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Gene-environment interactions differentially affect mouse strain behavioral parameters Valter Tucci, Heena V. Lad, Andy Parker, Sian Polley, Steve D.M. Brown, Patrick M. Nolan MRC Mammalian Genetics Unit, Harwell, Didcot, Oxfordshire, OX11 0RD, UK Received: 9 June 2006 / Accepted: 28 July 2006

Abstract

Systematic phenotyping of mouse strains and mutants generated through genome-wide mutagenesis programs promises to deliver a wealth of functional genetic information. To this end, the appropriation of a standard series of phenotyping protocols is desirable to produce data sets that are consistent within and across laboratories and across time. Standard phenotyping protocols such as EMPReSS (European Mouse Phenotyping Resource for Standardised Screens) provide a series of protocols aimed at phenotyping multiple body systems that could realistically be adopted and/or reproduced in any laboratory. This includes a series of neurologic and behavioral screens, bearing in mind that this class of phenotype is well represented in targeted mutants and mutagenesis screens. Having crossvalidated screening batteries in a number of laboratories and in a number of commonly used inbred strains, our group was interested in establishing whether subtle changes in cage environment could affect behavioral test outcome. Aside from unavoidable quantitative differences in test outcome, we identified significant and distinct genotype-environment-test interactions. For example, specific strain order in open-field center entries and total distance traveled can be reversed depending on the form of enrichment used, while prepulse inhibition of the acoustic startle response is, even quantitatively, unaffected by the enrichment condition. Our findings argue that unless systematically recorded, behavioral studies conducted under subtle variations in cage environment may lead to data misinterpretation, although this could be limited to

Correspondence to: Patrick M. Nolan; E-mail: p.nolan@har. mrc.ac.uk

particular behaviors. Further investigations into the extent and limits of genetic and environmental variables are critical for the realization of both behavioral and functional genomics endpoints.

Introduction The formation of a comprehensive association between gene and phenotype is a considerable goal in mouse functional genomics. Having established large catalogs of mouse mutants, one significant focus within the field has been to develop comprehensive phenotyping platforms, enabling one to categorize and compare mutant strains and allelic series using standard procedures. To this end, a consortium of European laboratories has worked on developing a collection of standard operating procedures for phenotypic analysis of mice in a number of body systems (Brown et al. 2005). The collection of screens, termed EMPReSS (European Mouse Phenotyping Resource for Standardised Screens), has been designed such that it can be introduced with relative ease into any laboratory and that it can be conducted quickly and efficiently. With this in mind, standard operating procedures for approximately 150 assays are available online (http://www.eumorphia.org/ EMPReSS/servlet/EMPReSS.Frameset). As part of this consortium, a subgroup was established to select a number of primary behavioral and neurologic screens that fit these criteria (S. Ho¨lter et al., unpublished). Consequently, a stepwise cross-laboratory validation strategy, based on the assessment of four inbred strains commonly used in the laboratory (C57BL/6J, C3HeB/FeJ, BALB/cByJ, and 129S2/ SvPas), was developed that considered test order, time of testing, and housing conditions. These four strains were selected because they are frequently

DOI: 10.1007/s00335-006-0075-x Volume 17, 1113 1120 (2006) Springer Science+Business Media, Inc. 2006

