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Non-Maternal Nesting

By July 8, 2019August 3rd, 2019No Comments


Non-maternal nesting is a nesting behavior carried out by a mouse when it builds its nest out of nesting materials. Nesting is a complicated behavior comprised of other behaviors that make it possible for a mouse to build its own safe, warm refuge. 


Non-maternal nesting is different from maternal nesting wherein a nest is built specifically for reproductive purposes. Another term for non-maternal nesting which is commonly used in the scientific literature is “thermoregulatory nesting” since the main function of this type of nesting behavior is to stay warm and regulate body heat. 

Non-maternal nesting, herein referred to as ‘nesting’, is comprised of a complex behavioral chain wherein several behaviors are performed in order to build the nest. 

The most commonly used outbred and inbred laboratory strains display nest-building behavior. 

Nesting Behaviors Mirror Humans’ Activities of Daily Living 

Activities of daily living (ADL) in humans are tremendously compromised by clinical conditions, such as stroke or chronic pain or Alzheimer’s. In these conditions, people cannot do the activities they did on a daily basis with the same ease as they had prior to their disabilities.   

In mice, nesting, due to its behavioral diversity and highly motivated nature, mirrors ADL in humans. Since nest building is associated with ADL, normal nesting behaviors are indicative of good performance, well-being, and healthy functioning. Thus, nest building behavior would be a desirable result (or behavior of interest) in experiments that are trying to restore ADL in disease models. 

Behaviors Comprising Nesting 

Nesting is a complex behavior that is made possible by a subset of other aligned behaviors. During nesting, the following behaviors are likely to be observed: 

  • Digging. When digging, a mouse is using its fore- and hind-limbs to displace materials. Digging is an essential behavior for nesting and is also often observed when a mouse is burrowing. 
  • Shoveling. Shoveling differs from digging in that the mouse does not use its forelimbs as it does when digging. Instead, when shoveling, the mouse uses its head and snout to move the bedding.  
  • Push-digging. Push-digging is performed in order to move adjacent materials away from the nest instead of towards it. So, a mouse will push-dig any material to the sides of the cage or somewhere away from the nest’s location. Push-digging may be observed in combination with forward movement or locomotion.   
  • Carrying. A mouse will carry materials to the nest location, in order to be able to use those materials to build the nest. This is an important behavior since it is implicated in the actual supply of materials that are necessary for composing a nest. 
  • Fraying. When a mouse is ripping apart material into smaller pieces by biting or gnawing at the material, it is known as fraying. The resulting torn materials are then used as pieces while nesting and constructing a nest. This resourceful behavior is useful because it demonstrates that a mouse can manipulate materials in order to form shelter and meet one of its basic needs. 
  • Sorting. Sorting is a deliberate action where a mouse is organizing its nesting material. When sorting, the mouse will place the material is specific locations. For example, a mouse may organize bedding material in order based on size, aligning bedding pieces from small to large. 
  • Pulling-in. When pulling-in, a mouse is pulling nesting material toward the nest. The mouse remains in the nest and does not move away from the nest in order to bring in material.
  • Fluffing. Fluffing is a means of enlarging the nest while the mouse is located inside the nest. When a mouse is fluffing, the nest will almost seem like it’s jumping or moving. 

The behaviors may occur in this order or they may follow a similar pattern in which some behaviors are repeated.maternal

Factors Influencing Nesting 

Some factors have been established as being influential on nesting behavior:

  • Temperature levels. One study showed that when C57BL/6 mice are held in cages with different temperatures (20, 25, or 30 °C), an effect on nesting behavior is observed. There is an inverse relationship between nest score and temperature. As temperature increases, the nest score decreases. The nest becomes more open and the nest’s walls are lower, thus leading to more heat loss. 
  • Amount of cage material. When mice are given about 6-10 grams of nesting material, mice will naturally initiate nesting behaviors. 
  • Type of cage material. When given different cage materials, there is a relationship between the materials that the mice are provided with and the outcome of the nest. For example, C57BL/6J mice, when provided with shredded paper strips, create a higher quality nest than they otherwise would with any other material. The nests that were made using tissues were of intermediate quality while the nest composed of cotton squares were poor in quality.

