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The laboratory environment existing outside the test situation itself can have a substantial influence on results of some behavioral tests with mice, and the extent of these influences sometimes depends on genotype. For alcohol research, the principal issue is whether genotype-related ethanol effects will themselves be altered by common variations in the lab environment or instead will be essentially the same across a wide range of lab environments. Data from 20 inbred strains were used to reduce an original battery of seven tests of alcohol intoxication to a compact battery of four tests: the balance beam and grip strength with a 1.25 g/kg ethanol dose and the accelerating rotarod and open-field activation tests with 1.75 g/kg. The abbreviated battery was then used to study eight inbred strains housed under a normal or reversed light-dark cycle, or a standard or enriched home cage environment. The light-dark cycle had no discernable effects on any measure of behavior or response to alcohol. Cage enrichment markedly improved motor coordination in most strains. Ethanol-induced motor coordination deficits were robust; the well documented strain-dependent effects of ethanol were not altered by cage enrichment.
The lab environment in which mouse behaviors are measured influences many but not all kinds of tests. Mouse inbred strain differences in open field exploration, for example, are very similar across labs and even between studies conducted decades apart that used substantially different task parameters [1-5]. Similarly, ethanol preference as assessed by the two-bottle test is highly reproducible in different labs . Anxiety-related behaviors and agonistic social interactions, on the other hand, sometimes show marked changes in relative strain differences across laboratories [1, 4, 6-8]. Although strain-specific measures of motor performance on the accelerating rotarod can differ substantially between labs, sensitivities to the disruptive effects of ethanol are nevertheless very similar on that task [9, 10].
If common variants in the lab environment nevertheless yield similar outcomes in genetic experiments on ethanol sensitivity, then the validity and generality of research in this field will be confirmed and enhanced. There are many differences in the ways that most labs arrange the local environmental context of tests [1, 3, 8, 11]. Although there is no way to equate lab environments completely, it is possible to assess the efficacy of some of the more common variants with controlled studies done in just one lab. Here we assess effects of two common variations in housing conditions in the mouse colony: the phase of the light-dark cycle when testing occurs and enrichment of the home cage .
Adding an independent variable such as light cycle or enrichment to an experimental design with many mouse strains doubles the number of groups to be tested. In order to accommodate the larger design with a substantial number of animals per condition, it would be helpful to reduce the amount of behavioral testing done with each individual. Accordingly, we first reduced a battery of seven tests to four that covered a reasonably wide range of the domain of motor coordination deficits induced by ethanol. All tests had previously been shown to detect strain differences in ethanol response . The goal was to identify a subset of those tests and ethanol doses that would provide an effective means to assess genetic differences in ethanol effects on behavior.
The studies reported here pertain to acute ethanol effects on the domain of motor performance. This domain is complex and no single test can adequately capture the extant genetic variation among standard inbred strains . For any one genotype, ethanol might disrupt performance on one kind of test while having minimal effects on another. Effective doses seem to depend on the sensory and motor requirements of the specific task. Thus, a test battery for use with mice should have a number of tests that can be given to one animal over a period of one to two weeks. Distributing individual tests across days allows sufficient time to recover from each ethanol injection while avoiding the lingering effects of any tolerance to prior doses of ethanol . The tests should show only modest correlations of strain mean values with each other; if there are two highly correlated, and therefore redundant tests, one can be dropped from the battery.
On the basis of prior studies, we devised a battery of seven refined tests, six of them involving motor coordination (Table 1), that spanned a major portion of the domain. When investigating a wide range of experimental treatment effects, however, a battery of seven tests is somewhat cumbersome. Consequently, the first step in the current series of experiments was to reduce the size of the battery and generate a more compact battery for testing effects of light-dark cycle and cage enrichment. Accordingly, we administered the larger battery of seven tests to 20 inbred mouse strains under the same lab environment conditions that were to be used in subsequent experiments, then used the data to inform our choice of items for a more compact battery .
The 20 strains used in this experiment were obtained from the Jackson Laboratory, Bar Harbor, Maine. They were chosen to be broadly representative of available strain genotypes as recommended by the Mouse Phenome Database (MPD; www.jax.org/phenome), avoiding those derived from wild species that we earlier found were very difficult to handle in behavioral studies . Eight were from the original MPD A list (129S1/SvImJ, A/J, BALB/cByJ, C3H/HeJ, C57BL/6J, DBA/2J, FVB/NJ, SJL/J), eight from the B list (AKR/J, C57L/J, C58/J, NOD/LtJ, NZB/BlNJ, PL/J, SM/J, SWR/J), one from the C list (LP/J), two from the D list (BTBR T+ tf/J, CE/J), and one not on any list (CBA/CaJ). Current assignments to priority Tiers are provided at the MPD website. Mice were received at about 6 weeks of age in four shipments of 80 mice each, with all 20 strains being represented by four mice of one sex in each shipment. Sexes were balanced across shipments for all strains. Location of cages on the shelves of a rack of cages was balanced so that no strain or sex was generally closer to ceiling lights or the room door.
Animals were housed four per cage in Animal Care Systems M.I.C.E. ventilated cages (920 L/min air through 56 cages) with free access to local tap water and Purina 5001 mouse chow. Each cage was lined with 6 mm Bed-o-cob bedding and provided with a fresh Nestlet after each weekly cage change. The colony room was maintained at 21 ± 1° C with humidity of about 70%. Fluorescent room lights (300 to 750 Lux at different levels of the rack) went on at 0600h and off at 1800h. All behavioral testing was done between 0800h and 1600h.
Each shipment of 80 mice was divided into two replications of 40 mice (two of each strain) that were tested eight days apart. Testing of the first replication began when mice were about eight weeks of age, after two weeks acclimatization to their new surroundings. Within a replication, one animal received saline injections on certain days while its cage mate in that replication received ethanol. The order of testing strains within a replication was randomized. Testing of one replication was done in any one day using 10 squads of four mice each, with each squad having one saline and one ethanol treated animal of each of two strains. About 1h before testing began, the four mice for a squad were removed from the home cage, placed individually in clean shoebox cages, and brought on a cart to the testing room, where they remained while the preceding squad was being tested. After all four mice had completed the day’s test, they were returned to their group cages in the colony room.
