A behavioral comparison of the common laboratory rat strains Lister Hooded, Lewis, Fischer 344 and Wistar in an automated homecage system

Authors


Abstract

Behavioral characterization is an important part of establishing novel animal models, but classical behavioral tests struggle to reveal conclusive results due to problems with both reproducibility and validity. On the contrary, automated homecage observations are believed to produce robust outcomes that relate more to natural animal behavior. However, information on the behavior of background strains from such observations, which could provide important reference material, is rare. For this reason, we compared the behavior of the commonly used Lister Hooded, Lewis, Fischer 344 and Wistar rats during 70 h of exposure to an automated homecage system at 2, 4 and 6 months of age. We found considerable strain differences in metabolic parameters, novelty-induced and baseline activity-related behavior as well as differences in the development of these parameters with age. The results are discussed in terms of advantages and disadvantages of the system compared to classical behavioral tests, as well as the system's ability to recreate common findings in literature.

Detailed behavioral characterization is an important part of establishing novel animal models in a variety of research fields. For this purpose, the animals' behavior is often assessed in a range of classical behavioral tests such as the open field, elevated plus maze or the Morris water maze. Although this enables the comparison with previously published results, there are known problems with classical tests of this kind. Many classical tests are based on running brief trials in non-homecage setups. This enables the researcher to control many aspects of the test, but it has also been suggested that the results mainly reflect the confrontation with an unfamiliar situation and environment rather than a baseline behavior (Tecott & Nestler 2004). Furthermore, many classical tests rely on non-standardized equipment and protocols. This low level of standardization can lead to difficulties in replication and generalization of study outcomes (Chesler et al. 2002; Izídio et al. 2005; Mandillo et al. 2008; Tucci et al. 2006; van der Staay & Steckler 2002; Vyssotski et al. 2002; Wahlsten et al. 2003a,b). Finally, classical tests involve direct handling of the animals by the experimenter prior to the test session. The experimenter has been shown to influence the outcome of some tests (Chesler et al. 2002) and might be considered a source of variation, even if equipment and procedures are carefully standardized (Crabbe et al. 1999).

Automated homecage observation has been promoted to provide a good solution to the problems stated above (Kas & van Ree 2004) and a series of systems for computer-based acquisition of homecage activities of rats and mice as well as analysis software are in use (e.g. de Visser et al. 2006; Goulding et al. 2008; Hübener et al. 2011; Jhuang et al. 2010; Voikar et al. 2010; Zarringhalam et al. 2012). Such systems offer a standardized testing environment, while also allowing standardized customization to run specific protocols. As the animals are housed in a homecage-like environment, their behavior is thought to better reflect a natural state (de Visser et al. 2006; Tecott & Nestler 2004). In addition, as the measurements are highly computerized, behavioral data is gathered objectively. The systems are further able to measure a broad spectrum of behaviors including activity, food and water intake as well as cognitive aspects. Thus, a well-functioning automated homecage system could in theory be used for the complete behavioral characterization of an animal model. But there is currently a lack of literature on automated homecage behavior of background strains, in contrast to classical behavioral tests (e.g. Berton et al. 1997; Brooks et al. 2004, 2005; Richards et al. 2013; Wilhelm & Mitchell 2009). Studies that focus on the behavior of background strains constitute important reference material and can aid researchers in their choice of both background strain and behavioral test setup. Due to this, we conducted a study aimed at evaluating the practicality and sensitivity of an automated behavioral testing system in differentiating the four commonly used rat strains Lister Hooded, Lewis, Fischer 344 and Wistar.

Animals and methods

Rats

We used 12 male rats from each of the four strains Lister Hooded (Crl:LIS), Lewis (Crl:LEW), Fischer 344 (F344/DuCrl) and Wistar (Crl:WI) (Fig. 1). The animals were obtained from Charles River (Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld, Germany) at the age of 4 weeks.

Figure 1.

Rat strains. For our study, the common laboratory rat strains Lister Hooded (a), Lewis (b), Fischer 344 (c) and Wistar (d) were obtained from Charles River Laboratories, Germany.

All experiments were approved by the commission for animal experiments at the Regierungspräsidium Tübingen in accordance with the guidelines of the German animal welfare act. The behavioral experiments were all carried out by the same experimenter, trained and experienced in laboratory animal research.