Functions of the Non-Maternal Nesting Behavior

Nesting serves many functions since nests: 

  • Provide shelter.  Nests protect mice from adverse weather conditions and thus prolong their survival time by shielding them from the elements, ultimately reducing their risk of becoming ill. 
  • Offer heat conservation. Since mice are sensitive to cold conditions, nests provide a means of heat conservation. Therefore, nests inadvertently reduce the stress that derives as a result of cold exposure. In laboratories, mice that are housed at standard laboratory temperatures (20 to 26°C) have increased tumor growth as well as reduced adaptive immunity when compared to mice that are housed in more thermoneutral conditions (about 30°C). 
  • Preserve the mice’s energy. If a mouse is cold, then its energy is used for keeping the organism and body warm. Thus, energy is used for the purposes of staying warm rather than for other homeostatic functions, such as restoration. Therefore, nests are a means for mice to stay warm and preserve their energy to be used for other bodily functions. 
  • Are protective. Nests, due to their shape and form, keep mice unseen and hidden from competitors and predators. If potential predators cannot see mice, this reduces their chances of detection and thus increases their chances of survival.  
  • May be a physiological need. In experimental studies, mice show a strong drive to collect and gather material. Even if the temperature is high enough and the mice may not need to insulate, they will still gather materials and create a nest. 
  • Facilitate homeostatic goals. Since nesting is so important to mice, it may be understood as a means of maintaining homeostasis which is for obvious reasons crucial for survival. 
  • Reduce exposure to stressors. In general, nests are able to shield mice from stressors, enabling them to maintain low-stress levels and rest adequately, ultimately helping them to survive for a longer. 

Based on these factors, the function of nesting is to help increase survival by benefiting the mouse across a variety of situations. 

Application of Nesting

Since nesting is such a vital part of mouse behavior due to its importance for survival, it will be observed under laboratory conditions. However, certain conditions are known to be able to elicit nesting, including: 

  • Cold conditions. Cold conditions and temperatures trigger nesting because nesting is a means for the mouse to keep warm. 
  • Access to materials. When materials are present, a mouse is very likely to use them for the purposes of creating a nest. 

Research Techniques Used for Studying Nesting

  • Genetic studies. Genetic studies can be used for the purposes of studying nesting in mice. Researchers can learn more about the relationship between genetics and nesting capabilities through the use of genetic manipulation and genetically modified mice. Well-designed genetic studies are able to reach the conclusion of how genetics are influential on nesting behaviors which may ultimately lead to insights of how to improve abnormalities which influence nesting. 
  • Pharmaceutical studies. Pharmaceutical studies are also a means of studying nesting. By giving mice a supplement of drugs or medicine, researchers are able to identify and study how nesting is affected by certain classes of drugs. Pharmaceutical studies are often combined with genetic studies, which offer genetic disease models, in order to study the impact of drugs on nesting behaviors.
  • Behavioral studies. In behavioral studies, researchers subject mice to behavioral tests which are carefully selected based on their validity of studying the desired behavior. Behavioral studies are often combined with elements of genetic and pharmaceutical studies, in order to assess specifically how genetics and drugs affect nesting behaviors.

Behavioral Tests for Assessing Nesting Behavior

Nest building performance can be used to monitor impairments (or progress) in a mouse’s behavior by means of quantifying and measuring the nest and the behaviors involved. 

Typically, scores with several grade levels may be used which will categorize the nest depending on whether a nest exists to whether the nest has high walls that surround the mouse. Some researchers will measure the nest’s height and calculate the percentage of used or unused nesting material. 

Scoring Nests

Nest scoring can occur in different ways, it is important to research and decide the protocol you want to follow based on your research question. Two common scoring methods include the 5-point scale and the 4-point scale.

4-Point Scale for Scoring Mouse Nests

  • 0: mouse has not created a nest at all
  • 1: mouse has created a primitive nest, mostly flat and contains almost the whole flat paper tissue
  • 2: mouse has created a slightly more complex nest by biting and warping the paper towel or nesting material
  • 3: mouse has created an accurate and complex cup-shaped nest by shredding the paper towel or nesting material and interweaving it in order to create the cup-shaped nest’s walls
  • 4: mouse has successfully created a complex hooded nest, including walls that create a ceiling and a single opening as the entrance. 

5-Point Scale for Scoring Mouse Nests

  • 1: the nestlet material remains largely untouched (>90% of the nestlet remains intact). 
  • 2: the nestlet is only partly torn up (50-90% of the nestlet remains intact). 
  • 3: the majority of the nestlet material is torn up, but an identifiable nest is lacking. The nestlet is basically scattered around the cage and there is not a properly formed nest in sight. 
  • 4: almost all of the nestlet is torn up (>90%) and there is an identifiable and flat nest present. Even though the nest is gathered up, the nest remains flat. 
  • 5: almost all of the nestlet is torn up (>90%) and there is a nearly perfect nest created in the cage.