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used in targeted and random mutagenesis phenotyping screens worldwide. As a complement to this cross-validation, an additional goal was to investigate whether subtle alterations in the mouse housing environment within a single test center could contribute to confounds in behavioral test outcomes. Systematic assessment of behavioral traits in mouse strains and mutants serves a dual purpose in scientific research. From a behavioral neuroscience perspective, catalogs of mouse mutants can be used as tools to understand the biological basis of behavior. Conversely, from a functional genomics perspective, effective series of mouse mutant behavioral screens can serve to categorize gene function. Unfortunately, neither is a simple task because test outcome is a product of complex gene-environment interactions. The expression of any mouse behavioral phenotype is dependent not only on background genotype (Morice et al. 2004; Young et al. 2002), but also on any number of environmental variables, including laboratory (Crabbe et al. 1999), experimenter (Chesler et al. 2002), and even mouse cage position before testing (Izidio et al. 2005). The nature of mouse home-cage enrichment can affect mouse behavioral phenotype, although it is generally established by comparing test outcomes from ‘‘standard’’ (nonenriched) and ‘‘enriched’’ housing conditions. For example, the use of cage enrichment can improve learning and reduce neuropathologic lesions in mouse strains and mutants (Jankowsky et al. 2005; Lazarov et al. 2005). Enrichment is desirable in the promotion of animal well-being and many institutes support the use of enrichment on welfare grounds. However, the use of enrichment for strain and mutant behavioral studies has raised concerns regarding the comparison of test outcomes across laboratories. Wolfer et al. (2004) recently concluded that cage enrichment and laboratory environment, although affecting a number of behavioral parameters, would not markedly affect the comparison of results across laboratories. However, if cage enrichment is used, are we to expect comparable test outcomes considering the wide variety of enrichment items that are available? Furthermore, can we identify specific parameters that are particularly sensitive to enrichment factors? In an effort to establish how sensitive mouse behavior is to cage enrichment condition, we carried out strain/behavior comparisons in groups of mice where only a single feature of enrichment condition is altered. In particular, we hypothesized that behavioral outcomes for different tests vary in mice housed under two different enrichment conditions (cardboard roll vs. mouse house). Although this study is limited in scale, it does imply that care

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ENVIRONMENT DIFFERENTIALLY AFFECTS STRAIN BEHAVIORS

should be taken when comparing specific mutant phenotypes to that of another laboratory, particularly where there are differences in genetic background and enrichment condition. Materials and methods Animals and husbandry. The study was conducted at the Medical Research Council, Harwell. All animal studies described in this article were carried out under the guidance issued by the Medical Research Council in Responsibility in the Use of Animals for Medical Research (July 1993) and Home Office Project Licence No. 30/1630. Ten males per enrichment condition from each of four inbred strains (C57BL/6J, BALB/cAnN, C3H/HeH, and 129/SvEvTac) were used for the study. All mice were imported to Harwell where they were maintained as inbred stocks. Mice were weaned at 3 weeks of age (5 per cage) into IVC cages (Techniplast) enriched with sawdust, shredded tissue (Datesand), and the addition of either a cardboard fun tunnel (Datesand) or a plastic mouse house (Techniplast). The former enrichment condition is the standard housing condition for all internal stocks. Mice were randomly selected from internal inbred colonies and littermates were distributed evenly across groups wherever possible. Access to an expanded breeder diet of food nuts (Special Diet Services) and water (chlorinated between 15 and 20 parts per million) was ad libitum. Additional conditions that remained uniform were as follows: temperature was 19 23C, humidity was 45% 65%, and light/dark cycle was 12:12 h with lights on at 0700. Each group of mice was housed in their environment until behavioral testing was completed. Cages, including enrichment material, were changed once per week. Behavioral testing. All mice between 8 and 10 weeks of age were subjected to a behavioral test battery that was previously validated and standardized in our group as part of a European phenotyping consortium (see www.eumorphia.org). A complete standard operating procedure (SOP) for each test can be accessed from the web (see EMPReSS at http://www. eumorphia.org/EMPReSS/servlet/EMPReSS.Frameset). Behavioral tests were always carried out in the order listed: open-field assessment, modified SHIRPA immediately followed by grip strength assessment, accelerating rota-rod, Y-maze spontaneous alternation, acoustic startle, prepulse inhibition, and swim ability. For all tests, equal numbers of mice from each group were tested at one time. Brief descriptions of each test are given below.

