSD), and anxiety2,3,4,5. This is important given that the levels of childhood poverty in the US have more than doubled recently, from 5.2% in 2021 to 12.4% in 20226, and although poverty itself is not necessarily ELA, it does increase the probability of various types of ELA7.
Animal models have long been essential for understanding the effects of early-life stress on brain development and adult outcomes. The two main animal models used in recent years to dissect this phenomenon are maternal separation (MS) and an impoverished environment induced by limited bedding and nesting materials (LBN). MS was developed as a model of parental deprivation8. In it, rodent dams are taken away from their pups, usually for several hours, every day until weaning8. The MS paradigm has been found to result in depressive- and anxiety-like behaviors in adulthood9, as well as an aberrant response to chronic stress10,11. On the other hand, the LBN model, first developed in the Baram laboratory12, does not separate the dam from the pups, but rather modifies the environment in which the pups are reared, mimicking a low-resource environment12,13. Decreasing the amount of nesting material and preventing direct access to the bedding in this model results in disrupted maternal care from the dams3. Since robust and predictable maternal care is required for the proper development of cognitive and emotional brain circuits14, fragmented maternal care from LBN can result in a range of outcomes, including an over-active Hypothalamic-Pituitary-Adrenal (HPA) axis, shifted excitatory-inhibitory balance in multiple brain regions, increased corticotropin-releasing hormone (CRH) levels, and depressive-like behavior in the offspring13,15,16,17,18,19.
The exact mechanism by which ELA results in increased risk for neuropsychiatric disorders is not completely understood. It is thought to be related to alterations in the HPA axis circuitry19,20, and recent evidence shows that this may be caused by changes in microglial synaptic pruning19. The LBN model has been shown to be a crucial tool for understanding the perinatal environment's impact on brain development and long-term behavioral outcomes. Although this model was initially developed for rats, it has also been adapted for mice in order to take advantage of the existing transgenic tools12,13. Notably, the model is very similar in both species and provokes highly convergent outcomes, such as alterations in the HPA axis, cognitive deficits, and depressive-like behavior, thus highlighting its cross-species utility and translational potential. This article will provide a detailed description of how to employ the limited bedding and nesting model in mice, collecting and analyzing maternal behavior and offspring outcomes to validate the model's efficacy and the expected results.
Protocol
Representative Results
Discussion
This article provides a detailed protocol to apply the LBN model in mice. This model is an important tool for understanding how an ethologically and translationally relevant form of chronic stress in early life contributes to the development of neuropsychiatric disorders in the offspring13. It is also useful for studying maternal behavior and any changes in the dams' brain from a molecular, neuroendocrine, or circuit-based perspective24. For these types of questions, mu…
Divulgations
The authors have nothing to disclose.
Acknowledgements
This work was supported by NIMH K99/R00 Pathway to Independence Award #MH120327, Whitehall Foundation Grant #2022-08-051, and NARSAD Young Investigator Grant #31308 from the Brain & Behavior Research Foundation and The John and Polly Sparks Foundation. The authors would like to thank the Division of Animal Resources at Georgia State University for providing exceptional care to our animals., and Ryan Sleeth for his excellent technical support in setting up and maintaining our video management system. Dr. Bolton would also like to thank Dr. Tallie Z. Baram for excellent training in the proper implementation of the LBN model during her postdoctoral fellowship.
Materials
| 2-inch 4 MP 4x Zoom IR Mini PT Dome Network Camera | Hikvision | DS-2DE2A404IW-DE3(S6) | |
| Amazon Basics Aluminum Light Photography Tripod Stand with Case – Pack of 2, 2.8 – 6.7 Feet, 3.66 Pounds, Black | Amazon | From Amazon | |
| Blue Iris | Blue Iris Security | Optional video management software | |
| CAMVATE 1/4"-20 Mini Ball Head with Ceiling Mount for CCTV & Video Wall Monitors Mount – 1991 | Camvate | From Amazon | |
| Corn cob bedding | The Andersons | 4B | |
| Cotton nestlet | Ancare | NES3600 | |
| Mesh divider | McNICHOLS | 4700313244 | Standard, Aluminum, Alloy 3003-H14, 3/16" No. .032 Standard (Raised), 70% Open Area |
| Tendelux DI20 IR Illuminator | Tendelux | From Amazon |
References
- Warhaftig, G., Almeida, D., Turecki, G. Early life adversity across different cell- types in the brain. Neurosci Biobehav Rev. 148, 105113 (2023).
