Here, we present a protocol to quantify nest building behavior in mice, which is known to be impaired in several neurological disorders and diseases. This protocol examines the utility of four materials and offers the opportunity to quantify the rater agreement in scoring, improving the validity and reliability of the assay.
Nest building is an innate behavior in male and female rodents, even when raised in laboratory settings. As such, many researchers provide rodents synthetic and/or natural materials (such as twine, tissue, cotton, paper, and hay) as a gauge of their overall well-being and as an ancillary assessment to predict the possible decline in cognition. Typically, changes in nesting behaviors, such as failure to create a nest, indicate a change in health or welfare. In addition, nesting behavior is sensitive to many environmental and physiological challenges, as well as many genetic mutations underlying pathological disease states. The following protocol describes a nesting behavior paradigm that explores the usage of four types of nesting material. In addition, the protocol utilizes intraclass correlations to demonstrate that inter-rater reliability is higher when nests are constructed out of shredded paper compared to other common nesting materials such as cotton squares, paper twists, and soft cob bedding. The chosen methodology and statistical considerations (i.e., intraclass correlation) for this assay may be of interest for those conducting experiments assessing the quality of living of mice.
Nest building is an innate behavior in many animals such as birds, fish, rodents, and great apes, and it is attracting more attention for its potential utility in the study of neurological diseases and disorders characterized by diminished well-being and impaired activities of daily living1. Mice, both male and female, build similarly-sized nests for reproductive purposes, heat conservation, and shelter; more importantly, they continue to do so even when raised in laboratory settings2. Hess, Petrovich, and Goodwin3 argue that biologically-appropriate stimuli are paramount to induce biologically-appropriate behaviors such as nesting; however, a variety of natural and artificial materials, such as hay, hemp, cotton twine, paper strips, cotton dental balls, and pressed cotton squares, have been used to assess nest building behavior2. Changes in these nesting behaviors (i.e., failure to create a nest out of the provided material) generally indicate a change in health or welfare. In most cases, a lack of nest building is attributed to several factors that negatively impact well-being. Such examples that have all effectively decreased the quality of nest building in mice include temperature extremes; painful stimulation; induced illness and infection; genetic mutations; and brain lesions in the medial preoptic areas, septum, and hippocampus4,5,6.
Alzheimer’s disease is a progressive neurodegenerative disease characterized by loss of brain tissue, accumulation of extracellular amyloid-β plaques, and intracellular neurofibrillary tangles composed hyperphosphorylated microtubule-associated protein, tau7. Additionally, Alzheimer’s disease is characterized by, most notably, deficits in learning and memory and impairments of activities of daily living. In mice, activities of daily living are commonly examined via circadian wheel-running8,9,10,11, although new alternatives, such as nesting, are growing in popularity. Nesting has been shown to be sensitive to manipulations (e.g., genetic mutations, environmental stressors) that have been identified as risk factors and/or causes of Alzheimer’s disease. As such, nesting can be used as an additional or complementary assay in many mouse strains that model those characteristics of Alzheimer’s disease. For example, Deacon and colleagues12 measured nest building of Tg2576 mice with the Swedish amyloid precursor protein (APPswe) and presenilin 1 (PS1) genetic mutations. The quality of nests constructed by group-housed Tg2576 mice was significantly worse than wildtype controls at both 3- and 12-months of age. In agreement with these findings, Filali et al.13 reported that singly housed APPswe/PS1 male mice given two pieces of 5 x 5 cm cotton built significantly poorer nests as rated on a scale of 1 - 5 (1 = nestlet not touched, 5 = a near perfect nest). Transgenic mice consistently built worse nests at 6-, 9-, and 12-months of age compared to their wildtype counterparts, and in some cases, the Appswe/PS1 mice completely failed to build a nest out of the provided cotton.