This system is often supplemented with weighing the amount of nestlet material that remained untorn. 

Scoring Nest Working Out Activity

It is also possible to measure how much activity took place during nest building. 

“Paperwork” Assessment

The “paperwork” assessment involves measuring how much paperwork was done by the mice while nest building. This assessment can gauge the amount of biting and shredding that the mice performed while nest building and is oftentimes used to supplement scoring in nest completeness. 

The following grading system is used for the “paperwork” assessment:

  • 0: the paper is largely intact and has little damage (<5% of the paper is destroyed)
  • 1: some paper damage is apparent (5-20% is destroyed)
  • 2: evident and pronounced paper damage (20-40% of the paper is destroyed)
  • 3: severe paper damage has occurred (>40%)

In order to know in which category the nest falls, each nest must be unwrapped, fixed by four corners, and glued to a sheet of black paper. 

Nesting behavior: A means for assessing hippocampus

Nesting behavior, in itself, can be considered as a test that measures cognition that is related to the hippocampus. Nesting behavior is a proxy for identifying and establishing the integrity of the hippocampus. 

Research findings, such as the study conducted by Lin et al. in 2007, have shown that mice have neurons located in the hippocampus which fire specifically when a mouse is perceiving a nest. Thus, the hippocampus is deeply involved in the nest-building process due to its functional role in perceiving nests.

Pharmaceutical Impact on Non-Maternal Nesting

Xamoterol Improves Nest Building in Down Syndrome Mice

A norepinephrine precursor, L-threo-3,4-dihydroxyphenylserine (xamoterol), improves nest building in Ts65Dn mice which model Down syndrome. Xamoterol, a partial agonist for the β1-adrenergic receptor, improves Ts65Dn mice’s nest performance by increasing the amount of material they used to form the nest and improving the nests’ form. 

Alzheimer’s Mice’s Nesting is Improved By Bexarotene

Alzheimer’s mice typically have problems in their nest-building abilities. One drug that reverses this inability is bexarotene. Bexarotene, also referred to as ‘Targretin,’ is a retinoid X receptor (RXR) agonist that can move across the blood-brain barrier and has been approved by the U.S. Food and Drug Administration. RXR agonists may be able to clear β-amyloid (Aβ) build up within the brain, which is typical in Alzheimer’s disease. When Alzheimer’s Tg2576 mice are given bexarotene, their nest construction is significantly improved just after 3 days of treatment, receiving bexarotene at 100 mg/kg/day. 

Donepezil Increases Nesting Behavior in Autistic Mice

Typically, ASD mice have very low scores in the nesting test, building poorer nests than control mice. Donepezil is an acetylcholinesterase inhibitor and therefore prevents the breakdown of acetylcholine, a neurotransmitter that is already low in patients with autism spectrum disorder (ASD). When autistic mice, induced with ASD via the well-validated method of valproic acid injection, are given a donepezil dose of 0.3 mg/kg daily and subchronically, nesting abilities are improved. ASD mice receiving donepezil roughly averaged a nesting score of 5 (the highest possible) while non-treated ASD mice received a score of 3.5, significantly lower than the treated ASD mice’s score. 

Mouse Strains


C57BL/6 mice are used as comparator or control mice in behavioral studies because these mice are considered to have normal nesting habits. The majority of C57BL/6 mice are expected to score 4-5 on nest construction using the 5-point scale system described previously. 

A hippocampus lesion (which will be discussed later under the “Abnormalities” section) alters nesting behaviors. C57BL/6 mice that have had a hippocampus lesion will have a median score of 1-2 on nest construction, also on a 5-point scale system, and are highly unlikely to exceed having a score of 3. 

Tg2576 mice

Tg2576 mice are transgenic mouse models of Alzheimer’s disease. These mice have the human mutation of the amyloid-β precursor protein (APP) overexpressed. These mice, when supplied paper towels to their home cages, fail to construct nests. This inability is further modulated by increasing age, an effect that is not present in their wild-type counterparts. Even when Tg2576 mice are subjected to a colder than normal environment, they do not begin to nest. 