All solutions were injected intraperitoneally using sterile 27 gauge hypodermic needles. Sterile saline was 0.7% sodium chloride. The ethanol solution was 20% v/v of 200 proof ethanol from Commercial Alcohols (Chatham ON) in saline. Three doses were administered on different days of the test battery: 1.25 g/kg, 1.75 g/kg and 3.0 g/kg. Time between injection and the start of a test depended on the specific test. Volume of solution was determined from mouse body weights measured at the start of the test day.
The sequence and timing of tests are indicated in Table 1. For mice in the first replication, Day 1 was Monday and Day 8 was the next Monday, whereas for those in the second replication Day 1 was Thursday and Day 8 was the next Thursday. Brief descriptions of each test are provided here, whereas detailed protocols for all tests are available from the authors on request and also the website for Mouse Behavioral Testing (www.elsevierdirect.com/companions/9780123756749). All mice were tested in the order indicated in Table 1, where abbreviations for the tasks are defined. Thus, on Day 1 there were no injections and all mice were tested in the same sequence of 4 pretraining trials on the BB, then 4 pretraining trials on the FSRR followed by 10 trials on the ARR. On Day 2, mice were injected with either saline or 1.25 g/kg alcohol and then given the FSRR test, followed immediately by the BB test and then the ARR test. On Day 5, mice were injected with either saline or 1.75 g/kg alcohol and then given an ACT test, followed by a GRIP, another FSRR test and finally the ARR test. On Day 8 all mice were placed into the hypothermia cages for 2h, then a baseline ORA rating was obtained and rectal temperature was recorded. All mice then received the 3.0 g/kg ethanol injection and were given another ORA test followed by two hypothermia measures.
The AccuRotor rod from Accuscan Instruments had a 6 cm diameter rod divided into four lanes with the rod in each lane covered with 320 grit sandpaper. The rod was 65 cm above a trough of bedding 3 cm deep. The rod turned at 6.5 rotations per min throughout the test. A mouse was placed onto the rod and a timer was started when its tail was released. Fall latency was recorded by the experimenter, unless the mouse remained atop the rod for the trial limit of 3 min on Day 1, 5 min on Day 2 and 3 min on Day 5. Pre-training on day 1 was concluded when the mouse remained on the rod for a full 3 min or after 4 training trials.
The beam was a grey PVC plastic beam (2 cm wide × 109 cm long) located 56 cm above the table top. Four pre-training trials were run, two from each end of the beam, so that the mouse learned to travel from one end to the other. No data were collected on Day 1. On Day 2 one test trial was run starting 10 min after injection of ethanol, requiring the mouse to traverse the beam in less than 10 min. The number of slips off the top of the beam by any paw was recorded by the experimenter.
The same rotarod was used for this test, with a slight procedural modification. Rather than spinning at an initial and constant RPM, the accelerating rotarod was held still until all four mice were placed into the separate lanes. If a mouse fell off before the start of a trial it was placed back onto the rod. A trial was started at 0 RPM and accelerated at 20 RPM/min to a maximum rotation speed of 99.9 RPM. A trial continued until all four mice had fallen. The latency from start of trial to fall was recorded by the experimenter. The maximum was never reached by any animal.
A clear plastic chamber 40 × 40 cm with 30 cm walls was placed in an enclosed cubicle that provided 75 Lux at the center of the field from fluorescent lights. Before each experiment, pink butcher’s paper was placed on the floor with the waxed side facing up. The path followed by the mouse was tracked with AnyMaze software (Stoelting Inc., Wood Dale, IL) using the automatic tracking option and a Panasonic CCTV camera (Model WV-BP334) with a 4mm lens. The experimenter noted the number of episodes of rearing. Following each trial, the plastic chamber was removed and wiped with S.O.X. (Rolf C. Hagen, Mansfield, MA - http://www.hagen.com), butcher paper was replaced with a fresh sheet.
Grip strength was measured using a 3 mm diameter stainless steel bar attached to an Extech 475040 Force Gauge (Waltham, MA). The experimenter held the mouse by the tail and lowered it so that the mouse could grasp the bar with both forepaws, then pulled the mouse horizontally until it lost its grip on the bar with both paws. Maximum force was recorded for each of three trials separated by a 30 sec intertrial interval.
Mice were placed in the center of a 39.5 × 39.5 grey PVC box with 6 cm high walls and observed for 1 min. The experimenter rated instances of wobble, splay, nose down and belly dragging behaviors, as previously defined. The ataxic behaviors were never seen during the one baseline observation period prior to ethanol injection.
Each mouse was weighed and placed into an individual plastic ventilated chamber one hour before the start of testing; no food or water was available. Its core body temperature was then recorded with a rectal thermometer probe (2 mm ball on 2 cm shaft) and digital thermometer (Sensortek TH-8, Suffolk, UK) shortly before an ethanol injection and then 30 min and 120 min post-injection.
All housing and experimental conditions were approved by the Animal Care Committee at the University of Windsor, following guidelines of the Canadian Council on Animal Care.
Preliminary explorations of the data suggested that influences of the control variables sex, replication and cage location were in most instances very small, and variance from these sources was therefore pooled with other sources of within-group variance to generate an error variance term. The design of the study precluded any confound of control variable effects with variables of principal interest. Analyses were then done with balanced factorial analyses of variance (ANOVA) using fixed effects models. A similar approach was employed for Experiments 2 and 3.