Previous research from our lab has demonstrated that wildtype C57BL/6J mice build significantly better nests out of cotton squares compared to CRND8 and CRND8/E4 mice9. However, the majority of experiments using pressed cotton squares appear to be variable, with wildtype mice failing to show expected high scores compared to transgenic mice that are expected to show very low scores in nesting2, which may in part lead to a lack of differences in estimated parameters (i.e., mean differences) and statistical significance. The lack of differences may be due in part to inadequate aging of mice or time allotted for nesting. Alternatively, nesting material may be an additional challenge, resulting in more variability due to researchers’ methodological preferences in the quantity and type of material, which may even interact with mouse strain. For example, Robinson-Junker and colleagues14 provided processed or unprocessed bedding material of different sizes (i.e., small or large flakes) to C3H/HeNCrl mice and BALB/cAnNCrl mice, which are commonly observed, respectively, as poor and strong nest builders. When provided unprocessed bedding, C3H mice built less complex, yet similar in quality nests compared to those of BALB/c mice. Likewise, Martin and colleagues15 compared nest complexity of different nesting materials given to deer mice, a distant relative of the Mus musculus species that have distinct evolutionary differences (i.e., more likely to burrow in trees and underbrush and are more active in captivity), but receive similar husbandry care as common laboratory mice and build nests out of any available soft, fibrous material16,17,18. Females and breeders with pups in the home cage built more complex nests than males, and the authors suggest that these behavioral differences may be due to associated changes in progesterone concentrations in deer mice15. More importantly, mice built more complex nests composed of brown paper followed by cotton squares and cotton cylinders, and the least complex nests were constructed out of white paper and dispersed mini-cotton squares.
Despite the growing popularity of nesting, considerations regarding scientifically valid, cost-effective, and time-sensitive practices are minimally discussed. Given the aforementioned methodological and economical challenges, this protocol investigates the utility of different nesting materials – cotton squares, paper twists, shredded paper, and processed bedding – in nesting behavior. Specifically, we provided all nesting materials to both aged C57BL/6J wildtype controls and Alzheimer’s disease APOE e4 mice in order to investigate any potential genotype by material interactions in nesting quality. Additionally, the experiment sought to assess inter-rater reliability of nests constructed out of different materials. Taken together, this protocol demonstrates the superiority of one nesting material in this sample – shredded paper – in terms of nest quality and scorer agreement, with the intention of improving the validity and reliability of the nesting assay.
All procedures were approved by the George Mason University Institutional Animal Care and Use Committee and are in accordance with guidelines set forth by the Assessment and Accreditation of Laboratory Animal Care.
1. Animals and considerations prior to the assessment
2. Room and nesting set-up
Figure 1: Cage set-up for each material. All mice completed one trial with each type of material for a total of four trials. Corncob bedding lined the entire bottom of the cages containing a paper twist, a pressed cotton square, and shredded paper. Soft cob bedding was evenly dispersed across the cage to encourage mice to separate out the small cotton squares from the corncob. Please click here to view a larger version of this figure.
3. Nesting trial
4. Scoring and assessing inter-rater reliability
Figure 2: Example of scoring using the criteria for shredded paper, the preferred material. From left to right: 1 – the shredded paper remained untouched; 2 – some of the paper was constructed into a nest but most of the pieces remained scattered; 3 – a noticeable nest was constructed, but several pieces were still scattered; 4 – most pieces were used for the nest, but some pieces were scattered near the nest; 5 – all pieces were used to make the nest. Note that in the photographs, the animal’s assigned number is shown to avoid unblinding. The color of the card is random. Researchers should allow the mouse to remain in the cage in order to avoid startling the mouse, which could potentially disperse the nest. Please click here to view a larger version of this figure.
5. Statistical considerations
Results from the four different nesting materials provided to wildtype and APOE e4 mice are explained as follows. Based on existing criteria, the ICC showed a strong agreement among all three coders for all four nesting materials (shredded paper ICC = 0.94; square ICC = 0.91; bedding ICC = 0.87; twist ICC = 0.87); therefore, the three scores were averaged together to create a single score for each material provided. A 2 x 4 mixed ANOVA yielded a significant main effect of genotype, F(1, 19) = 31.30, p < 0.01, ηp2 = 0.62. Across all four provided materials, wildtype mice scored higher on nest quality (3.18 ± 0.20) compared to APOE e4 mice (1.98 ± 0.16). In addition, the mixed ANOVA yielded a significant main effect of material, F(3, 57) = 57.48, p < 0.01, ηp2 = 0.75. Pairwise comparisons with Bonferroni correction showed that the shredded paper (4.11 ± 0.20) was rated significantly higher (p < 0.05) in quality than the square (1.95 ± 0.21), bedding (2.21 ± 0.21), or twist (1.94 ± 0.20) materials, with no differences seen between the square, bedding, and twist materials (all p > 0.99). There was no significant interaction between genotype and material. Data are shown in Figure 3.