- Duffy, K. A., Mclaughlin, K. A., Green, P. A. Early life adversity and health-risk behaviors: Proposed psychological and neural mechanisms. Ann N Y AcadSci. 1428 (1), 151-169 (2018).
- Molet, J., et al. Fragmentation and high entropy of neonatal experience predict adolescent emotional outcome. Transl Psychiatry. 6 (1), e702 (2016).
- Garvin, M. M., Bolton, J. L. Sex-specific behavioral outcomes of early-life adversity and emerging microglia-dependent mechanisms. Front Behav Neurosci. 16, 1013865 (2022).
- Andersen, S. L. Neuroinflammation, early-life adversity, and brain development. Harv Rev Psychiatry. 30 (1), 24-39 (2022).
- Shrider, E. A., Creamer, J. . Poverty in the United States: 2022. , P60-P280 (2023).
- Roos, L. L., Wall-Wieler, E., Lee, J. B. Poverty and early childhood outcomes. Pediatrics. 143 (6), e20183426 (2019).
- Ader, R., Tatum, R., Beels, C. C. Social factors affecting emotionality and resistance to disease in animals: I. Age of separation from the mother and susceptibility to gastric ulcers in the rat. J Comp Physiol Psychol. 53 (5), 446-454 (1960).
- Nishi, M. Effects of early-life stress on the brain and behaviors: Implications of early maternal separation in rodents. Int J Mol Sci. 21 (19), 7212 (2020).
- Trujillo, V., Durando, P. E., Suárez, M. M. Maternal separation in early life modifies anxious behavior and fos and glucocorticoid receptor expression in limbic neurons after chronic stress in rats: Effects of tianeptine. Stress. 19 (1), 91-103 (2016).
- Yu, S., et al. Early life stress enhances the susceptibility to depression and interferes with neuroplasticity in the hippocampus of adolescent mice via regulating miR-34c-5p/SYT1 axis. J Psychiatr Res. 170, 262-276 (2023).
- Walker, C. D., et al. Chronic early life stress induced by limited bedding and nesting (LBN) material in rodents: Critical considerations of methodology, outcomes and translational potential. Stress. 20 (5), 421-448 (2017).
- Rice, C. J., Sandman, C. A., Lenjavi, M. R., Baram, T. Z. A novel mouse model for acute and long-lasting consequences of early life stress. Endocrinology. 149 (10), 4892-4900 (2008).
- Glynn, L. M., Baram, T. Z. The influence of unpredictable, fragmented parental signals on the developing brain. Front Neuroendocrinol. 53, 100736 (2019).
- Karst, H., et al. Acceleration of GABA-switch after early life stress changes mouse prefrontal glutamatergic transmission. Neuropharmacology. 234, 109543 (2023).
- Demaestri, C., et al. Resource scarcity but not maternal separation provokes unpredictable maternal care sequences in mice and both upregulate CRH-associated gene expression in the amygdala. Neurobiol Stress. 20, 100484 (2022).
- Breton, J. M., et al. Early life adversity reduces affiliative behavior with a stressed cagemate and leads to sex-specific alterations in corticosterone responses in adult mice. Horm Behav. 158, 105464 (2023).
- Bath, K. G., Manzano-Nieves, G., Goodwill, H. Early life stress accelerates behavioral and neural maturation of the hippocampus in male mice. Horm Behav. 82, 64-71 (2016).
- Bolton, J. L., et al. Early stress-induced impaired microglial pruning of excitatory synapses on immature CRH-expressing neurons provokes aberrant adult stress responses. Cell Rep. 38 (13), 110600 (2022).
- Dahmen, B., et al. Effects of early-life adversity on hippocampal structures and associated HPA axis functions. Dev Neurosci. 40 (1), 13-22 (2018).
- Bolton, J. L., Short, A. K., Simeone, K. A., Daglian, J., Baram, T. Z. Programming of stress-sensitive neurons and circuits by early-life experiences. Front Behav Neurosci. 13, 30 (2019).
- Yang, M., Lewis, F., Foley, G., Crawley, J. N. In tribute to Bob Blanchard: Divergent behavioral phenotypes of 16p11.2 deletion mice reared in same-genotype versus mixed-genotype cages. Physiol Behav. 146, 16-27 (2015).
- Vegetabile, B. G., Stout-Oswald, S. A., Davis, E. P., Baram, T. Z., Stern, H. S. Estimating the entropy rate of finite Markov chains with application to behavior studies. J Educ Behav Stat. 44 (3), 282-308 (2019).