Useful data were obtained from 312 mice of all 20 strains, including equal numbers of males and females for almost all strains. When averaged over all strains and both sexes, ethanol effects were large for almost all tests (Fig. 1). Except for the accelerating rotarod at the lowest dose (1.25 g/kg), the ethanol effects ranged from d = 0.7 to d = 2.0 standard deviation at the 1.25 and 1.75 g/kg doses and were unquestionably significant (all P < .00001; see Table 2). Both the balance beam and fixed speed rotarod revealed obvious impairment at the lower dose, while accelerating rotarod and grip strength were impaired at the 1.75 g/kg dose, a dose that also produced activation of locomotion in the open field. The decline in grip strength was so great (d=2.0) that we opted for doing this test with the lower 1.25 g/kg dose in Experiment 3. The 3.0 g/kg dose caused overt ataxia apparent to a human observer that was not seen at the lower doses, and that high dose also caused mild hypothermia.
Strain differences were large and clearly significant for all tests (Table 2). The strain effect accounted for only about 10% of total variance on the balance beam and fixed speed rotarod tests, whereas strain differences were quite substantial on all other tests. Statistical interaction between strain and ethanol could not be meaningfully assessed for three tests (balance beam, ataxia, hypothermia) where there was virtually no variance among the normal controls. Among the other tests, only the accelerating rotarod and open field activity showed significant strain x ethanol interaction.
The strain profiles of baseline performance on each test are shown in Fig. 2A. On each test following ethanol injection, there was a wide range of impairments (Fig. 2B), to the extent that there was usually a strain that showed almost no ethanol effect at all. No strain was highly impaired on every test, nor was there a strain that failed to show substantial impairment on any test. Among all 20 strains, only PL/J was above the median for impairment on every test. A simple index of impairment for the purpose of this study was constructed by comparing each strain to the most impaired strain on each test, such that the most impaired was assigned a score of 100%. On that index, only PL/J showed a remarkably high average index of more than 75% (Fig. 3), whereas every other strain averaged 30 to 60% relative impairment. The correlations of strain mean ethanol effects across all tests at three doses (Table 3) indicated that at 1.75 g/kg the fixed speed and accelerating versions of the rotarod yielded very similar results (r = 0.67, P < .01), whereas correlations among several other tests were considerably lower. Thus, only the two versions of the rotarod test revealed any noteworthy redundancy in the battery of seven kinds of tests.
Not only was the fixed speed rotarod afflicted by redundancy, but it also showed high variability of behaviors on the two days (Fig. 4). On each day and dose, half or more of the mice receiving saline never fell from the rod within the time limit, and 10 to 20% of mice receiving ethanol also failed to fall, whereas many mice in the same group took the plunge very quickly (Fig. 4A). There was little consistency in which mouse remained on the fixed speed rod the full time on the two days (Fig. 4B), especially for the ethanol-treated mice where the correlation of fall latency at 1.25 and 1.75 g/kg was a modest r = 0.40 for 153 mice. Repeatability on the accelerating rotarod over the two days under ethanol (Fig. 4C) was considerably greater (r = 0.64).
On the basis of data for all nine measures on seven tests with 20 inbred strains, it was possible to reduce the original battery to a more compact battery of four tests for future studies. The fixed speed rotarod yielded highly variable behavior from day to day, and at the 1.75 g/kg dose it was also redundant with the accelerating version of the task. Thus, we felt that little information on genetic sensitivity to ethanol would be lost by utilizing only the accelerating rotarod in subsequent experiments. The accelerating rotarod was insensitive to ethanol effects at the 1.25 g/kg dose and therefore was removed from the battery at the lower dose as well. At 3.0 g/kg, hypothermia was clearly a consequence of ethanol, but was not a motor behavior at all, and mice were so severely ataxic that they could not meaningfully be assessed on any of the other tests. Thus, the first experiment provided good reasons to employ a battery consisting of balance beam and grip strength at 1.25 g/kg and accelerating rotarod and open field ambulation at 1.75 g/kg in subsequent experiments.
For all of the tests studied here, valid data were obtained for every strain and there were very few instances of missing data. On the two rotarod tests, only five of 312 mice failed to produce a complete data set. No mouse failed to complete the grip strength test, while only three yielded missing values for open field activity because of apparatus failure. On the balance beam, only one mouse could not be scored for missteps, while 39 could not be scored for latency to cross the beam because they never fully traversed it. On the basis of this first experiment, all four tests used in the abbreviated battery appeared to be satisfactory and did not require further refinement [8, 20].
The data from the present survey of inbred strains were reasonably similar to earlier tests with the same strains. Here we evaluated 17 of the same strains that were assessed previously in two labs on the accelerating rotarod as part of the Mouse Phenome Project . In that earlier study, the strain correlation of ethanol impairment was r = 0.82 between data collected in Edmonton, Alberta and Portland, Oregon. Correlations of those two data sets with the current strain mean impairment ranks collected in Windsor were r = 0.45 and r = 0.50, respectively. All three data sets found FVB to be least impaired and AKR, BALB and NZB to be only mildly impaired. BTBR was most impaired in the 2003 data and substantially impaired in the current study, although not to the extent seen more recently in PL/J. The methodology in the two studies was somewhat different, in that the impairment scores in the 2003 study were based on pre- and post-injection measures of fall latency in mice that all received ethanol, whereas in the present study separate groups of mice received ethanol and saline.
Every mouse lab makes a difficult choice about the light-dark cycle. Mice are nocturnal mammals that show relatively short periods of activity during the light phase . Hence, a good argument can be made for maintaining a behavioral test facility on a reversed light-dark cycle . At the same time, animal care and test lab personnel prefer to do their jobs in the light phase. Perusal of the literature on mouse behavior genetics indicates that some labs use a reversed cycle and others do not. Levels of expression of many genes change with the time of day , as does activity of enzymes involved in ethanol metabolism . Inbred strains differ substantially in the way they apportion activity between light and dark periods (Mouse Phenome Database entries for Mogil2, Seburn1; http://phenome.jax.org). Voluntary consumption of ethanol is greater in the dark phase [25-27], while ethanol-induced increases in motor activity are also greater and loss of righting reflex lasts longer in the dark phase [28, 29]. Abrupt changes in the lighting cycle can markedly alter many processes, including memory and mood [30, 31]. In most reports, however, the light-dark cycle itself was not altered in a way that modified the association of light phase with other events in the laboratory (e.g., ). One exception is Clenet et al. , who subjected Swiss mice to five different lighting schedules, including normal and reversed light-dark periods but found little or no influence on levels of motor activity and anxiety-related behavior.
