Maintaining Mice for Neurobehavioral Examination

Type of ventilation (references)

Strain

Behavioral effect

Three different types of IVC racks [17]

BALB/c

Avoid high intra cage ventilation but preference counteracted by nesting material

Prefer larger cages with air supply in the cover

IVC housing and ambient environment caging (AEC) [18]

C3HeB/FeJ (C3H) and C57BL/6 J (B6)

IVC reduces activity and enhances anxiety; reduces grooming latency reduced in B6

IVC increases startle response in C3H and not in B6J

Forced-air IVCs and motor-free individual ventilation [19]

4-Week-old C57Bl/6 J

Forced-air IVCs: greater water consumption than in motor free

Forced-air IVCs: mice move more frequently in the front halves of their cages

IVC housing and ambient environment caging (AEC) [1]

C57BL/6 JArc

IVC does not modify cognition and locomotion

IVC increases anxiety in elevated + maze

IVC increases social behavior

Socially more active than mice of filter-IVC increases locomotor sensitivity to MK-801

IVC housing and ambient environment caging (AEC) [20]

DBA/2

Reduced number of pups in IVC in interaction with enriched environment

IVC housing and ambient environment caging (AEC) [21]

Heterogeneous

Increased saccharin preference and increased fluid consumption in AEC

Similar locomotion, food intake, social exploration, and novel object recognition in an AEC

Depressive-like increased in IVC

Individually ventilated cages have a long-term effect [1]. While this does not mean that the system should be rejected, it limits the scope for comparisons and meta-analyses. Authors are therefore urged to include a description of the type of ventilation in the method section of papers to be published.

Should the cages be opaque or transparent? With a transparent cage it is possible to make rapid checks and have early detection of any undesirable behavior such as aggression or excessive grooming, and also to monitor births without disturbing the mothers. A transparent cage giving mice a view of the room and movements in it is well accepted by most strains provided that staff does not make any sudden movements. For certain strains, however, opaque cages seem more suitable. Studying castaneus mice, Roubertoux (unpublished) observed earlier sexual maturation and more frequent copulation in animals reared in opaque cages, and more NZB/BlNJ pups survived in opaque cages.

Attempts to establish guidelines stipulating the floor area per mouse and increasing the allotted area invariably trigger protests in the mouse research community because of strategic and financial issues. Anne Fawcett’s report (http://www.animalethics.org.au) examining 16 published papers dealing with the impact of floor area on animal welfare found no clear results because of several factors. In the studies examined, a larger floor area often correlated with a higher population density; the ventilation systems were different, and measurements of well-being were not standardized. The report concluded that no consensus could be drawn from the results, thus highlighting the complexity of the question.

“(1) There is no consensus in the scientific literature about the minimum cage floor area or maximum stocking density for housing laboratory mice. Different strains may have significantly different space requirements, which may be altered by in-cage furnishings or enrichment items (…). (2) Living area: The living area should be large enough to allow mice to compartmentalize their space. At the same time, cages with large quantities of open, empty space without hiding places should be avoided, as these may be stressful to mice. (3) In terms of physical movements, mice should be able to turn freely without twisting their heads and bodies, walk at least a few steps, stand on their hind limbs, and stretch up. They should also have room to shelter and rest. The floor area should ensure that no part of a mouse’s body is unavoidably distorted by contact with the cage in any of the postures that mice may adopt. However, this does not imply that a larger cage is necessarily better. Mice exhibit thigmotaxis, and may therefore not respond to an increase in living area in the same way as other species.”

There is clearly a need for well-planned studies to address the question before considering any new rules. Europe and the USA have however reached consensus on the need to increase the floor area per mouse. Appendix A to the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (2006, http://conventions.coe.int/Treaty/EN/Treaties/PDF/123-Arev.pdf) stipulates the minimum floor areas for mice (and other organism models). For a group of mice the basic floor area is 330 cm², plus 80 cm² per mouse. For 10 male mice housed together, the allotted area would be 330 cm² + 80 cm² × 10 = 1,130 cm². A 24 × 46 cm cage (1.104 cm²) should not hold more than 9 mice (25 g each). The floor area allotted per individual increases according to the weight of the mouse, as detailed in Appendix A to The Guide for the Care and Use of Laboratory Animals—NIH published by The National Academies Press (2011, http://grants.nih.gov/grants/olaw/Guide-for-the-care-and-use-of-laboratory-animals.pdf) gives different indications for the USA. The calculation based on 330 cm² has been replaced by an individual allocation of 96.7 cm². A female mouse alone with her litter, in both the USA and the European Union, should have 330 cm² floor area. In the European Union, if the litter is maintained with the father (monogamous pair plus the litter), the floor area must be 330 + 180 cm². The height of the cage, which is crucial to allow vertical jumping, is set at 12 and 12.7 cm in the European Union and the USA, respectively.