Housing conditions followed the recommendations of the European Union (ETS 123 A). The environmental conditions in the housing room were kept at 21–23°C ambient temperature, 55 ± 10% humidity and a 12/12 h light/dark cycle with lights off at 1400 h and lights on at 0200 h. Animals were kept in groups of four animals of the same genotype in type IV autoclavable plastic cages with high lid (38 × 55 cm wide, 24.5 cm high) (Fig. 2b). Cages contained 1000 g of autoclaved wooden bedding and were cleaned twice a week. Food (ssniff V1534-000 standard rat chow) and tap water were delivered ad libitum.

Figure 2.

PhenoMaster metabolic cage and homecage. The homecage-like PM cages (a, c) differ from standard housing cages for rats (b, d). Differences concern cage dimensions, constitution of the lid and presentation of water and food. The PM cages have a moderately smaller floor area and a considerably lower height due to their flat lids. The airtight lids of the metabolic cages further isolate the inner of the cage to a larger degree from the surroundings. Water and food bins are hanging lower than in the homecage and the food baskets offer a substantially smaller surface for feeding.

The animals were weighed and inspected weekly in order to assess their general health status and accustom them to human handling. The rats were given 2 weeks of acclimatization in our animal facility prior to the start of the experiments.

Measurements

Body weight

The animals were weighed weekly using a kitchen balance (accuracy was ±1 g) in order to record body weight development.

Body weight and body length at 10 weeks of age

At 10 weeks of age, we measured the body length and body weight of the animals in order to assess differences in body size. For the measurement of body length, the animals were briefly anesthetized with isoflurane and the length from the tip of the nose to the base of the tail (head-trunk length) was measured, using a ruler (accuracy was ±0.1 cm).

Automated homecage observations

Measurements were carried out at the age of 2, 4 and 6 months. For this purpose, the animals were transferred to the testing cages, where they were housed individually. The measurements started 40 ± 5 min before the onset of the dark phase. Data were collected in 20-min sample intervals during a total of 70 h of recording. The animals were assigned to one of the 12 cages in a pseudo-randomized manner. Each run of 12 animals contained four animals from three strains, respectively, so that each time point comprised four runs and lasted 12 days in total.

During testing, the animals were inspected daily for proper food and water intake and the system for accurate functioning.

In between runs, the system was cleaned thoroughly and new bedding as well as fresh water and food pellets was supplied.

Automated homecage system

For the behavioral analysis, we used the automated homecage system PhenoMaster (PM) (TSE Systems, Bad Homburg, Germany) (Fig. 2a) with the software version 1.3.7 (2010). Our customized setup included a set of 12 homecage-like autoclavable plastic cages (37.5 × 48 cm wide, 20 cm high) plus a reference cage. All cages were equipped with a frame with infrared light beams for activity detection (Actimot2, 302020 series). Our setup further included metabolic units for measurement of water and food consumption (Drink/Feed, 259980 series) and the analysis of respiratory gases (CaloSys, 994600 series). For this purpose, the cages contained a drinking bottle and a food basket suspended from high-precision sensors. The cages were further sealed with an airtight plastic lid containing an input tube for air supply and an output tube for gas sampling.

Activity

Infrared light beams were installed at two different height levels around the cage and spaced1.5 cm apart from each other. The lower set was located at a height of 3 cm (2-month-old animals) or 4 cm (4- and 6-month-old animals) to measure activity in the x- and y-axis (locomotion). A higher set of beams at 8 cm (2-month-old animals) or 12 cm (4- and 6-month-old animals) detected activity in the z-axis (rearing). The height of the beams was initially evaluated using Sprague Dawley rats. We decided on having fixed setups for all rat strains, but differing between the ages, since the biggest difference in size did not occur between strains but between the youngsters at 2 months of age and the adult animals at 4 and 6 months of age. For more accurate measurements, the amount of bedding placed in the cages during the measurements was set to 250 g.

Locomotion was further subdivided into overall ambulatory activity (ambulation), fine movements and ambulatory activity in the periphery and the center of the cage. The system recorded a movement as ambulatory activity, when three or more different light beams were disrupted consecutively, whereas it was recorded as fine movement, when adjacent light beams were disrupted alternately. In order to analyze activity in specific parts of the cage, a peripheral and central area was defined with the software. The center accounted for the inner 16.5 × 12 cm (198 cm2) of the total 48 × 37.5 cm (1800 cm2). From this, we calculated the ratio of periphery to center activity (per/cen). The sensors were scanning activity with a rate of 100 Hz and were programmed to a refractory period of 0.8 seconds. The activity measurement relied on the number of beam breaks (counts) made by the animal. It is important to note that as each animal was always breaking several light beams at a given time, the number of beam breaks was much higher than the number of events underlying the recorded counts. The software also provided the option to count all beam breaks detected at a time as one single event. However, having it counted as only one event would disregard the quality (i.e. duration, intensity) of the event. For this reason, we decided to have all beam breaks counted separately.