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Open-field activity. Open-field activity was completed using a Tru-Scan arena (Coulbourn Instruments) made from clear perspex (26 · 26 cm, 37 cm high). To acclimate animals to the testing room environment, each mouse was subjected to a 30-min period of habituation in individual cages before testing. The test session lasted 30 min with data recorded in 3-min bins. Modified SHIRPA. Modified SHIRPA is a method based on the screen developed by Irwin (1968) that uses functional and observational criteria to assess a mouse. A modified SHIRPA version was developed within the EUMORPHIA consortium. Briefly, each mouse was weighed and then placed into a viewing jar (typically for 5 min) and assessed for unprovoked behavior, after which the mouse was transferred to the test arena for a series of observations and manipulations. Score sheets were used to record data semiquantitatively. Grip strength. A commercially available Bioseb grip strength meter was used to measure the grip strength of mice. Two types of measurement were performed in the grip strength test: (1) forelimb measurement and (2) forelimb and hind limb measurement. Five measurements of each were taken from which means were calculated. Accelerating rota-rod. The rota-rod test is used to assess motor coordination and balance using the latency of the mouse to fall from the rotating rod at accelerating speed as an indicator. The Ugo Basile rota-rod apparatus was used. Following a 30-min habituation period, mice were subjected to four trials with 15-min intertrial intervals. During each trial, the rota-rod was set to accelerate from 4 and 40 rpm in 300 sec. Latencies to fall from the rotating rod were recorded. Y-maze spontaneous alternation. A custombuilt Y-maze was made of plexiglas with three identical arms (40 cm long · 9 cm wide · 16 cm high) placed at 120 with each other and with different motifs on the walls of each arm. Mice were placed into the maze facing the end of an arm and behavior was observed for 5 min. Total arm entries, latency to exit the starting arm, and spontaneous alternations were recorded. Spontaneous alternation performance percentage was determined as the ratio of actual (total alternations) to possible alternations (total arm entries ) 2) · 100. Acoustic startle response. The apparatus was custom-built and consisted of an outer soundproof chamber and an inner perspex chamber in which the

mouse was placed for the duration of the test. The inner chamber contained a grid floor attached to an accelerometer and a roof that enclosed a loudspeaker that generated sound. Mice were presented with ten sound pulses of white noise at 110 dB, in pseudorandom order. The duration of each pulse was 40 msec with an interval between 20 and 30 sec. The startle response was recorded every millisecond for 65 msec after the onset of startle. Prepulse inhibition. In the same chamber, mice were presented with eight different prepulse trials of 70, 80, 85, or 90 dB white noise stimuli of 10-msec duration, presented alone or preceding the pulse (as above) by 50 msec. A trial was presented in which only background noise of 65 dB was used to measure baseline movement of the animal in the chamber. Swim ability. The swim ability test is a useful indicator of mice that have balance defects. A container large enough to allow free movement and swimming was filled with water to a depth of approximately 15 cm (at 24 26C). Each mouse was gently placed into the center of the container and observed for 1 min to assess their ability to swim. A score system was used to indicate specifically the mode of swimming. Statistical analysis. A two-way factorial analysis of variance (ANOVA) model with betweensubject factors of enrichment condition (cardboard vs. mouse house), strain (C57BL/6J, C3H/HeH, BALB/cAnN, and 129/SvEvTac) and enrichment condition · strain was performed. Data analyses were performed with commercially available statistical software (SPSS v11.0, SPSS, Inc., Chicago, IL). Bonferroni correction for multiple comparisons and the Scheffe´ test for post hoc comparisons were used when appropriate. The statistical significance level was p < 0.05. Results and discussion Our results confirm that environmental condition can have a significant effect on specific quantitative behavioral parameters in all mouse strains tested, whereas other parameters are primarily unaffected. Parameters affected include those that measure emotionality (open field test) and sensory responsiveness (acoustic startle response), while those unaffected include tests of neurologic and neuromuscular function (grip strength, the majority of SHIRPA parameters), working memory (Y-maze spontaneous alternation), and sensorimotor gating. In addition, significant behavioral differences

27.3 (0.6) 133.3 (1.4) 347.9 (19.5) 5 (0.8) 18.6 (1.5) 32.4 (2.4) 771.5 (315.5) 56.2 (7) 50.6 (9.7) 36.3 (13.7) 93.3 (1.7) 50.2 (7.7)

889.3 (152) 69.9 (2.5) 62.5 (4.4) 55.1 (5) 108 (2.4) 62.3 (5)

BALB/c

26.6 (0.7) 137.6 (2.4) 446.05 (40.6) 10.2 (0.9) 23.7 (1.2) 52.6 (3.5)

C3H

55.9 (2.9)

90.4 (1.2)

64.2 (4.4) 59.2 (4) 49.7 (5.3)

907.2 (140.5)

25.4 (0.7) 142.6 (5.6) 710.2 (26.6) 15.3 (0.8) 29.1 (1.01) 52.6 (2.32)

C57

62.3 (3.03)

101.2 (1.5)

68.5 (5.1) 65.8 (2.6) 56.4 (5.3)

1285.5 (217.8)

23.3 (0.7) 140.2 (1.8) 439.7 (26.9) 11.6 (1.4) 23.5 (1.4) 50.9 (3.6)

C3H

59.8 (8.4)