Other experiments from our laboratory have yielded similar results in early-onset models of Alzheimer’s disease. For example, as demonstrated in Figure 4, 5.5-month-old P301L rTg4510 (htau) mice built significantly worse nests out of shredded paper compared to their age-matched wildtype counterparts31. Likewise, dual J20 (hAPP)/htau and single htau mice built poorer nests out of shredded paper compared to their wildtype and CAMKIIa-promoter only counterparts, both at 3.5- and 7-months of age32 (data not shown).
Figure 3: Rated quality of nests made from different materials provided to wildtype and APOE e4 mice. A main effect of genotype (*p < 0.01) demonstrated that wildtype mice consistently built better nests made from the shredded paper, pressed cotton squares, paper twist, and soft cob bedding compared to APOE e4 mice. The main effect of material (**p < 0.01) also demonstrated that nests constructed from the shredded paper were rated higher in quality compared to the three other materials. The shredded paper had the highest inter-rater reliability measure as assessed by ICC. Please click here to view a larger version of this figure.
Figure 4: Shredded paper nests built by wildtype and htau mice, on or off zinc water. (A) Representative nests constructed by 5.5-month-old wildtype and htau mice, on control (tap) water or water supplemented with 10 parts per million zinc (a biometal implicated in Alzheimer’s disease). Nests were scored on the 1 - 5 scale using the specified criteria. From left to right: wildtype + zinc water (4.54±0.52); wildtype + control water (4.15±0.80); htau + control water (3.08 ± 0.64); htau + zinc water (2.46±0.97). (B) A genotype*water interaction demonstrated that htau mice on zinc water constructed significantly worse nests compared to the other groups (*); in addition, simple effects showed that htau mice on zinc water built worse nests than htau mice on control water. Wildtype mice on control water and zinc water-built nests of higher, and similar quality, compared to their htau counterparts. Reprinted from Journal of Alzheimer’s Disease, Vol 64, Craven, KM, Kochen WR, Hernandez CM, Flinn JM, Zinc Exacerbates Tau Pathology in a Tau Mouse Model, 671-630, Copyright (2018), with permission from IOS Press31. The publication is available at IOS Press through http://dx.doi.org/10.3233/JAD-180151. Please click here to view a larger version of this figure.
Supplementary File 1: Supplemental Coding File – nesting.R Please click here to download this file.
Supplementary File 2: AD Study nesting scores.csv Please click here to download this file.
Nesting is an evolutionarily important rodent behavior and has been used to assess the activity of daily living and general well-being in mice2. The ease to conduct the test, its reliability, and its face validity make nesting a practical complement to many behavioral tests such as burrowing, circadian rhythm, and grooming. But, as nesting becomes more commonly utilized in the laboratory, the various combinations to conduct, quantify, and interpret nesting increase. As such, further research is needed to explore the best methodologically and practically sound procedures for nesting, such that the validity and reliability of the assay are not sacrificed for costs, time of testing, and other procedures that reduce testing burden.
Nest building quality is indeed sensitive to the type of bedding provided as well as genotype. Overall, wildtype mice built significantly better nests compared to APOE e4 mice regardless of the nesting material; however, both wildtype and APOE e4 mice build significantly higher quality nests out of shredded paper compared to the three other nesting materials. Other studies provide corroborating evidence regarding the shredded paper: mice constructed more complex nests out of shredded paper strips than with pressed cotton squares, tissue, and aspen bedding33. Furthermore, nests constructed out of shredded paper strips have been qualitatively evaluated as more "naturalistic" than those made of other materials, which are characterized by the shape of the nest itself and the height of the walls around the nest cavity in order to form a dome6,33. As such, selection of proper material for this assay is critical in order to better observe natural behavior in a relatively controlled environment, i.e., the laboratory setting. More importantly, although this protocol assessed nesting once at 9 – 12 months of age, we emphasize that nesting should be conducted at several ages. The simplicity of this protocol permits it to be conducted several times, ideally before the onset of deficits that accompany neurodegeneration. Repeated measurements afford the opportunity to document the likely causative role of neurodegeneration in decreased nesting ability.