3xTg-AD mice

3xTg-AD mice have triple mutations in the APP, PS1 and Mapt genes and represent rarer forms of Alzheimer’s disease. 3xTg-AD mice have Aβ plaques and neurofibrillary tangles which are similar to those in human patients who have Alzheimer’s disease. 3xTg-AD mice do not display any differences from non-transgenic controls when it comes to nest building, although they do perform worse than the non-transgenic controls in other behavioral tests, such as Passive/Avoidance and the T-Maze Reversal tests. 

Ts65Dn mice

Ts65Dn mice are predominantly used for modeling Down syndrome and display cognitive deficits similar to those found in humans with Down syndrome, such as impairments in contextual learning. Ts65Dn mice are genetically manipulated to have Down syndrome and are trisomic for a fragment of the mouse chromosome 16 which extends from Mrpl39 to Znf295. These mice are also noted to have advanced degeneration of locus coeruleus neurons. Ts65Dn mice have poorly formed nests and are likely to use only a small quantity of any nesting material provided. 

PLCβ1-/- mice

Schizophrenic patients have been found to have lower levels of phospholipids in their brains. Phospholipase C (PLC) β1 is specifically expressed in the amygdala, hippocampus, olfactory bulb, and cerebral cortex and schizophrenics have been found to have possible shortages of PLCβ1. PLCβ1-/- knockout mice lack PLCβ1, an enzyme associated with G-protein coupled receptors that performs hydrolyzation that eventually leads to the formation of secondary messengers. Perhaps due to these molecular changes, PLCβ1 knockout mice have compromised nesting abilities. PLCβ1 mice do not initiate nesting as quickly as normal, healthy mice and they are not as responsive to the presence of nesting materials as healthy mice are.

MECP2308/Y mice 

Rett syndrome, a type of mental retardation observed predominantly in females, can be modeled in mice that have the methyl-CpG binding protein 2 (MECP2) gene mutated. MECP2 mice are used to model Rett syndrome. This is an X-linked disorder caused by a faultiness of the MECP2 gene. These mice show large impairments in nest building and cannot build nests in the way that normal, healthy mice can.

Abnormalities in the Behavior

Hippocampus Lesion

One study showed that mice with a complete hippocampal lesion have inhibited, disrupted nesting habits. Lesions to the ventral or dorsal hippocampus do not have such effects, suggesting that these regions are sub-threshold but play an additive role in nesting behaviors.

Pain and Sickness 

Mice that are sick are reported to have minimal (if any) nesting behavior. Also, mice that are experiencing pain have altered nesting behavior, but this returns to normal levels when the pain has passed.

Autism Spectrum Disorder

Autism spectrum disorder has been associated with abnormal abilities of nesting behaviors. Mice that have autism spectrum disorders are not able to build quality nests compared to normal or healthy mice, implying that autism is associated with poorer nesting abilities. 

Rett Syndrome

Rett syndrome is an abnormality that affects nesting behaviors. Prior to its genetic discovery, it was often classified as a type of autism spectrum disorder. In mice, Rett syndrome is associated with poorer nesting outcomes. 


Schizophrenia is another abnormality in mice that is associated with poorer nesting outcomes. Mice that model schizophrenia do not build nests of good quality, demonstrating that schizophrenia compromises nest-building abilities. 

Down Syndrome

Down syndrome is associated with poorer nest building and thus is a nesting abnormality. Mice that model Down syndrome will not have properly formed, complex nests. 

Alzheimer’s Disease

Alzheimer’s disease is an abnormality that affects normal cognitive processing in mice. Typically, mice are not able to build nests that are of comparable quality to chosen controls’ nests. However, there are some transgenic mouse models of Alzheimer’s disease, such as the 3xTg-AD mice, which build nests that are not significantly different from controls’.

Disease Models

Rett Syndrome in MECP2308/Y Mice

The gene encoding methyl-CpG binding protein 2 (MeCP2) is linked as the leading cause of mental retardation with autistic features in females. This is an X-linked disorder caused by faultiness of the MECP2 gene. Interestingly, ten-week-old MECP2308/Y mice do not have abnormal anxiety responses, locomotion deficits, or issues with grooming and ataxia. However, these mice do display impairments in nest building and utilization. 