100.7 (1.3)

52.2 (8.7) 39 (12.1) 31.4 (15)

871 (151.2)

22.8 (0.7) 142.3 (2.1) 515.6 (34.08) 10.6 (1.4) 22.8 (2.2) 34.9 (4.04)

BALB/c

59.4 (5.4)

100.8 (1.4)

81.05 (2.8) 81.1 (4) 65.8 (5.7)

1170.3 (151.2)

22.4 (0.5) 132.8 (2.4) 371.1 (15.7) 6 (0.5) 10.4 (0.6) 15 (1.9)

129

0.000 0.000 0.000 0.000

0.03 0.05 NS NS

0.006

NS

NS

NS

0.001

0.000

NS

NS

0.000

NS

0.000

NS

NS

0.000

0.003

Strain effect (P)

0.01

Enrichment effect (P)

NS

NS

NS

NS

NS

0.01

NS

NS

0.01

0.000

0.001

0.01

Interaction enrichment · strain (P)

ET AL.:

63.3 (7.9)

105.9 (4.3)

83.2 (2.8) 74.8 (4.7) 61.3 (3.9)

907.2 (444.3)

20.9 (1.2) 146.9 (2.6) 504.2 (35.6) 8.9 (0.6) 10.4 (1.2) 12.7 (1.6)

129

M/H

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PPI = prepulse inhibition. Mean values and standard errors (in parenthesis) are reported for each parameter. The statistical p value for each parameter is reported for the enrichment principal effect (cardboard, C/R vs. mouse house, M/H), for the inbred strain (C57BL/6J, C3H/HeH, BALB/cAnN, and 129/SvEvTac), and for the interaction of the two.

25.8 (1.17) Move time 140.3 (sec) (2.3) Distance 540.9 (cm) (24.2) Centre entries 13.05 (0.6) Rearings 24.7 (1.01) Rearing time 46.9 (2.9) Acoustic startle response Response 635.7 (124) PPI (% of inhibition) 80 dB prepulse 59.1 (2.8) 85 dB prepulse 48.4 (6.4) 90 dB prepulse 36.9 (4.8) Grip strength Grams 94.8 (1.4) Y-Maze Spontaneous 50.78 alternation (%) (3.5)

Open field Movement episodes

C57

C/R

Table 1. Environment · enrichment effects on behavioral parameters

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A

C

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1000.0 800.0

B

1000.0 800.0

600.0

600.0

400.0

400.0

200.0

200.0

0.0

0.0

1000.0 800.0

D

1000.0 800.0

600.0

600.0

400.0

400.0

200.0

200.0

0.0

0.0

Fig. 1. A comparison of distance traveled by mice in the open-field arena according to the cage enrichment condition: cardboard roll (C/R) and mouse house (M/H). Mean values and standard errors are plotted for each graph. Distances (in cm) are plotted over 3, 6, 9, and 12 min for (A) C57BL/6J, (B) C3H/HeH, (C) BALB/cAnN, and (D) 129/SvEvTac strains. Inset panels paired the average total distance for each enrichment condition within the same inbred strain. Levels of statistical significance are *p < 0.05, **p < 0.01.

between all strains were identified in all tests regardless of whether an enrichment effect was identified. Extensive studies on strain differences in behavioral parameters have been detailed elsewhere and are not presented as novel findings in this study. However, our most noteworthy finding shows that significant enrichment effects are invariably associated with significant gene · enrichment interactions. This can manifest itself in either one of two situations. The first is a differential unidirectional shift in strain responses according to enrichment condition with an associated alteration in the order of strain performances. The second is represented by opposing directional shifts in strain responses according to enrichment condition, again resulting in an altered order of strain performances. Behavioral data from the four mouse strains housed under one of two enrichment conditions are listed in Table 1. Data for a number of representative behavioral parameters within the test battery are shown. In addition, significance values for enrichment effect, strain effect, and enrichment · strain interaction effect are shown. Several parameters, measured in the open-field test and the acoustic startle response, indicate a significant interaction effect. Comparison of raw data values in both