In order to assess the possible influence of the lighting cycle in the colony room on behavioral test results, we took advantage of two identical, adjacent colony rooms that contained identical cages and ventilation. The two rooms and the test room were contained in a small suite of rooms that was separate from all other rooms in the building, so that we could achieve complete control over lighting in all parts of the lab. Our own lab personnel did all cage changing and washing, which gave us total control over entry into the rooms for routine animal care. It is possible that subtle vibrations from elsewhere in the building or vehicles on nearby streets were correlated with the actual time of day.
Equal numbers of males and females of eight strains from the MPD A list (129S1/SvImJ, A/J, BALB/cByJ, C3H/HeJ, C57BL/6J, DBA/2J, FVB/NJ, SJL/J) were obtained at 6 weeks of age from the Jackson Laboratory and then tested at 8 to 10 weeks, which entailed at least two weeks of acclimatization. On the day when a new shipment arrived in the lab, mice were unpacked, tail marked, assigned randomly to normal or reversed cycle (different colony rooms), and then placed into clean cages in the appropriate colony room, where they were housed throughout the study. The location of cages on the rack in a room was balanced across upper and lower shelves for sex and the eight strains. There were 2 shipments of 128 mice, and each shipment was tested as 4 successive, complete replications of the experiment to yield a sample size of eight mice per each strain-sex-lighting group with half of the mice in each cage receiving ethanol injections. Each replication involved one mouse from every strain, sex and lighting combination. Whether the male or female in a strain received ethanol or saline for a given housing condition was balanced across two successive replications involving 64 mice. Which specific mouse within a cage received ethanol or saline injection was determined randomly.
Caging, food and water were the same as in Experiment 1. In the room with a normal light cycle, room fluorescent lights went on at 0600h and off at 1800h, whereas in the other room lights went off at 0600h and on at 1800h. Light from an adjacent utility room was attenuated by covering the small window in each door and placing a towel across the gap at the bottom of the door. Light levels at the center of each room were from 300 to 750 lux depending on the cage location on the rack during the bright phase and less than 1 Lux during the dark phase. The unaided eye of the experimenter could not read any information on the cage tag when the room was dark.
The experimenters worked with mice during the dark phase in their colony room while wearing Night Owl Optics NOTG1 night vision goggles (El Paso, TX) that illuminated the environment with infrared light and detected objects with an infrared sensing video camera that created a visible image on two miniature LCD screens in the goggles. Prior to entering a dark colony room, the person first turned off all lights in the adjacent room and put on the goggles. The daily inspections for adequate food and water and the weekly cage changes were done at about the same time of day for both colony rooms, aided by the night vision goggles in the dark room. Shortly before testing, when the four mice for a squad were removed from the group cages and placed individually into clean shoebox cages, they spent the 30 min acclimatization period in the colony room rather than the test room. The test room was maintained at 5 Lux or less for all mice during testing, a light level that made it possible for the experimenter to observe slips on the balance beam, falls from the rotarod, and grips on the steel bar. When moving mice to or from the test room to the colony room, lights in the utility room between the two were always turned off for the reversed cycle mice.
As in experiment 1, half received saline and half received ethanol. Mice were weighed at the start of any day when injections were given. Tests were given on Monday (Day 1), Wednesday (Day 3) and the next Monday (Day 8) for one replication and Tuesday, Thursday and the next Tuesday for the next replication. On Day 1 there were no injections and each mouse received 4 pretraining trials on BB followed by 10 pretraining trials on ARR. On Day 3, trials were given both prior to and after injections (saline or 1.25 g/kg alcohol) for the BB and GRIP tests, which yielded change scores of impairment for mice that received ethanol and test-retest comparisons for the saline group. On Day 3 prior to injection, one BB trial was given, followed promptly by 2 GRIP trials. An injection was then given. One BB trial was given 5 min post-injection and two GRIP trials 7 min post-injection. On Day 8, three ARR trials were given with a 30 sec intertrial interval shortly before the injection. Mice then received an injection (saline or 1.75 g/kg alcohol), and two GRIP trials were given 2 min later, one 5 min ACT trial was given 5 min after injection, and three ARR trials were given 30 min post-injection. Each mouse was scored on each test on each day for ease of handling , and an average score was determined across all tests.
Data were analyzed under two different ANOVA models. One examined response to ethanol as a within-subjects effect based on comparison of pre- and post-ethanol behavioral scores (Table 4A). The other estimated ethanol sensitivity as a between-subjects factor using data from all animals tested in the experiment (Table 4B). The table shows the degrees of freedom (df), the significance level of each effect as an approximate P value, and the F ratio for effects where P < .05. For the between-subjects analysis, the relative magnitude of the combined effects of all predictors is shown as the multiple R2. For many variables, the effects were statistically very large (R2 > 0.4). Table 4 provides information on all measures, whereas Fig. 5 shows results only for the three principal measures where pre- versus post-injection comparisons were meaningful.
There were no significant main effects of the light-dark cycle or interactions of light cycle with strain on any behavioral test in this experiment. There were marginally significant interactions of light cycle with strain for body weight and ease of handling during weighing. Neither were the effects of ethanol modified by the kind of light cycle used in the colony room.
Strain differences, on the other hand, were large and clearly significant (Fig. 5; Table 4) for all measures except grip strength. Ethanol effects were very large for all measures except body weight and capture scores. The results for the open field test showed strong evidence of strain-dependent effects of ethanol, whereas on other tests the interaction effects failed to achieve significance, and the patterns of impairment by ethanol were quite similar across all eight strains.
