P18 ATLA 42, P18 P22, Assessing the Effects of Environmental Enrichment on Behavioural Deficits in C57BL Mice

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1 P18 ATLA 42, P18 P22, 2014 DISCUSSION Assessing the Effects of Environmental Enrichment on Behavioural Deficits in C57BL Mice Ellen J. Coombs A study on the effects of environmental enrichment on behaviour and cognitive and motor functioning in a standard mouse model and a strain known to have behavioural deficits, suggests that environmental enrichment can positively influence natural functioning and natural behaviour For many years, rodents have been used as animal models for investigating human diseases such as Alzheimer s disease (AD) or Huntington s disease (HD). 1 The mouse genome (specifically that of the C57BL/6 strain) has about 30,000 genes, 99% of which have direct counterparts in humans. 2 This makes the mouse a good model for studying the function of human genes, with particular emphasis on disease. Furthermore, mice with their small size and short generation time make a very suitable test species. 3 Despite the obvious welfare concerns that arise when testing on animals, this practice is likely to continue until reliable and effective alternatives can be employed. In the meantime, it is believed that by enriching the environment in which a test species is housed, we can take steps toward improving their welfare. Thus, the aim of the present study was to firstly assess whether environmental enrichment (EE) can influence the behaviour of the standard laboratory mouse (here, the C57BL/6 strain) and, if so, whether the effects are strain dependent. Secondly, the goal was to determine whether its close cousin, the C57BL/10 mouse, when housed in a standard environment, would show the motor and cognitive deficits that would actually make it a suitable AD model. The mouse model The most widely used mouse strain in behavioural studies is the C57BL/6 (or B6). Alternatively, the C57BL/10 (or B10), which shares a common ancestry with the B6, is used in non-behavioural studies of immunology and inflammation (as a result of stimuli such as prion diseases). 4 Thus, B6s and B10s are seemingly identical in appearance and general behaviour, but are used in different studies. 4 Observations on the differences of cognition, species-typical behaviour and motor-coordination have shown deficits in B10s. 4 In the majority of these tests, B6 mice consistently functioned better. Limited work on B10 mice has suggested deficits in hippocampal functioning and anatomy. 5 There is a paucity of literature available on behavioural experiments documenting the B10 behavioural phenotype, and only one paper directly on a comparison of B10 and B6 strains, so most comparisons between the strains are based upon the research of Deacon et al. 4 Abnormalities shown by B10s are possibly representative of some neurodevelopmental and neurodegenerative disorders, such as dyspraxia and AD. Mice expressing different mutant forms of amyloid precursor proteins and/or presenilin-1 may develop functional or cognitive defects resembling the symptoms observed in human AD patients. 6 It has been suggested that B10s might be a useful model for aspects of certain neurological disorders, such as autism 4 and potentially AD. The latter proposal was tested in the study described in this paper. Determining the effects of environmental enrichment An environment is considered to be enriched or not, when it is compared with standard laboratory conditions. 7 Standard (S) cages (with bedding and ad libitum access to food and water) are commonly used when housing mice, but it has been shown that enrichment increases brain weight 8 and reduces anxiety. Enrichment can be supplied in the form of extra nesting material, plastic tubing, etc. Furthermore, it has been documented that mice (often of the B6 strain), housed in standard cages show impaired and abnormal brain development, repetitive behaviour and an anxious behavioural profile. 9 Mice housed in standard cages were hyperactive, with reduced dendrite branching and length. These behavioural and neuronal abnormalities were greatly

2 P19 reduced in environmentally-enriched mice.10 It was found that mice from enriched housing have significantly more neurons (~15%) in the dentate gyrus (part of the hippocampus) than those of S littermates. Enriched mice were also found to have a larger hippocampal granule cell layer.11 Environmentally-enriched mice also have reduced levels of corticosterone (a hormone associated with anxiogenic situations) following exposure to predator cues such as scent or visual presence.12 Hutchinson et al.13 hypothesised that enriched mice produce less endogenous cortisol and exhibit higher levels of proinflammatory cytokines in response to antigenic immune attacks, suggesting that environmentally- Figure 1: enriched mice have a greater anti-inflammatory cytokine profile than their S cohorts. The study Housing and test models We wanted to assess whether the provision of EE could enhance the functioning of B10s to a level similar to that of the more-widely studied B6s. The subjects were 20 female C57BL/6 and 20 female C57BL/ 10 mice (Harlan, Shardlow, Derbyshire, UK). Half of the mice were housed in enriched cages (Figure 1a Environmentally-enriched and standard cages a) b) c) d) a) Aerial view of environmentally-enriched cage; b) side view of environmentally-enriched cage (show here without bedding); c) aerial view of standard cage; d) front view of standard cage.

