Definition

Digging involves the removal or scraping of material from a certain spot by the fast, coordinated alternation of the paws. 

Overview 

Digging is a nesting behavior in which mice use their paws to pull dirt or cage material backwards, thus creating a pile under their bellies. Every now and then, in the midst of this behavior, the mice will also use their hind feet to kick away the accumulated pile, sending it backwards. It is also common to observe the use of the head, in addition to the forepaws, as a means of pushing dirt or cage material. 

It is possible to classify digging as an exploratory behavior, given the frequency of this behavior during exploratory behavioral assessments. 

Facts About Digging in Mice

  • Digging is best exhibited by young adult mice, aged 2-4 months, due to their high energy levels. 
  • Mice will occasionally display the dig behavior in their own cages, especially when the cages are cleaned and new, fresh bedding is applied. However, since the typical bedding depth is only 1 cm, the digging behavior elicited in such circumstances is not very vigorous. 
  • When more bedding is applied, reaching a depth of 5 cm, mice will naturally exhibit robust digging behaviors.
  • Wild-type mice will frequently exhibit the digging behavior when searching for food. Digging is also commonly observed when mice are burrowing or storing food. 
  • When mice are nesting, or building a home, digging may also be noted. When exhibiting nesting behaviors, digging is a part of a wider behavioral sequence which also includes complimentary behaviors such as pulling, sorting, and fluffing.  
  • In other situations, such as when the mouse is digging very frantically or quickly, digging can be interpreted as abnormal behavior, a form of stereotypy.

Function of the Digging Behavior in Mice

  • To search or store food: Digging serves a purpose when mice are searching or storing food pellets or items. When digging is used to search or store food, the behavior helps the mouse to feed itself (or save food for a later time), in order to have enough energy to survive and do its daily tasks. 
  • To build a nest: Digging serves the function of building a nest which protects the mouse and prolong its life expectancy by sheltering it from the cold and potential predators, as well as creating a place for reproduction. 

If the mouse stops digging, for one reason or another, it will be unable to create a nest and to find food, thus will become unhealthy quickly, increasing the chances that it will be caught by predators or get sick and die.  

Application

Under the following conditions, a mouse is very likely to display the digging behavior:

  • In the presence of abundant materials: When a mouse has plenty of cage material, bouts of digging will increase. 
  • While nesting: When a mouse is in the process of creating a nest, digging is bound to be observed.

Behavioral Tests for Assessing Digging Behavior

  • The Marble Burying Test: The Marble Burying/Digging Test is a behavioral test in which mice bury or dig the marbles which are placed on top of the cage material. Through this test, digging is easily quantified. Typically, the control group is expected to bury or dig the highest amount of marbles. For example, the C57BL/6 strain is expected to dig or burry as much as 75% of the marbles in a 30 minutes time frame. However, this benchmark is markedly reduced if the mice are on selective serotonin reuptake inhibitors (SSRI’s) or have hippocampal lesions. 
  • The Burrowing Tube Test: In addition to the Marble Burying Test, the Burrowing Tube Test is another common assessment tool for assessing the digging behavior in mice. In the burrowing tube, substances such as food pellets are placed and the amount that is dug out after 2 hours is measured. The Burrowing Tube Test is simple to run, provides quantified data, and is sensitive to conditions such as prion disease and brain lesions. 
  • The Digging Test: This test has a 3 minutes duration. To prepare, 5 cm of bedding is evenly placed in the cage. Data is gathered by recording the number of digging bouts, the total duration of the digging, and the latency to start digging. 

Pharmaceutical Studies Examining Digging Behavior

Vitamin B6 Attenuates Clomipramine or Venlafaxine’s Effects

Clomipramine or venlafaxine (serotonin-norepinephrine reuptake inhibitors) are both able to reduce digging behaviors significantly in mice as revealed by performance in the marble burying test. These drugs combined with the addition of vitamin B6 have an even stronger effect in reducing digging behaviors. 

Diazepam’s Varying Effects on Digging

Diazepam, a drug with a calming effect which belongs to the benzodiazepine family, has various effects on digging behavior. For example, in one experiment, female MF1 mice were assessed in the burying and digging behaviors using the marble test. The mice that were given 0.1 mg/kg of diazepam had increased digging behaviors while 1.0 mg/kg did not affect digging behaviors. However, digging was reduced at a diazepam dose of 5.0 mg/kg.

Mouse Strains Exhibiting Digging Behavior

Digging can be observed in the majority of mice, since digging is fundamentally related to other behaviors which are crucial for survival such as nesting or searching for food. When observing digging behavior, researchers typically choose inbred or outbred mice. Below is a summary of commonly used mice in laboratory research and their digging profiles. 

C57BL/6J Mouse Strain 

C57BL/6 mice are a suitable strain for closely studying digging behavior. C57BL/6 strain is expected to dig (or bury) as much as 75% of the marbles in a 30 minutes time frame. 

In a digging test, C57BL/6 mice (which often serve as controls) will begin to dig at about 16 seconds into the test and will dig for a total of about 58 seconds. 