PLCβ1-/- Mice Modeling Schizophrenia Rarely Nest

Nesting is very rarely observed in mice which lack the phospholipase C β1 gene (PLCβ1-/-), a particular mouse strain used to model schizophrenia since they also demonstrate deficiencies in this gene. One experiment showed that PLCβ1-/- mice are much less responsive towards building a nest out of provided cage materials when compared to wild-type mice. When given materials, all wild-type mice featured in the experiment built their nests within the hour, a stark contrast to the tested PLCβ1-/- mice out of which not one mouse built a nest. 

Transgenic Mouse Models of Alzheimer’s Disease

In most, but not all, transgenic mouse models of Alzheimer’s disease, deficits pertaining to nesting can be observed. While some mice, such as 3xTg-AD mutant mice do not have nesting affected due to their disease, other mice modeling Alzheimer’s disease, such as Tg2576 mice, do. These mice either have decreased nest quality or prolongation of latency before initiating nest building. Such observations indicate that deficient nesting patterns may be a characteristic of Alzheimer’s disease mouse models, but continue to display variability across transgenic mouse models.

Down Syndrome

The most common cause of mental retardation in children is Down syndrome. It is characterized by deficits in memory, as well as contextual learning. As mentioned previously, in mice, Down syndrome alters nesting behavior and significantly compromises the quality of the nest. Down syndrome is predominantly modeled using Ts65Dn mice. These mice are also noted to have advanced degeneration of locus coeruleus neurons. Although the locus coeruleus has not been established to serve a role in contextual discrimination, it is required for contextual learning and deficits in its neuronal population have been correlated with neurodegenerative diseases.

Autism Spectrum Disorder

Mice that model autism spectrum disorder have their nesting abilities compromised. Mice that have been induced with autism spectrum disorder using the well-validated method of valproic acid injection will show deteriorated nesting abilities. 

Nesting Affects Behavioral Assessment 

Nesting Lowers Mice’s Corticosteroid Levels

Mice that are provided with plenty of extra nesting material have less circulating corticosterone levels than mice provided standard levels. Since corticosterone modulates the stress response and stress is associated with changes in behavior, the fact that nesting can lower corticosteroid levels in laboratory mice means that it will affect behavioral assessment.

Therefore, behavioral researchers should be cautious and aware of the decisions they make when housing mice. If they fail to provide mice with adequate nesting material, this could affect the mice’s overall health and subsequently lead to chronically increased stress levels. Such a factor could ultimately confound and affect any experimental results which are derived as a result of using stressed mice without access to bedding material.

Nesting May Elicit Aggression

One experiment demonstrated that mice which built a nest have high levels of aggression towards an intruder in the Resident-Intruder Test when compared with mice that are provided with sparse nesting materials or mice that are provided with both nesting materials and structural objects (wherein they built their nest inside the structural tube or box). The mice which received only nesting material (without any structural objects) had an increased number of attacks and a shorter latency time to attack. However, the combination of structural objects with nesting material may prevent aggression. Such findings demonstrate that the types of cage and nesting material provided to mice can modulate aggression which can, in turn, confound experiments designed to assess agonistic behaviors.


  • Nesting is a goal-directed, complex behavior with multiple actions and functions wherein a mouse builds a safe, warm place out of nesting materials. 
  • Nesting is comprised of a complex behavioral chain wherein several behaviors are performed in order to build the nest.
  • In mice, nesting, due to its behavioral diversity and highly motivated nature, mirrors ADL in humans.
  • Non-maternal nesting is different from maternal nesting wherein a nest is built specifically for reproductive purposes.  
  • Nests provide shelter, heat conservation, protection from competitors and predators, and permit for successful reproduction.
  • There are numerous protocols available for assessing nest-building behavior in mice.
  • Xamoterol improves nest building in Down syndrome mice. Bexarotene improves nest building in mice modeling Alzheimer’s disease. Donepezil increases nesting behavior in ASD mice. 
  • Nesting is affected by many factors and conditions including brain lesions, sickness, pain, temperature levels, the presence of extra cage material, and the type of cage material. 
  • MECP2308/Y mice modeling Rett syndrome display poor nest building. 
  • PLCβ1-/- mice modeling schizophrenia rarely nest. 
  • The majority of transgenic mouse models of Alzheimer’s disease have impairments in their nesting abilities.  
  • Nesting can be used as a measure of established well-being and health in mice. 
  • Nesting may affect behavioral assessment by lowering corticosterone levels. However, in an agonistic encounter, mice that built nests are more likely to be aggressive than mice that did not build nests. 
  • Nest may influence mice’s aggression levels. 


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