enrichment conditions indicates that BALB/cAnN and 129/SvEvTac strains primarily contribute to the interaction effect in open-field measures (movement time, distance traveled, and center entries). C57BL/ 6J mice also contribute to the interaction effect in the distance traveled parameter. Similar trends were seen in the semiquantitative measurements of transfer arousal and locomotor activity in the modified SHIRPA test (data not shown). In contrast, the C57BL/6J and C3H/HeH strains primarily contribute to the interaction effect in the acoustic startle response. Differential responses of all four strains in the open-field distance traveled parameter are shown in Figure 1. This pattern is representative of a number of the open-field parameters recorded. Data for the first four 3-min bins and averaged cumulative values over the same time period have been used for statistical comparisons because the rest of the recording was neither informative nor discriminative. Enrichment condition differences in this parameter over all four bins are evident for three of the strains. The analysis of the distance traveled by mice in the arena according to enrichment condition revealed a statistical main enrichment effect [F(1,62) = 4.46; p < 0.03], a strain effect [F(3,62) = 19.8; p < 0.0001] and an enrichment

1118

A

C

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30.0

B

ET AL.:


30.0

25.0

25.0

20.0

20.0

15.0

15.0

10.0

10.0

5.0

5.0

0.0

0.0

30.0 25.0

D

20.0 15.0

30.0 25.0 20.0 15.0

10.0 5.0 0.0

10.0 5.0 0.0

Fig. 2. A comparison of entries into the center zone of the open-field arena according to the cage enrichment condition: cardboard roll (C/R) and mouse house (M/H). Mean values and standard errors are plotted for each graph. Number of entries are plotted over 3, 6, 9, and 12 min for (A) C57BL/6J, (B) C3H/HeH, (C) BALB/cAnN, and (D) 129/SvEvTac strains. Inset panels paired the average total entries for each enrichment condition within the same inbred strain. Levels of statistical significance are **p < 0.01.

· strain significant interaction [F(3,62) = 8.83; p < 0.0001]. While C3H/HeH mice show no differences associated with enrichment condition, C57BL/6J and BALB/cAnN travel longer distances when housed in the M/H condition and, remarkably, 129SvEvTac mice travel shorter distances when housed in this condition. In a direct comparison, C57BL/6J distance traveled is almost twice that of 129SvEvTac in the M/ H condition, whereas in the C/R condition, 129SvEvTac distance traveled is only slightly less than that of C57BL/6J. Additional strain-specific behaviors are evident. C57BL/6J and 129SvEvTac mice coming from either enrichment condition habituate to the open field; only BALB/cAnN M/H mice habituate and neither group of C3H/HeH mice habituate. Furthermore, the analysis of cumulative center entries (commonly regarded as a measure of anxiety in mice) over a 12-min period revealed a few interesting differences. For center entries, enrichment affects the mean number of entries into the center zone [F(1,62) = 3.74; p < 0.03]. Furthermore, enrichment condition has an opposite effect on open-field center entries in BALB/cAnN and 129/SvEvTac mice, while C3H/HeH parameters are relatively unaffected (Fig. 2). Figure 3 shows the acoustic startle response for all mouse groups. In this instance there is a tendency

toward an increased startle response in all strains housed under the M/H enrichment condition, but this increase is significant in only two of the four strains, C57BL/6J (Scheffe´, p = 0.04) and C3H/HeH (Scheffe´, p = 0.014). The magnitude of effect in C3H/ HeH mice is largest (44.6% increase in response), even though open-field parameters for this strain were virtually identical for both enrichment conditions. Prepulse inhibition values for all strains and enrichment conditions are shown in Figure 4. Of all behaviors measured, this would appear to be one of the most robust with little or no sensitivity to

Fig. 3. A comparison of acoustic startle response (ASR)

values in response to a 110 dB sound pulse according to the cage enrichment condition: cardboard roll (C/R) and mouse house (M/H). Mean values and standard errors are shown for all groups. Levels of statistical significance are *p < 0.05, **p < 0.01.