Novelty-induced behavior

Analysis of activity levels during the first hour served as a measure for the behavioral response to a novel environment. Ambulatory and rearing activity were regarded as exploratory-related behaviors, whereas per/cen was considered to be anxiety-related.

Water and food intake

Water and food intake was measured with high-precision sensors. The water bottle contained 150 ml of fresh tap water and the food basket was loaded with 150 g of ssniff V1534-000 standard rat chow. Water and food intake are given in ml per hour (ml/h) and g per hour (g/h) or in ml per hour per kg of body weight (ml/h/kg) and g per hour per kg of body weight (g/h/kg), respectively in order to account for body weight differences between the individuals and the groups. Containers were filled before the start of the measurement and the amount of food and water was sufficient for the 70-h measurement. A minimum consumption of 0.01 ml of water and 0.01 g of food as well as a maximum consumption of 0.1 ml of water and 0.1 g of food were set in order to exclude values caused through spillage or leaky bottles.

Indirect calorimetry

In order to obtain data on respiratory gases, the PM cages were fed with room air via an in-bound tube with a constant air flow of 1.8 l per min. Every 20 min, an out-bound tube conducted 0.25 l of sample air first to a cooling unit, where the sample was dried, and further to the measuring unit containing O2- and CO2-electrodes. Subtraction of the respective gas pressure from that of a reference cage revealed the VO2 and VCO2 (carbon dioxide production). From these values, the software further calculated the respiratory quotient (RQ = VCO2/VO2). VO2 and RQ are only presented for the age of 2 and 4 months, since a problem with the gas calibration occurred at the last age.

Data analysis

Processing of raw data with R statistics

The PM software itself includes an analysis and graphing function. However, in order to conduct a detailed statistical analysis, we decided to export the data table and use additional software.

By means of R statistics (R: A Language and Environment for Statistical Computing, R Development Core Team, R Foundation for Statistical Computing, Vienna, Austria, 2011, ISBN 3-900051-07-0, http://www.R-project.org), we developed a script that sorted the data from all recordings according to animal number (1–12), study groups (strains) and time point of measurement (age 2, 4 or 6 months). From this, it created either an hourly sum of the respective parameter (ambulation, fine movements, rearing, per/cen) or an hourly mean (VO2, RQ). The data were separated into a habituation period (first hour of measurement) and a long-term measurement of additional 69 h. For this 3-day observation period, the script further calculated mean values for light and dark phase separately.

Graphs

Graphs were created using GraphPad Prism 6.00. Illustrations on the basis of photographic images were arranged with Adobe Photoshop CS3.

Statistical analysis

For statistical evaluation of the development of the four strains over time, we performed analyses of variance (anova). Body size at 10 weeks of age was compared with a one-way anova (anova1) and Tukey's multiple comparison test (Tukey post-test), while all the parameters obtained at more than one age were analyzed with a repeated measurements two-way anova (rmanova2). Differences between strains in these parameters were investigated using Tukey post-test. Age effects were analyzed with Dunnett's multiple comparisons test (Dunnett post-test), comparing the second and third measurement to the first one. Body weight gain before and during the PM were compared using Sidak's multiple comparisons test (Sidak's post-test).

Statistics were performed with GraphPad Prism version 6.00 for Windows (GraphPad Software, San Diego, CA, USA, http://www.graphpad.com). Values given in the text refer to group mean and standard deviation. The α-level was set to 0.05. Respective P-values are given in the text or in the figures down to a level of 0.0001 as reported by GraphPad Prism.