3 Enriched Versus Standard Environment

A review of 40 studies investigating preferences for an enriched environment published up to and including 2000 concluded that mice prefer a complex environment, particularly for finding material for nesting [2]. Mice actively look for nesting material or use material provided. As Anne Fawcett (2013) observed, the organization and quality of the space are more important than the amount of space. We analyzed studies on enrichment published between 2001 and 2013 (Table 2). The criteria were sufficient information on (1) enrichment strategy, (2) strains, and (3) behavioral testing. The results are reported in Table 2. The main finding was consensus on enrichment and its effect on the brain and behavior. The failures were due to either very short exposure or an inappropriate enrichment. This tallies with the analysis of the previous period [2]. Several other points may be noted:

Table 2

Standard versus enriched environment in laboratory mice

Condition, strain (references)	Brain	Behavior
Enriched environment/C57BL/6 and 129S6/SvEv [22, 23]		Increases exploratory activity in the plus-maze and reduced habituation in the locomotor activity test in B6 mice, whereas in 129 mice increased hot plate latencies and reduced aggression were observed
Physical enrichment, BDNF(+/−) mice [24, 25]	Rescues hippocampal BDNF expression in males	Not done
Enrichment from weaning to 4.5–6 months/PS1/PDAPP [26]	Modifies proteins related to alpha beta sequestration and synaptic plasticity	Cancel the difference across mutants and controls
Enriched environment during 5 weeks previous the tests [27]	Increases the survival of newborn cells	Decreases the distance traveled and an increase in the amount of time spent in the center of open field, the startle response, reduces immobility time in a forced swim test
Physical enrichment/LSAMP [23, 28]		Enrichment abolished differences between the genotypes in body weight, social behavior, increases anxiety No changes for whisker trimming, locomotor activity, marble burying
2 months’ enrichment in CF1 low and high explorers [29]	Increases BDNF level in hippocampus	Enrichment enhanced exploratory behavior, memory performance both in low and high explorers
Enriched environment in heterozygous Mecp2+/− females and Mecp2-/year males [30]		Improves motor coordination in heterozygous Mecp2+/− females but not Mecp2-/year males
Enrichment combined with social isolation (from 3 to 8 weeks) C57BL/6J [31]		Isolation increased locomotion, anxiety, reduces fear conditioning performances and Morris water maze performance. In contrast, absence of nesting material increased anxiety-like behavior Enrichment improves water maze performances, rotarod, nociception, and prepulse inhibition remains unchanged
Enriched environment and physical activity/C57Bl/6J [32]	Increases synaptophysin levels in the neocortex and hippocampus	Exercise, but not cognitive stimulation or acrobat training, improved spatial working memory Spatial reference memory was unaffected by enrichment Physical activity has no effect on memory or synaptophysin levels
6-Week-old CD-1 (running wheel, novel objects, and social interaction for 2 months) [33]	Increases opioid signaling, acetylcholine release cycle, and postsynaptic neurotransmitter receptors Decreased Na+/Cl−-dependent neurotransmitter transporters	Increases motor (rotarod) and learning (passive avoidance)
Running wheel, novel objects, and social interaction in NMRI mice [34, 35]	Activation of the hippocampus and the infralimbic cortex	Improves memory
Physical enrichment of the mother [36]	Cell proliferation in the hippocampus of females	Increases motor activity and time in the open field center in female offspring
Enrichment (2 weeks)/CD-1 [37]	Increases corticosterone and 3-methoxy-4-hydroxyphenylglycol in cortex and hippocampus after mild stress	Increased activity and aggressiveness
Physical enrichment, BALB/cByJ [38]	Prevents corticosterone elevations and altered hippocampal norepinephrine utilization occurring after defeat	Not done
Running, enrichment, running + enrichment C57BL/6J [39]	Environmental enrichment alone does not significantly increase hippocampal neurogenesis	Improves spatial learning under broad conditions
Enrichment from 3 to 11 weeks of age; switched from enriched to standard environment at week 12/C57Bl/6J [40]	Increased mRNA levels of corticotropin-releasing factor (CRF) in stria terminalis and increases in CREB phosphorylation in stria terminalis and in n. accumbens	Switching increases the rewarding effects of cocaine
Enriched environment/FMR1-KO [3]	Increases alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor subunit 1 (GluR1) levels in both genotypes, does not increase FMRP levels in the FMR-KO	Rescues behavioral abnormalities in open field and contact of new objects
7-Week-old BTBR T + tf/J mice during 30 days [41]	Not done	Reduction in time spent grooming
Shelter, shelter + running wheel, and shelter + novel objects/BALB/cJ [42]		Shelter increased longevity, reduces aggression Adding a running wheel increased aggression over shelter alone, changed behavior in the elevated plus (EP) and open field (OF) Novel objects impacted behavioral measures
Enrichment combined with social isolation (from 3 to 8 weeks)/nNOS [43]		Social + nonsocial enrichment reduces locomotor behavior and anxiety (open field), depressive responses in the forced swim test Social housing increased open arm exploration in the elevated + maze. Both social + nonsocial enrichment reduces aggressive behaviors
Enrichment from weaning to 4.5–6 months/C57Bl/6 [44]	Changes on neurotrophin levels	Reduces anxiety as measured in elevated + maze
Physical enrichment during 8 weeks/BDNF+/− [45]	Increase in dendritic spines in the hippocampal CA1 region and DG of the wild-type mice. The effect is less pronounced in mutants	Increases BDNF+/− activity in the open field and not in the hole board
Enriched environment [46]		Attenuates the acute morphine-induced hyperlocomotion and repeated morphine-induced behavioral sensitization Blocks the conditioned place preference induced by morphine