Natural nest building has been shown to differ among mice of different background strains34, and as such, the overall quality and shape of the nest may differ not due to the transgene of interest, but due to the background strain. For example, the ancestors of C57BL/6 mice were considered "hole" nesters, whereas the ancestors of BALB/C mice were considered "surface" nesters35. Mice on the C3H background or crosses with this strain, such as the hybrid C3H/He-C57BL/6 with E4 used in Graybeal et al.9 are also considered to be poor nesters; thus, researchers should strongly consider using control mice on the same background as transgenic mice, which would overall improve the direct, causative role of the transgene(s), rather than the background, in neurodegeneration and subsequent deficits in nest building behavior.
Some experiments often utilize a single scorer to qualitatively judge the complexity of the nest; however, we make the argument to include more scorers, and more importantly, scorers blind to experimental conditions. Through this approach, we utilized three independent, blind scorers and an intraclass correlation to assess the agreement among the scorers, who, with basic training, yielded high intraclass correlations which were indicative of high inter-rater reliability and strong agreement regarding nest quality. Furthermore, scores of nests composed of shredded paper and cotton squares had the highest intraclass correlations, an indication of stronger agreement and greater consistency among the scorers. The strong agreement also provides justification to average the multiple scores together, a strategy haphazardly implemented in behavioral research. Although this strategy requires more individuals and, presumably, more time to score nests, it effectively reduces bias in qualitative assessments such as nesting.
The nesting materials used in this assay were approximately equal in price except for the soft cob bedding. Commercial bedding for nesting may be economically resourceful for some experimenters; however, Martin et al.15 note that cotton squares, when compared to other materials such as crinkled paper, is one of the most expensive materials available for purchase. This may be due to easy availability, storage, and administration, but other researchers may opt for similarly valid and reliable materials, especially when challenged by large numbers of animals in a facility, the number of nesting trials, time restrictions for scoring nests, and high cage costs. Thus, shredded paper may be a more feasible and appropriate option. In addition, data capture for our method can be conducted immediately (i.e., scoring in person), although photographs should be highly considered in order to record, save, and re-quantify nests, if desired for the purposes of assessing inter- and intra-rater reliability at later times. As noted, we strongly emphasize the inclusion of multiple scorers for "test, re-test" practices to assess for agreement, as these methodologically sound procedures are often overlooked.
In conclusion, we believe that the methodology and statistical considerations (i.e., intraclass correlation) for this assay may be of interest for experiments assessing the quality of living, general well-being, and activities of daily living in mice.
We would like to thank Kevin Quant, Mario Martinez, and Edwin Portillo for scoring nests, Rachel Barkey for assisting in the preparation for filming, and Stephen Lippi and Deborah Neely for proofreading this manuscript. We would also like to thank the Department of Psychology for covering the costs of cages for this experiment, and the Krasnow Institute Animal Facility caretakers for their support.
Name | Company | Catalog Number | Comments |
Corncob bedding | Envigo | 7092 | 1/8 in bedding for cotton squares, shredded paper, and paper twist trials |
Cotton Squares | Envigo | Iso-Blox | |
Diamond Twists | Envigo | 7979C.CS | Paper twists used in protocol |
Mouse - APOE4 e4 | Jackson Laboratories | #012307 | Homozygous APEO4 e4 mouse bred with hemizyous J20 mouse |
Mouse - C57BL/6J | Jackson Laboratories | #000664 | Wildtype mouse for controls |
Mouse - J20 | Jackson Laboratories | #006293 | Hemizygous mouse bred with the homozygous APOE e4 mouse to generate cross |
Rstudio | R Core Team | V1.1.463 | Run with R version 3.5.3 (2019-03-11) -- "Great Truth". Note: additional R Packages are included in provided code and can be installed from CRAN |
Soft Cob | Envigo | 7087C |
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