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A

B

C

D

Fig. 4. A comparison of percentage prepulse inhibition values using 80, 85, and 90 dB prepulse and a 110 dB sound pulse according to the cage enrichment condition: cardboard roll (C/R) and mouse house (M/H). Mean values and standard errors are shown for (A) C57BL/6J, (B) C3H/HeH, (C) BALB/cAnN, and (D) 129/SvEvTac strains.

enrichment condition. C57BL/6J mice show the largest differences across all prepulse intensities but none of these differences reach significance. The results of our current experiment within a single laboratory informs us that specific behavioral measures in mouse strains are differentially sensitive to particular enrichment conditions. Is it possible that mouse behavior is sensitive to any mere change in housing condition? We consider this unlikely because the mice had spent at least 5 weeks in their new enrichment condition before behavioral testing. Furthermore, we had previously observed no differences in behavior in mice weaned into a number of enrichment condition variants (unpublished data). The most significant inference from our study is that enrichment can affect strain ranking in a number of measured parameters, moreover the differential effects on strain ranking may implicate complex gene-environment-behavior interactions. For example, enrichment condition has opposite effects on open-field center entries in BALB/cAnN and 129/SvEvTac males and comparable minor effects on acoustic startle response. While open-field center entries are unaffected in the C3H/HeH strain, enrichment condition has a dramatic effect on the acoustic startle response. Although our study focused on the effects of environmental enrichment on the mouse performance in standard behavioral tests, an even more interesting set of data would come from the observation of mouse behavior in the home cage before any testing. This was beyond the limitations of our facility.

Being aware of and exploiting gene-environment interactions can only further our understanding of neurobiological gene function. The behavioral differences between the two conditions in our study, for example, could be related to mouse strain preference for a particular form of enrichment. In a recent study (Van Loo et al. 2005), mice, when given a preference, spent significantly less time in cages containing a plastic mouse house identical to the one used in our study than in cages with a cardboard nest box. The results do not prescribe the use of one particular enrichment condition in the analysis of mouse mutants. Rather, our report illustrates two confounds in enrichment studies. First, in behavioral phenotyping, the researcher should be aware that interactions of strain and test with environment may continue to account for specific behavioral differences in cross-laboratory comparisons. Second, in test batteries that purportedly measure components of complex behaviors such as anxious behavior, the differential order of strain performances in two enrichment conditions suggests that individual behavioral assays may measure distinct domains of complex behaviors.

Acknowledgments This work was funded by the MRC and by the EUMORPHIA project (QLG2-CT-2002-00930) which is supported by the European Commission under FP5.

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References 1. Brown SD, Chambon P, de Angelis MH; Eumorphia Consortium (2005) EMPReSS: standardized phenotype screens for functional annotation of the mouse genome. Nat Genet 37, 1155 2. Chesler EJ, Wilson SG, Lariviere WR, Rodriguez-Zas SL, Mogil JS (2002) Influences of laboratory environment on behavior. Nat Neurosci 5, 1101 1102 3. Crabbe JC, Wahlsten D, Dudek BC (1999) Genetics of mouse behavior: interactions with laboratory environment. Science 284, 1670 1672 4. Irwin S (1968) Comprehensive observational assessment: Ia. A systematic quantitative procedure for assessing the behavioral and physiologic state of the mouse. Psychopharmacologia 13(3), 222 257 5. Izidio GS, Lopes DM, Spricigo L, Ramos A (2005) Common variations in the pretest environment influence genotypic comparisons in models of anxiety. Genes Brain Behav 4, 412 419 6. Jankowsky JL, Melnikova T, Fadale DJ, Xu GM, Slunt HH, et al. (2005) Environmental enrichment mitigates

7.

8.

9.

10.

11.

ET AL.:


cognitive deficits in a mouse model of AlzheimerÕs disease. J Neurosci 25, 5217 5224 Lazarov O, Robinson J, Tang YP, Hairston IS, KoradeMirnics Z, et al. (2005) Environmental enrichment reduces Abeta levels and amyloid deposition in transgenic mice. Cell 120, 701 713 Morice E, Denis C, Giros B, Nosten-Bertrand M (2004) Phenotypic expression of the targeted null-mutation in the dopamine transporter gene varies as a function of the genetic background. Eur J Neurosci 20, 120 126 Van Loo PL, Blom HJ, Meijer MK, Baumans V (2005) Assessment of the use of two commercially available environmental enrichments by laboratory mice by preference testing. Lab Anim 39, 58 67 Wolfer DP, Litvin O, Morf S, Nitsch RM, Lipp HP, et al. (2004) Laboratory animal welfare: cage enrichment and mouse behavior. Nature 432, 821 822 Young KA, Berry ML, Mahaffey CL, Saionz JR, Hawes NL, et al. (2002) Fierce: a new mouse deletion of Nr2e1; violent behavior and ocular abnormalities are background-dependent. Behav Brain Res 132, 145 158