3 P20 and 1b), and half in standard cages containing aspen shavings and sizzle-nest bedding (Figure 1c and 1d), for approximately six weeks before the experimentation commenced, and throughout the experimentation. Food and water were available ad libitum, temperatures were controlled at ~21.1 ±1 C, and a dark cycle commenced at 7pm for 12 hours. In order to gain a sound idea of whether EE had any effects on mouse behaviour and functioning, a variety of experiments were used. The tester remained blind as to which mice were which. Figure 2: 60W anglepoise lamp The light dark box dark section box 20cm Test methods The effects of EE include increased socio-positive behaviour (e.g. grooming) and species-typical behaviour, and a reduction in agonistic behaviour. 14 Therefore we assessed spontaneous semi-naturalistic behaviour, such as digging, grooming, burying objects, climbing, and nest construction. EE has been attributed to an increase in locomotor and exploratory activity, object exploration and learning ability. 14,15 Therefore, we also wanted to assess whether EE affected motor skills, so we carried out tests of physical ability, including locomotor activity and strength. These included watching the mouse in an open field (OF) box (assessing how reluctant the mouse was to stay in the open), and assessing the mouse on a horizontal bar (jumping and climbing/balance abilities along static bars). We also carried out activity tests, by measuring locomotor activity and counting the number of times the mouse crossed back and forth across a boundary. This logically led to anxiety tests, since environmentally-enriched mice have reduced levels of corticosterone (a hormone associated with anxiogenic situations) following exposure to predator cues such as scent or visual presence. 12 As previously stated, Hutchinson et al. 13 hypothesised that environmentallyenriched mice produce less endogenous cortisol than their S cohorts. We believed that it was important to test the effects of EE on anxiety levels, so the following behaviours were assessed: a) the willingness of the mouse to exit an enclosed dark space and enter a brightly lit one (light dark box; Figure 2); b) the willingness of the mouse to explore the open, exposed arms of a maze, as compared to walled arms (the plus-maze [PM]); c) the movement of the mouse in a black white alley (similar to the light dark box, where the black section of the alley is where a more anxious mouse will predominantly reside); and d) the movement of the mouse on the hole board (a board with holes in it), to test the number of head-dips into a hole, where a confident mouse will exhibit more multiple head-dips than an anxious one. Throughout the trials, faecal boli were counted. Boli or urea are more often excreted by anxious animals. We also tested for hyponeophagia, i.e. the time it takes for a mouse to investigate a novel food item. The more anxious mouse will take longer to do this. 20cm light section box Studies have shown that EE may induce experience-dependent neuroplasticity. 16 Thus, cognitive ability trials were also performed. These included tests of mental processes, such as spatial reference and ability to alternate. 17 In particular, EE has been shown to mitigate cognitive deficits in a mouse model of AD, with learning and memory deficits (associated with AD) also being ameliorated. 18,19 Mice provided with environmental stimulation were significantly improved in several measures of cognitive performance, with EE strongly modulating the pathological and behavioural progression of AD in the mouse model. This was tested by assessing whether mice would spontaneously investigate one arm of a maze on the first trial, then another arm of the same maze on the second trial, exhibiting memory functioning (spontaneous alternation in the T-maze; Figure 3) and similarly spatial novelty in the Y-maze (Figure 4). Statistical analysis For tests with multiple factors (the majority comparing strain and environment), two-way ANOVA was performed. Numerical data, such as the number of faecal boli, were subjected to Chi-Squared analysis. Values of p 0.05 were considered statistically significant. Results Overall, regardless of strain, environmentallyenriched mice showed enhanced functioning, exhibiting greater exploration and reduced anxiety-like behaviour. In tests on anxiety, environmentallyenriched mice showed potential reductions in levels of nervousness by making more transits, investigatory head-dips, and having the lowest latency to leave enclosed areas.