Of particular interest for future studies was the comparison of ethanol effects between subjects to the change from pre-injection trials to post-injection trials within subjects. For the mice receiving saline injections, there was little or no change across trials on three tasks, whereas those receiving ethanol showed clear and significant changes, especially for the balance beam and grip strength. The absolute amount of change, as well as the statistical significance levels, was very similar for the between- and within-subject comparisons, despite the fact that the within-subjects data involved half as many mice. Impairment of grip strength after 1.75 g/kg ethanol was more severe than after 1.2 g/kg, but even at the lower dose the change was substantial. These data suggest that the test battery in future studies could be made even more compact and efficient by eliminating the 1.75 g/kg dose for grip strength and giving all mice an injection of ethanol, evaluating them before and after the injection, for three of the tests.
While results showed no significant main effect for the light-dark cycle on any of the tests, there are a few potential reasons for this result. Wild mice (Mus musculus) are known to be nocturnal and crepuscular, but for laboratory mice (Mus musculus laboratorius) the majority of stimulating environmental activity takes place during the daytime hours. It is possible that in some mouse facilities the daily activities outside the colony rooms were stimulating enough to overcome circadian rhythms most commonly synchronized to levels of light. Noteworthy daily events included the daily cage inspections, cage changes and washing, and general laboratory and building noises (e.g., elevators) that could not be negated or controlled. Those in addition to other geophysical events could also have functioned as external time cues or zeitgeber . Another potential problem could be the duration of circadian clock re-entrainment with inbred strains of mice. Takamure et al.  found that a rat circadian clock re-entrains within three days, while Bosgraaf and Toth  reported a similar time course for shifting the phase of mouse body temperature. Three days is considerably less than the minimal two week habituation time period of the present study. Because it is possible to verify re-entrainment to a new light-dark cycle and thereby circadian cycle by measuring motor activity or core body temperature, in future studies verification using those measures may prove to be worthwhile. The one other lab-environment factor that may have had an effect was the 5 lux lighting during testing. Alternatively, the indifference to these conditions could stem from a simple methodological parameter; it is possible that the 30 min acclimation period was sufficient to foment awakening and arousal in all mice before each behavioral test. Light cycle effects might be more apparent if tests were given shortly after the animal is removed from its home cage and colony room, but this is not the usual procedure in most labs where acclimatization is commonplace.
Based on these results, the light-dark cycle had no effect on outcome of motor performance tests or on levels of impairment following an injection of ethanol. Although it might be possible to detect a light-dark cycle effect on motor tests using other procedural parameters, those used in this study were the ones employed in our previous work and in many other labs. Several other behavioral responses are impervious to the light phase in which testing is performed; for example, certain social behaviors  and light cycle effects on motor activity and anxiety-related behavior in the elevated plus maze are minimal .
Labs often differ in the use of home cage enrichment devices, and the deployment of such devices is becoming more common. According to the construct of brain and cognitive reserves (i.e. the reserve theory), environmental enrichment promotes the development of factors that enhance nervous system vitality . For example, enrichment alters protein expression [39-41], and many proteins promote a more complex brain by signaling the development of more dendritic branches, the formation of more synapses, and induction of neurogenesis [39, 42-45]. Enrichment improves motor coordination and complex learning [46-48] and decreases the effects of some drugs [49, 50]. High levels of enrichment may help to ameliorate effects of prenatal or neonatal alcohol exposure , but there is little information on the consequences of enrichment for acute effects of ethanol given to mature animals. There is insufficient information available concerning enrichment influences on a wide variety of common behavioral tests  or how much and what kind of enrichment of a mouse cage is needed to create a noteworthy improvement . While it has been suggested that cage enrichment will not compromise many kinds of tests [5, 54], we could find no published source on cage enrichment in relation to the acute behavioral effects of alcohol.
We therefore compared ethanol effects on mice housed in either a sparsely furnished plastic cage or the same cage filled with as many commercially available enrichment devices as could be fit into the available space. Particularly in the behavioral domain of animal research, there is a growing need to evaluate the implications of moderate levels of home cage enrichment on behavior in response to growing demands that enrichment of home cages be the rule. In the present study, we wanted to know if moderate levels of enrichment would alter the effects of ethanol intoxication on tests of motor behavior. To do so, we compared mice living in standard laboratory cages to mice living in enriched cages for the entire 4.5 week period prior to testing.
It is likely that cage enrichment would have a greater effect if applied from birth rather than beginning with arrival in the lab at 6 weeks of age. In this study, however, we wanted to apply the treatment in the same way it would occur if done routinely in our labs when mice are delivered post-weaning from a commercial supplier. Despite the restricted period of enrichment, the treatment did indeed have an effect on behavior. Although the study did not address directly the broader question of whether cage enrichment should be routinely employed through the life of a mouse to enhance animal welfare, the results are relevant to such discussions. Neither does the study address the question of what should be considered a normal environment for lab mice or a minimally adequate environment. We use the term “standard” condition to describe the common kind of cage environment that currently prevails in most labs that have not deliberately added enrichment devices. Perhaps our enrichment condition will become standard practice in the future, but it certainly is not standard practice now.
Equal numbers of males and females of the same eight strains used in Experiment 2 were obtained at 5 to 6 weeks of age from the Jackson Laboratory. There were two shipments, each of 64 mice, four males and four females of each strain, and the total sample size was eight mice per group, the same as in Experiment 2. In this experiment there were not separate saline and ethanol groups; each mouse received an ethanol injection and was tested before and after the injection. On the day when a new shipment arrived in the lab, mice were unpacked, tail marked, assigned randomly to standard or enriched housing, and then placed into clean cages of the appropriate kind in the same colony room, where they were housed throughout the study. Animals were housed two per cage, and their tests began after 32 to 33 days in their assigned housing condition. Apart from behavioral testing, mice were handled once per week when tail marks were reinforced and cages were cleaned. They remained in the assigned housing throughout testing.