- Rincón-Cortés, M., Grace, A. Postpartum scarcity-adversity disrupts maternal behavior and induces a hypodopaminergic state in the rat dam and adult female offspring. Neuropsychopharmacology. 47 (2), 488-496 (2022).
- Gallo, M., et al. Limited bedding and nesting induces maternal behavior resembling both hypervigilance and abuse. Front behav neurosci. 13, 167 (2019).
- Manzano Nieves, G., Bravo, M., Baskoylu, S., Bath, K. G. Early life adversity decreases pre-adolescent fear expression by accelerating amygdala pv cell development. eLife. 9, e55263 (2020).
- Johnson, F. K., et al. Amygdala hyper-connectivity in a mouse model of unpredictable early life stress. Transl Psychiatry. 8 (1), 49 (2018).
- Demaestri, C., et al. Type of early life adversity confers differential, sex-dependent effects on early maturational milestones in mice. Horm Behav. 124, 104763 (2020).
- Reemst, K., et al. Molecular underpinnings of programming by early-life stress and the protective effects of early dietary ω6/ω3 ratio, basally and in response to LPS: Integrated mRNA-miRNAs approach. Brain Behav Immun. 117, 283-297 (2024).
- Reemst, K., et al. Early-life stress and dietary fatty acids impact the brain lipid/oxylipin profile into adulthood, basally and in response to LPS. Front Immunol. 13, 967437 (2022).
- Reemst, K., et al. Early-life stress lastingly impacts microglial transcriptome and function under basal and immune-challenged conditions. Transl Psychiatry. 12 (1), 507 (2022).
- Wang, T., et al. The nucleus accumbens CRH-CRHR1 system mediates early-life stress-induced sleep disturbance and dendritic atrophy in the adult mouse. Neurosci Bull. 39 (1), 41-56 (2023).
- Knop, J., Van, I. M. H., Bakermans-Kranenburg, M. J., Joëls, M., Van Der Veen, R. Maternal care of heterozygous dopamine receptor d4 knockout mice: Differential susceptibility to early-life rearing conditions. Genes Brain Behav. 19 (7), e12655 (2020).
- Bennett, S. N., Chang, A. B., Rogers, F. D., Jones, P., Peña, C. J. Thyroid hormones mediate the impact of early-life stress on ventral tegmental area gene expression and behavior. Horm Behav. 159, 105472 (2024).
- Parel, S. T., et al. Transcriptional signatures of early-life stress and antidepressant treatment efficacy. Proc Natl Acad Sci U S A. 120 (49), e2305776120 (2023).
- Julie-Anne, B., et al. Reactivation of early-life stress-sensitive neuronal ensembles contributes to lifelong stress hypersensitivity. J Neurosci. 43 (34), 5996 (2023).
- Bolton Jessica, L., et al. Maternal stress and effects of prenatal air pollution on offspring mental health outcomes in mice. Environ Health Perspect. 121 (9), 1075-1082 (2013).
- Block, C. L., et al. Prenatal environmental stressors impair postnatal microglia function and adult behavior in males. Cell Rep. 40 (5), 111161 (2022).
- Peña, C. J., et al. Early life stress alters transcriptomic patterning across reward circuitry in male and female mice. Nat Commun. 10 (1), 5098 (2019).
- Lapp, H. E., Salazar, M. G., Champagne, F. A. Automated maternal behavior during early life in rodents (amber) pipeline. Sci Rep. 13 (1), 18277 (2023).
- Madison, F. N., Palin, N., Whitaker, A., Glasper, E. R. Sex-specific effects of neonatal paternal deprivation on microglial cell density in adult California mouse (Peromyscus californicus) dentate gyrus. Brain, Behav. Immun. 106, 1-10 (2022).
- Walker, S. L., Sud, N., Beyene, R., Palin, N., Glasper, E. R. Paternal deprivation induces vigilance-avoidant behavior and accompanies sex-specific alterations in stress reactivity and central proinflammatory cytokine response in California mice (Peromyscus californicus). Psychopharmacology. 240 (11), 2317-2334 (2023).
- Molet, J., Maras, P. M., Avishai-Eliner, S., Baram, T. Z. Naturalistic rodent models of chronic early-life stress. Dev Psychobiol. 56 (8), 1675-1688 (2014).
- Tsuchimine, S., et al. Comparison of physiological and behavioral responses to chronic restraint stress between C57BL/6J and balb/c mice. Biochem Biophys Res Commun. 525 (1), 33-38 (2020).
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