4 P21 Figure 3: *novel arm The T-Maze, with the novel arm partitioned off during the first trial start arm = Removable partitions/doors. *This arm can be inter changed with the other arm, depending on the trial. 10cm In tests of cognition, environmentally-enriched mice also made faster and more correct choices. Environmentally-enriched B10s were found to leap out of the apparatus with ease, being highly agile and able to escape confinement. Environmentally enriched B10s ran from between the open arms of the PM with high speed and agility (despite higher body mass), and showed increased investigatory behaviour. Both environmentally-enriched strains showed higher levels of burrowing. In species-typical behaviour tests, latency to start grooming and the number of Figure 4: start arm The Y-Maze, with the novel arm partitioned off during the first trial 8cm *novel arm = Removable partition. *This arm can be inter changed with the other arm, depending on the trial. marbles buried, were improved in the environmentally-enriched B6s. The enrichment of the B6 housing may explain why this group also obtained a higher nesting score, and burrowed more material after two hours than the S B6s. EE may raise B6 performance above that of S B6s. For example, in the holeboard, more investigatory rears (up the sides of the apparatus) and head-dips into the holes were made by environmentally-enriched B6 mice than by S B6 mice. Environmentally-enriched B10s showed greater exploratory behaviour, and were less anxious than S mice of the same strain. In many cases, environmentally-enriched mice were the highest performers. Contrary to expectations, the S B6 mice functioned at a lower level relative to S B10s in most, but not all, of the species-typical behaviour tests. The marked deficits of B6s were lower in tests of nesting and burrowing. B6s functioned at a lower level on a number of motor tests, such as the static rods and OF, and were impaired in most tests of anxiety. Cognitive tests also revealed lower functioning in B6 mice, with B6s making fewer correct alternations in the T-maze, and taking longer to do so. Both S B6 and B10 mice showed deficits in the majority of, but not all, species-typical behaviours (including climbing and burrowing less than their environmentallyenriched counterparts), and in motor, anxiety and cognitive tests. Environmentally-enriched mice of both strains appeared to function at the highest levels, with S mice of both strains generally having the longest latencies to leave the closed arms and enter the open arms of the apparatus. Although the B10 brain appeared to be influenced by EE, S B10s did not show the deficits expected of their strain, suggesting that the B10 would not provide a suitable mouse model for AD. It was also apparent that the behaviour of B10 mice differed significantly from that of B6 mice, suggesting that the strains could not be used interchangeably in trials. The aim of this study was to assess whether EE influences the behaviour and cognitive and motor functioning of test animals. We used a standard mouse model (B6) and the B10 strain, known to have behavioural deficits. The study aimed to determine whether the B10 mouse housed in an enriched environment would show lesser motor and cognitive deficits, consequently asking whether EE can be influential on brain and behavioural functioning in the adult B10. Contrary to the results of previous studies, the B10s functioned at a higher level than the B6s in a number of species-typical behaviour, motor, anxiety and cognitive tests. Unlike in previous studies, B10 mice, particularly those that had been raised in an enriched environment, functioned at the highest level of all groups in the majority of tests. Environmentally-enriched mice generally functioned at higher levels than those of their S counterparts, suggesting that EE may be the cause of raised functioning in environmentally-enriched mice, and of enhanced behaviour in both strains, particularly the B10 strain. This may suggest that EE is not only ben-