Food, water, bedding, light-dark cycle (300 to 750 Lux on at 0600h and off at 1800h), temperature and humidity were the same as in Experiment 1. Cages from the Standard and Enriched conditions were distributed across the shelves of the same rack of cages in a balanced manner, and every shelf had cages from both conditions, all eight strains and both sexes. In the Standard condition, the cage environment was made as simple as could be achieved with the available ACS caging. It was actually less stimulating for the mice than what they routinely experienced in studies not involving enrichment, because we wanted to maximize the difference between the two cage conditions. In order to minimize possibilities for climbing, the food hopper was removed and food cubes were placed in a small pile on the bedding. Mice could still climb on the water bottle spout and the filter housing at the front of the cage. Cardboard partitions were positioned so that mice in the Standard condition could not see animals in the adjacent cages on the same shelf. The only novelty was the weekly cage changes in which animals received new bedding, but without a Nestlet. In the Enriched condition, mice could see into an adjacent Enriched cage, and the food hopper allowed extensive climbing. A fresh Nestlet (Ancare) was placed on the floor each week. Enrichment devices added to the ACS cage almost filled the cage. There was a stainless steel running wheel (Otto Environmental), a Techniplast mouse hut, two 6 mm nylon balls (Otto Env.), a gumma bone, a 13 cm plastic tunnel (Bio-Serv), and two elevated platforms that attached to the food hopper (Otto Env.). The enrichment devices were very similar in number and kind to those recommended recently by Sztainberg & Chen . All devices were removed and cleaned weekly along with the cages. Devices were always placed in the same location in a fresh cage, but the mice often moved them.
Testing began at about 10 weeks of age, after mice had been in the lab at least four weeks. Testing was done using squads of four mice. Each squad contained equal numbers of both sexes and housing conditions, and every two successive squads contained all eight strains. Strain, sex and housing combinations were balanced across all squads and replications. Order of testing within a squad was randomized. Three tests that were suitable for pre- versus post-injection evaluation were used in this study (BB, balance beam; GRIP, grip strength; ARR, accelerating rotarod), and for each task the apparatus was the same as that used in Experiments 1 and 2. For half of the mice, Days 1, 3 and 8 were on Monday, Wednesday and the following Monday, whereas for the other half they were Tuesday, Thursday and the following Tuesday. Procedures on those days are described briefly.
Day 1 - No injections; 4 pre-training trials on BB, 10 pre-training trials on ARR.
Day 3 - Prior to injection, 1 BB trial and 2 GRIP trials served as baseline. Then 1.2 g/kg ethanol was injected. One BB trial was given 5 min after injection and two GRIP trials 7 min after injection.
Day 8 - Prior to injection, 3 ARR trials served as baseline. Then 1.75 g/kg ethanol was injected. Two min after injection, mice received 2 GRIP trials, and then 30 min after injection there were 3 ARR trials.
As shown in Fig. 6A, depicting baseline performance prior to ethanol injection, mice living in enriched cages made fewer slips on the balance beam, had a stronger grip on the bar, and remained longer on the accelerating rotarod. On the balance beam, only three of 64 enriched mice made even one slip, whereas 12 of 64 mice in standard cages slipped at least once (χ2 = 7.3, P = .013, 1-tail). Half of strain A/J made at least one slip, while the other strains were nearly perfect. For baseline grip strength on Day 2, the effects of enrichment and sex were pronounced (F = 24.0 and F = 21.3, respectively, both with P < .00001), while strain differences were rather small but significant (F = 3.2, P = .004). Enrichment did not affect some strains more than others; no strain by enrichment interaction term approached significance. For baseline rotarod performance on Day 3, both the effects of enrichment and strain differences were quite large (F = 23.1 and F = 28.2, respectively, both P < .000001), whereas other factors failed to produce significant effects on performance.
Ethanol had major effects on performance in all three tests (Fig. 6B, Table 5), but the extent of the change observed between baseline and post-ethanol injection tests was not influenced by enrichment. Neither was there any indication that enrichment affected the response of some strains to ethanol more than others; a strain by enrichment interaction for ethanol’s effects on behavior was not observed (Table 5). On the balance beam, there was a large strain difference seen in the increase in slips caused by ethanol, but enrichment did not mitigate the effect of ethanol seen overall (no enrichment effect) or across the different strains of mice (no strain by enrichment interaction). Small but significant strain differences were noted in the ethanol-induced decline in grip strength. Strain differences in the degree of impairment due to ethanol on rotarod performance were substantial.
The aim of experiment 3 was first to assess the effects of home cage enrichment on individual performance using several common behavioral tests and then to determine whether or not enrichment interacted with the effects of ethanol on mouse motor behavior. We found that enrichment did improve performance on motor behavioral tests but did not alter the responses to ethanol. In fact, the effects of ethanol were so robust that animals from enriched and standard conditions were equally impaired on all three motor tests.
This study revealed the power of the within-subjects approach for assessing ethanol effects on three tests. Despite the modest sample size of eight mice per treatment group (combining males and females), ethanol effects were highly significant on all three tests and strain by ethanol interactions were clearly significant for two of the tests.
While enrichment usually is taken to mean addition of toys and other objects to the cage, other changes to the cage environment may also affect outcomes. For example, Oliva et al.  observed that, following a move to a new animal facility, their animals seemed to be fighting more. The only obvious change in husbandry conditions was the use of high ventilation cages. In a controlled comparison with their previous lower ventilation cages, they found that high ventilation increased fighting behavior and changed the number and volume of P2 glomeruli in the olfactory bulb. That kind of olfactory influence could not have been a major factor in our study of enrichment because the standard and enriched ACS cages both involved the same flow rate of fresh air through each cage, and enrichment devices were cleaned thoroughly each week when the cages were changed.
It is informative to compare the results of the same tests conducted several months apart in the three experiments, all of which were done with the same eight inbred strains in the same lab with the same apparatus but different experimenters at the University of Windsor (Fig. 7). Patterns of strain differences in response to ethanol indicate genotype by treatment interactions, whereas overall mean scores on a test indicate consistent environmental differences between experiments.