Studies performed between 2000 and 2013. See [2] for previous studies

Enrichment, genotype, and sex may interact.
Mice naturally look for nesting items.
Correlated brain modifications may occur.
A compensatory effect may be detected (well described in [3]). Enrichment increases alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor subunit 1 (GluR1) levels in mouse models of fragile X disease but does not increase FMRP levels in FMR-KO mice. Here the enrichment does not rescue the deficient protein but stimulates a compensatory neurochemical process.
The mechanisms by which changes in the environment can modify behavior or its brain correlates were recently analyzed. An enriched cage environment improved performances in fear conditioning tests and the improvement is greater in mice lacking the CREB-binding protein [4]. An association was observed between the effect and an increased number of dendritic spines in the mutant mice. In other words, the benefit of environmental enrichment was greater in mutants. The most interesting finding was that differential transcription induced by the enriched environment. In a standard environment, wild-type mice and mutant mice did not have similar brain transcripts, and the under- or over-transcription generated by the enriched environment was not the same in wild-type mice and mutant mice reared in the same enriched environment.

The challenge is to produce standardized enrichment for easy replication in different laboratories. Several items are available on the market and provide a simple way of producing replicable rearing conditions. The dome house provides a shelter for a group of same-sex mice or for a dam and litter. It can be made of cardboard that can be gnawed, thus supplying nesting material (then, obviously, has to be replaced); or it may be made of solid, lasting plastic, with nesting material added. Special Diet Distribution (http://www.sdsdiets.com/contact_and_distributors) or Otto Environmental (www.ottoenvironmental.com) has a range of mouse housing options. We tested a cardboard version with hiding and climbing possibilities. A mouse house leaves scope for self-regulating light. The outside lighting is 245 lux and can go down to only 30 lux on the opposite wall, and nearly zero inside the house. Effects observed on reproduction, litter survival, and aggressive behavior are shown in Fig. 1. An enriched environment thus appears to be better for mouse welfare if the criteria considered are better reproductive performance, a higher survival rate, and more peaceful relationships.

Fig. 1

Effect of a mouse house on well-being. (a) Percentage of surviving pups (number of pups born/number of pups at weaning) in 4 inbred strains; 18 to 22 litters per strain was observed; (b) percentage of male mice living in groups (3–5 males) with tail injuries after 10 days; observed under standard conditions (SC) and mouse house conditions

Rearing a mouse in an enriched environment generates a different reaction raising a number of questions. What is the best model for pervasive disorders of brain development—a mouse reared in a standard environment or an enriched environment? The answer mirrors the response we gave some years ago to the question on the best background for generating mutant mice. A mutation should be tested on different genetic backgrounds. A mouse model of pervasive disorders should be assayed in different environments [5, 6].

4 Handling

Repeated handling generates physiological modifications. The measurement by telemetry shows that the heart rate increased during a handling procedure and took more than 1 h to return to normal [7]. An increase in body temperature is attributable to the stress of handling [8]. Systematic or extended handling generated molecular reactions in the brain that are similar to the symptoms of stress [9, 10]. Both occasional and systematic handling generate anxiety [5, 6, 11–15]. According to Gouveia and Hurst [16] “Handling stress is often pointed out as a potential source of unexplained variation within and between animal studies. This is because handling stress is known to influence both the behavior and physiology of animals.” Systematic handling or gentling should be excluded, as it is an uncontrollable source of variation. Hurst and West suggested an interesting solution, using the spontaneous tendency of the mouse to enter a cylinder to catch it, preferably by the tail. Variants of this method will be cited in the chapters on behavioral testing of adult mice.

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