5 P22 eficial to animal welfare, but can also positively influence natural functioning and natural behaviour. Ellen J. Coombs Currently at: WSPA 222 Gray s Inn Road London WC1X 8HB UK ellenjcoombs@gmail.com [This work was carried out in the Department of Zoology, Oxford University, UK] References 1 Smithies, O. (1993). Animal models of human genetic diseases. Trends in Genetics 9, Gunter, C. & Dhand, R. (2002). The mouse genome. Nature, London 420, European Commission Workshop (2010). Of mice and men are mice relevant models for human disease? Outcomes of the European Commission Workshop Are mice relevant models for human disease? held in London, UK, on 21 May 2010, 10pp. Brussels, Belgium: Health Directorate, DG Research, European Commission. 4 Deacon, R.J., Thomas, C.L., Rawlins, J.N.P. & Morley, B.J. (2007). A comparison of the behaviour of C57BL/6 and C57BL/10 mice. Behavioural Brain Research 179, Wimer, R.E., Wimer, C.C., Chernow, C.R. & Balvanz, B.A. (1980). The genetic organisation of neuron number in the pyramidal cell layer of hippocampal regio superior in house mouse. Brain Research 196, Van Leuven, F. (2000). Single and multiple transgenic mice as models for Alzheimer s disease. Progress in Neurobiology 61, Van Praag, H., Kepermann, G. & Gage, F.H. (2000). Neural consequences of environmental enrichment. Nature Reviews Neuroscience 1, Henderson, N.D. (1970). Brain weight increases resulting from environmental enrichment: A directional dominance in mice. Science, New York 169, Wolfer, D.P., Litvin, O., Morf, S., Nitsch, R.M., Lipp, H-P. & Würbel, H. (2004). Cage enrichment and mouse behaviour. Test responses by laboratory mice are unperturbed by more entertaining housing. Nature, London 432, Restivo, L., Ferrari, F., Passino, E., Sgobio, C., Bock, J., Oostra, B.A., Bagni, C. & Ammassari-Teule, M. (2005). Enriched environment promotes behavioural and morphological recovery in a mouse model for the fragile X syndrome. Proceedings of the National Academy of Sciences of the USA 102, 11,557 11, Rampon, C., Jiang, C.H., Dong, H., Tang, Y.P., Lock - hart, D.J., Schultz, P.G., Tsien, J.Z. & Hu, Y. (2000). Effects of environmental enrichment on gene expression in the brain. Proceedings of the National Acad - emy of Sciences of the USA 97, 12,880 12, Roy, V., Belzung, C., Delarue, C. & Chapillion, P. (2001). Environmental enrichment in BALB/c mice: Effects in classical tests of anxiety and exposure to a predatory odor. Physiology & Behaviour 74, Hutchinson, E., Avery, A. & Vandewoude, S. (2005). Environmental enrichment for laboratory rodents. ILAR Journal 46, Marashi, V., Barnekow, A., Ossendorf, E. & Sascher, N. (2003). Effects of different forms of environmental enrichment on behaviour, endocrinological, and immunological parameters in male mice. Hormones & Behavior 43, Prior, H. & Sachser, N. (1995). Effects of enriched housing environment on the behaviour of young male and female mice in four exploratory tasks. Journal of Experimental Animal Science 37, Kempermann, G., Kuhn, G.K. & Gage, F.H. (1997). More hippocampal neurons in adult mice living in an enriched environment. Nature, London 386, O Keefe, J. & Nadel, L. (1978). The Hippocampus as a Cognitive Map, 504pp. Oxford, UK: Clarendon Press. 18 Arendash, G.W., Garcia, M.F., Costa, D.A., Cracchiolo, J.R., Wefes, I.M. & Potter, H. (2004). Environmental enrichment improves cognition in aged Alzheimer s transgenic mice despite stable beta-amyloid deposition. NeuroReport 15, Jankowsky, J.L., Melnikova, T., Fadale, D.J., Xu, G.M., Slunt, H.H., Gonzales, V., Younkin, L.H., Younkin, S.G., Borchelt, D.R. & Savonenko, A.V. (2005). Environmental enrichment mitigates cognitive deficits in a mouse model for Alzheimer s disease. Journal of Neuroscience 25,