Slips on the balance beam after ethanol were remarkably similar in the three experiments, always being highest in A/J and lowest in FVB. Every strain showed a clear and significant (P < .05, one-tailed) increase in slips under the influence of ethanol in all three experiments, with the sole exception of FVB in Experiment 2. Baseline counts of slips showed differences between experiments, whereas slips after an ethanol injection were quite similar in all experiments. The differences among experiments appeared to arise from slightly different definitions of a slip used by the experimenters. The person counting slips was always the same one who gave the injection and therefore knew the treatment condition, and that could have influenced judgments of slips. The test might be improved by concealing information about treatment condition during observations, but this would require the services of a second experimenter. Greater consistency of scoring slips might be achieved by some kind of electronic touch circuit or video observation. That kind of automation would not address the challenge of mice that yield no data because they do not traverse the beam or engage in other non-compliant behaviors . Despite these limitations, strain-specific results on the balance beam were clearly replicable across experiments and experimenters. The test is a good indicator of impairment of motor behavior by ethanol.
Baseline grip strengths were much higher in Experiments 1 and 3 than in the second study, but in all three studies, strain differences were relatively small, while ethanol effects were consistently very large. Only strain C3H in Experiment 3 failed to show a significant degree of impairment by ethanol. The grip strength test entails an intimate relation between the mouse and its human handler. Exactly how it is held, in the transfer from its holding cage and during the test itself, could influence the maximum strength of pull. In the present configuration, the handler must pull the mouse away from the strain gauge after it grasps the bar, and the rate of pull could be very important. A mechanical device that always pulls the gauge away from the mouse at a constant rate could improve consistency of the test. Pressure of the experimenter’s fingers on the tail may also play a role, and some kind of artificial cuff might provide a more consistent grasp on the tail. In any event, the current version of the test is very sensitive to effects of moderate doses of ethanol, while it indicates that strain differences in grip strength are generally quite small and therefore less consistent across experiments. The strain difference was not even statistically significant in one experiment (light-dark cycle), even though the ethanol effect on grip strength was large and unquestionably significant.
Mean scores on the accelerating rotarod were substantially but not entirely consistent across the three experiments. Over all groups, fall latencies were very similar in Experiments 1 and 2 but lower in Experiment 3, which could be a consequence of the sandpaper wearing down over time . The sandpaper is a likely source of a difference between successive shipments of mice reported in Fig. 8.2 of ; more frequent renewal of the 320-grit sandpaper could address this problem. Strain rank orders under the saline condition showed some consistency, with C57BL/6J always being the best and BALB/cByJ ranking at or near second best, while C3H/HeJ was at or near the bottom rank. Once the overall mean fall latency in the three experiments is taken into account, substantial consistency of ethanol effects is apparent across experiments for strains SJL/J, C57BL/6J, C3H/HeJ, A/J, and 129S1/SvImJ. Strain FVB/NJ strain showed no impairment in two experiments but a slight improvement in another, which ranks them as least impaired in all three experiments. DBA/2J and BALB/cByJ showed clear impairment in two cases but no impairment in another. By comparing the magnitude of ethanol effects across the three experiments, it is evident that they were relatively smaller for the accelerating rotarod than for any other behavioral test. Generally speaking, it is expected that smaller effects will show less consistency across experiments.
It was interesting to compare the extent of ethanol-induced activation in the open field in Experiment 2 for the same eight strains that were also tested in the first experiment (Fig. 7A, B). Under saline, strains A/J and 129S1/SvImJ were consistently low in activity while C57BL/6J was relatively high. The strain by ethanol interaction effect in Experiment 2 was an almost perfect replication of the interaction effect for the same strains in Experiment 1; the only exception being the absence of ethanol-induced activation for DBA/2J in the second experiment. This was a surprising finding in view of the well-documented sensitivity of this strain to ethanol’s locomotor stimulant effects . The nature of the interaction effect depended on the specific phenotype; open field rearing was greatly reduced by ethanol for six of eight strains in Experiment 2 but not for SJL/J that clearly showed an activation effect on distance traveled (data not shown).
Distance traveled in the open field was considerably greater across the same eight strains in Experiment 1 than Experiment 2, even though the same video camera and automated tracking software were employed. Recent tests have shown that illumination of the apparatus can markedly influence measured path distances . It appeared to the experimenters that illumination was very similar in the two experiments. The experimenters themselves differed, however, and they could have influenced activity levels [59, 60], especially after the injection of saline or drug that entails human handling. It cannot be concluded that experimenters were the source of activity difference, although they could have been. A controlled study wherein different people test mice in a balanced order within the same study might provide data that are more convincing. Strain differences in locomotor activity have been an extremely stable characteristic of inbred mouse strains across decades of testing in multiple laboratories . A recently completed study in the Wahlsten laboratory found a large difference between activity levels of mice following injection of ethanol by different experimenters but not before injection (see ).
The sample sizes for most of the groups shown in Fig. 7 were eight mice per group, and it can be seen from the standard error bars that individual variation was substantial for many measures. Some of the failures to replicate specific strain or ethanol effects across experiments could arise from sampling error. Larger sample sizes are generally needed to detect interaction effects than to detect main effects [61, 62]. Even larger sample sizes would be needed to ensure replication of an interaction effect itself, unless the interaction is very large.
It might be argued that our sample size was too small to detect strain by treatment interactions involving light-dark cycle and cage enrichment. This situation is of greatest concern, however, when there appears to be an interaction in the data but the significance test does not detect it. In the present study, there was not even a hint of an interaction effect. Thus, sample size cannot account readily for the absence of interaction effects involving light-dark cycle or cage enrichment.