We excluded a total of 88 of 1968 data sets from the two-way analysis of variance (ambulation (7/384), rearing (9/384), per/cen activity (20/96), fine movements (2/144), drinking (16/144), feeding (10/144)), because 88 out of the 5904 individual values in these data sets were most likely erroneous due to hardware problems. For activity, we found the system to register very high counts for some animals. This might have been due to bedding that was shifted around by the animal. We only excluded values that were more than three times higher than the group mean and did not occur repeated times for the same animal. Measuring per/cen activity revealed a special problem: After the initial exploration of the test cages, we found some of the rats to lie down and sleep, usually in a corner of the cage, leading to low ambulatory activity counts in general and even lower or zero activity counts for the central area. Thus, such an animal displayed either a very high per/cen value or no value at all in case of zero center activity. For food and water intake, it was detected that drinking bottles were leaking from time to time (leading to unreasonably high values for water intake) and the food got stuck in the food basket, so that the animal was not able to reach it (leading to unreasonably low values for food intake). For activity, drinking and feeding, the values that were excluded are spread equally among groups and testing cages, suggesting that they are not the result of a systematic error or having a biological meaning. In contrary, the number of values that had to be taken out of the analysis of per/cen activity differ between rat strains and are clearly related to a difference in behavior.

Results

Because of the large amount of data obtained, values for group means and standard deviation as well as most of the statistics obtained from post hoc analysis are listed in separate tables. Only the statistical results from the anovas are given in the text, together with the description of the results.

Part I: morphology

The four rat strains showed morphological differences. Lister rats were the only pigmented rats, while the others were albinos (Fig. 1).

Furthermore, there were differences in body weight and size (Fig. 3). At 10 weeks of age, body weight was highest in Wistar followed by Lister and Lewis rats and Fischer rats weighed least (F3,44 = 106.3, P < 0.0001, Fig. 3a).

Figure 3.

Body size. Body length (a), body weight (b) and weight/length ratio at 10 weeks of age (c) as well as body weight development from 4 to 28 weeks of age (d) are shown for the four rat strains Lister, Wistar, Lewis and Fischer. The (a)–(c) show values for individual animals with the group mean being indicated by a horizontal line. (d) Displays weekly mean and standard deviation for the four rat strains. Statistics: anova1 (a–c), P-values for group comparison are given as lowercase letters with one letter = P < 0.05, two letters = P < 0.01, three letters = P < 0.001, four letters = P < 0.0001 and meaning a = significantly different from Lister rats, b = significantly different from Wistar rats, c = significantly different from Lewis rats and d = significantly different from Fischer rats. rmanova2 (d–e), P-values for strain, time and strain*time differences (d) and P-values for the Bonferroni post-test (e) are given as asterisks with *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. For the group comparison (e), the additional information on the time point, the P-value was observed for is added (w = weeks).

Similarly, body length at 10 weeks of age was highest in Wistar rats, Lister and Lewis rats were medium sized and Fischer rats were smallest (F3,44 = 57.12, P < 0.0001, Fig. 3b).

Further investigation showed that significant differences between strains remained after relating body weight to body length (F3,44 = 79.93, P < 0.0001, Fig. 3c).

The weekly measurements of body weight revealed significant differences between the strains (F3,44 = 178.6, P < 0.0001) as well as a significant development of body weight with age (F72,1056 = 70.68, P < 0.0001, Fig. 3d,e). Wistar rats weighed significantly more than the other strains at any time throughout the study, while the three other strains did not differ at young ages. From 8 weeks on, Fischer rats showed a significantly lower body weight than all other strains. At the age of 25 weeks, Lister rats separated from Lewis rats, with Lister being the heavier ones. The exact level of significance for these comparisons varied with age (Fig. 3e).

Part II: behavioral observations in the automated homecages

As described above, we used an automatic homecage system to monitor activity and metabolism in Lister, Wistar, Lewis and Fischer rats. The rats' behavior was assessed during a 70-h experiment. The first hour of observation was used to investigate the response to the novel environment, while activity-related and metabolic parameters were followed for another 69 h. Group means and standard deviation are given in Table 1 for activity-related parameters and in Table 2 for metabolic parameters; P-values from post-testing are displayed in Tables 3 (activity) and 4 (metabolism).