Non-Vigorous Strains Have Low Digging Performance 

129S2/Sv Mouse Strain

129S2/Sv are a non-vigorous mouse strain, therefore behaviors which require a lot of energy, such as digging, will only be performed when absolutely necessary (such as in the context of nesting).  

CBA Mouse Strains

Also, the CBA mouse strains display low digging performance. As a non-vigorous strain, these mice may be totally inactive during the duration of behavioral testing. 

C58/J Displays Digging Less Than C57BL/6J Mice

C58/J mice are said to model autism spectrum disorder. However, these mice display other repetitive behaviors more frequently than digging, such as jumping. 

In an experiment comparing the C57BL/6J strain (serving as the control) with C58/J mice, it was established that C57BL/6J mice displayed digging twice as much as the C58/J mice. 

BTBR Mice Exhibit High Digging Levels

BTBR T+Itpr3tf/J (BTBR) mice exhibit very high levels of digging. This rapidly performed behavior is also coupled with high levels of self-grooming behavior and cognitive deficits. 

Abnormalities Associated With Digging Behavior 

Mixed Findings Across ASD Mouse Models

In ASD animal models, some strains will show a marked decrease in marble burying and digging behaviors (such as the Shank1-/-, Ephrin-A-/-, Ephrin-A3-/-, and the C58/J strains)  while other ASD mouse strains will show an increase (such as the BTBR, Eif4ebp2-/-, and the FRM1-/- strains). 

Therefore, more research is needed, in order to gain a stronger understanding of the digging behavior in mice.  

Hippocampal Lesions Reduce Digging 

Mice with hippocampal lesions perform poorly in the digging test. Compared to C57BL/7 mice, mice with a hippocampal lesion spend less time digging and are much slower to initiate digging behaviors. 

Excessive Digging Behaviors Are Associated with Stereotypy

When digging is expressed in redundancy, as demonstrated by repetitive digging and overly rapid alterations of the fore and hind limbs, then digging may be associated with stereotypy. Current research efforts are beginning to investigate this as a possible behavior for creating disease models. 

Summary

  • Digging involves the removal or scraping of material from a certain spot by means of fast alternation of the paws. 
  • Digging can be associated with nesting behaviors, burrowing, and/or searching for food. 
  • The Marble Burying/Digging Test and the Burrowing Tube Test are behavioral assessments commonly used to assess digging behaviors in mice. 
  • Certain serotonin-norepinephrine reuptake inhibitors, such as clomipramine or venlafaxine, can significantly decrease digging. When coupled with vitamin B6, this effect is even more pronounced.
  • Diazepam can either decrease, increase, or have no effect on digging behavior, depending on the dose. 
  • The C57BL/6 strain is expected to dig or burry as much as 75% of the marbles in a 30 minutes time frame 
  • The C57BL/6 strain is typically used as a control when comparing multiple mouse strains. 
  • Non-vigorous mouse strains, such as 129S2/Sv and CBA strains, will have poor digging performance due to their temperament.
  • BTBR mice exhibit very high levels of digging.
  • Digging in extreme repetition is abnormal and is a stereotypy.  
  • ASD models show different frequencies of digging behavior, depending on the mouse strain being used. 

References 

  1. Deacon, Robert MJ. “Digging and marble burying in mice: simple methods for in vivo identification of biological impacts.” Nature protocols 1.1 (2006): 122.
  2. Mesripour, Azadeh, Valiollah Hajhashemi, and Athar Kuchak. “Effect of concomitant administration of three different antidepressants with vitamin B6 on depression and obsessive compulsive disorder in mice models.” Research in pharmaceutical sciences 12.1 (2017): 46.
  3. Handley, Sheila L. “Evaluation of marble-burying behavior as a model of anxiety.” Pharmacology Biochemistry and Behavior 38.1 (1991): 63-67.
  4. Deacon, Robert MJ. “Digging in mice: marble burying, burrowing, and direct observation reveal changes in mouse behavior.” Mood and anxiety related phenotypes in mice. Humana Press, Totowa, NJ, 2009. 37-45.
  5. Amodeo, Dionisio A., et al. “Differences in BTBR T+ tf/J and C57BL/6J mice on probabilistic reversal learning and stereotyped behaviors.” Behavioural brain research 227.1 (2012): 64-72.
  6. Deacon, Robert MJ, and J. Nicholas P. Rawlins. “Hippocampal lesions, species-typical behaviours and anxiety in mice.” Behavioural brain research 156.2 (2005): 241-249.
  7. Ryan, Bryce C., et al. “Social deficits, stereotypy and early emergence of repetitive behavior in the C58/J inbred mouse strain.” Behavioural brain research 208.1 (2010): 178-188.
  8. Kim, Hyopil, Chae-Seok Lim, and Bong-Kiun Kaang. “Neuronal mechanisms and circuits underlying repetitive behaviors in mouse models of autism spectrum disorder.” Behavioral and Brain Functions 12.1 (2016): 3.

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