The present experiments were designed to assess the influences of two common variations in the laboratory environment that might alter results of studies of ethanol effects on behavior by carefully controlling the variations within an experiment in one lab. They were not explicitly designed to detect causes of different outcomes between experiments in one lab or between different labs in any more global way, but the results have some relevance to these questions. It is has been argued that replicability of results in different labs will be enhanced if each lab employs more than one housing condition within a single experiment [63, 64]. This question has been addressed in a general way using computer simulation  with a factorial design involving several mouse strains and an experimental treatment studied independently in different labs. The critical issue in judging replicability is the strain by treatment by lab interaction effect. If it is very small, then results of the strain by treatment experiment are substantially the same across labs. The simulation shows how the outcome of the statistical analysis depends on the properties of the error term in the analysis. If the two housing conditions are included as a separate factor, the strain by treatment by lab interaction effect is not altered in any noteworthy way. If, on the other hand, variance attributable to housing condition is pooled with other sources of within-group variation into a global error term, power to detect the strain by treatment by lab interaction effect is reduced. The analysis is then less likely to find a significant interaction effect when such an effect is indeed present in the model that generates the data.
Another possible influence on the strain differences reported here could be that the strains differ in alcohol absorption, distribution, and/or elimination after equal intraperitoneal doses based on g/kg body weight. We did not measure blood ethanol levels in these studies. Nonetheless, we have done so in many other studies with these and other strains using these and other tests after giving alcohol doses throughout the range employed here. A global analysis of the role of blood ethanol levels in ethanol intoxication found that it was not an important factor in explaining strain differences in behavioral sensitivity to ethanol intoxication, so we do not believe it was important here .
It is noteworthy that results of the experiments on light-dark cycle and enrichment that used the abbreviated test battery with eight strains rather than the longer battery with all 20 strains detected many alcohol effects that were large and highly significant. In our view, little information was lost because of the more compact test battery and abbreviated list of inbred strains, while the overall efficiency of subsequent experiments was greatly increased. Nevertheless, the larger sample of 20 strains did reveal some interesting facts that may warrant further study, such as the markedly greater sensitivity of strain PL/J to alcohol impairment of motor performance. PL/J was never among the least affected strain on any test. The notably greater sensitivity of C57L/J than C57BL/6J is also intriguing, but it arose mainly from greater sensitivity of C57L/J on just two tests, grip strength and hypothermia, that showed a low strain of correlation of only r = 0.28 across the full set of 20 strains.
The use of a within-subject design to evaluate alcohol effects also increased the efficiency of tests of strain differences. In Experiment 2, the sizes of the ethanol main effects were large and significance levels were high for both the between-subject and within-subject comparisons. In Experiment 3 that utilized only the within-subject design with the same eight strains, alcohol effects were very large and highly significant, as were several strain by alcohol interaction effects. Results from a within-subject design can be more difficult to interpret if the direction of a trend across trials for an untreated subject is similar to the effect of a treatment. In the present tests, however, the trend for untreated mice was clearly an improvement in performance across trials because of learning to balance on the beam or rotarod or to grip the bar. In the open field, activity of untreated mice tends to habituate across trials. Alcohol, on the other hand, tends to increase slips on the balance beam, decrease grip strength, decrease latency to fall from the rotarod, and increase open field activity, at least at moderate doses. Thus, reduced performance that was evident from pre- to post-injection levels of performance in this study must represent a genuine impairment of performance by alcohol. This interpretation may not be valid for other tests or higher doses of alcohol. Preliminary evaluations of within- versus between-subject designs would be well advised when working with different kinds of tests. There could be situations where the experience with a test during pre-injection testing attenuates the effects of an alcohol injection that might be evident when alcohol is given to a naïve animal.
In the present series of experiments, the abbreviated battery of four tests applied with a within-subjects design proved to be both efficient and effective for the purposes of the study. Those methods were particularly well adapted to experiments designed to evaluate possible interactions with an environmental treatment factor, because the sample size within a group could be reasonably large. Between-subject designs with many tests in a battery are likely to suffer a loss of statistical power when spreading fewer mice thinly over more test conditions.
In conclusion, these studies provide further evidence for the influence of cage enrichment on certain behaviors in mice. Interestingly, testing mice in their most active circadian phase appeared to be no different than testing them during a period when they normally sleep. It was reassuring to find that ethanol had potent intoxicating effects regardless of circadian phase or whether mice had been reared in standard or enriched cages. Thus, there appears to be little danger of misinterpreting genetically based differences when employing common variations of these two environmental factors.
When a battery of seven tests of ethanol effects on behavior was given to 20 inbred strains, the wide range of phenotypic scores made it possible to eliminate tests that were redundant or especially difficult to interpret. The result was a compact battery of four tests (balance beam, accelerating rotarod, grip strength, open field activity) that was very sensitive to genetic differences in ethanol effects and spanned a wide range of the domain of motor behavior. That compact battery was then employed in studies of the effects of two common variants in the laboratory environment, light-dark cycle and cage enrichment, on differences among eight of the most commonly studied inbred strains in ethanol effects. Mice maintained with a normal light cycle (lights on at 0600, off at 1800) were housed in an identical colony room adjacent to mice with a reversed cycle, while other aspects of husbandry and testing were the same for the two groups. There were no statistically significant effects of light-dark cycle on any behavior, and strain differences were very similar under both lighting regimes. Data from that experiment also indicated that the large ethanol effects on behavior were very similar when separate groups were compared (saline versus ethanol injections) and when the same animals were compared before and after ethanol injection. That result showed that the test battery could be made even more compact by using a pre- versus post-injection design. An experiment was then done with this method using the same eight strains and the battery of four tests to evaluate effects of home cage enrichment. Cage enrichment improved test scores on all four tests but did not ameliorate ethanol effects or alter strains differences in ethanol effects. Thus, for two common variations in lab environment, light-dark cycle and cage enrichment, genetic differences in sensitivity of motor behavior to alteration by ethanol were not influenced by the local environment. Cage enrichment changed the overall mean scores without affecting the patterns of strain differences.
Research reported in this paper was supported by grant AA12714 from the National Institute of Alcoholism and Alcohol Abuse. JC also received support from grants AA13519, AA10760, and a grant from the US Department of Veterans Affairs. The sponsoring agencies were not involved in the planning, execution or analysis of the experiments and did not contribute to writing the report.
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