Table 1. Activity-related parameters
 Ambulation (counts)Rearing (counts)Per/cen (ratio)Ambulation (counts)Fine movements (counts)Rearing (counts)
 AdaptationAdaptationAdaptationLight phaseDark phaseTotalLight phaseDark phaseTotalLight phaseDark phaseTotal
  1. Group mean and standard deviation for rat strains in novelty-induced behavior as well as activity-related parameters during the three-day observation period are given for 2, 4 and 6 months of age.
2 months
Lister6067 ± 18481505 ± 6642.6 ± 1.174250 ± 20949104312 ± 25916178562 ± 1694029255 ± 687937499 ± 577566754 ± 506323909 ± 832936585 ± 2228760493 ± 27512
Wistar4405 ± 16551436 ± 6062.9 ± 1.741526 ± 8306163442 ± 21241204968 ± 2344117985 ± 283849467 ± 416367452 ± 574616567 ± 673369395 ± 1634185962 ± 22151
Lewis5274 ± 17691275 ± 5763.8 ± 1.549421 ± 9078135590 ± 12322185011 ± 1606420256 ± 394544544 ± 336464800 ± 506615400 ± 495845015 ± 968859268 ± 12931
Fischer5222 ± 25131281 ± 5762.8 ± 1.045449 ± 6853155415 ± 27776201017 ± 2589219897 ± 200347076 ± 538567158 ± 520611377 ± 397043666 ± 744755552 ± 8310
4 months
Lister5434 ± 10511296 ± 1681.7 ± 0.438307 ± 6078105400 ± 12112143707 ± 1018117703 ± 231037133 ± 313154836 ± 34686117 ± 215020052 ± 426326170 ± 5872
Wistar4366 ± 1659905 ± 3421.9 ± 0.426447 ± 3910113585 ± 22812140032 ± 2497915803 ± 321438652 ± 440754455 ± 65153375 ± 176918324 ± 589421699 ± 7476
Lewis3504 ± 808918 ± 4862.7 ± 0.827868 ± 4817104299 ± 8356132167 ± 922715118 ± 315737295 ± 212652413 ± 35724233 ± 362821436 ± 1232322444 ± 8932
Fischer4311 ± 1064688 ± 2182.0 ± 0.530310 ± 4152115154 ± 27025145270 ± 2664816215 ± 330138533 ± 465354936 ± 56123266 ± 137617110 ± 647620375 ± 7383
6 months
Lister3612 ± 801904 ± 1801.6 ± 0.328095 ± 360478098 ± 7063106193 ± 713815058 ± 222430271 ± 246945598 ± 34985416 ± 282918174 ± 702623589 ± 9477
Wistar2796 ± 1433740 ± 6251.7 ± 0.518927 ± 270987040 ± 16662105966 ± 1711313821 ± 224832923 ± 364846328 ± 45753916 ± 363917944 ± 1023221860 ± 13697
Lewis2285 ± 904473 ± 2392.4 ± 0.924960 ± 347878695 ± 6529103655 ± 685213685 ± 164930987 ± 221444996 ± 34574057 ± 245915352 ± 538619698 ± 7186
Fischer3248 ± 799410 ± 1501.6 ± 0.328792 ± 697698357 ± 20191126640 ± 2292317422 ± 384734863 ± 364651288 ± 66473595 ± 233715122 ± 473118717 ± 6891
Table 2. Metabolic parameters
 Body weight (g)Water intake (ml)Relative water intake (ml/kg)Relative VO2 (ml O2/kg/h)
 Before testAfter testLight phaseDark phaseTotalLight phaseDark phaseTotalLight phaseDark phaseTotal
2 months
Lister224 ± 15231 ± 2027.2 ± 7.834.1 ± 10.661.5 ± 6.442.9 ± 15.252.9 ± 14.795.7 ± 11.42058 ± 1382136 ± 1972097 ± 106
Wistar293 ± 20311 ± 1812.4 ± 4.082.1 ± 12.893.9 ± 11.113.9 ± 4.793.8 ± 17.4107.3 ± 16.21723 ± 1142133 ± 1541928 ± 130
Lewis210 ± 28226 ± 3114.7 ± 7.354.6 ± 12.069.6 ± 11.224.7 ± 13.487.6 ± 13.3113.1 ± 19.22108 ± 2302339 ± 1372224 ± 179
Fischer195 ± 20206 ± 187.0 ± 3.345.0 ± 7.252.1 ± 5.612.6 ± 777.3 ± 10.190.2 ± 11.91848 ± 1892101 ± 1241974 ± 150
4 months
Lister420 ± 11413 ± 1813.6 ± 3.057.6 ± 8.570.7 ± 7.011.0 ± 2.846.2 ± 6.357.0 ± 5.31165 ± 381442 ± 621304 ± 35
Wistar520 ± 35521 ± 3412.7 ± 4.980.7 ± 15.892.0 ± 16.38.1 ± 2.951.8 ± 11.958.9 ± 12.11064 ± 551394 ± 721229 ± 60
Lewis398 ± 21402 ± 225.9 ± 3.261.8 ± 5.867.1 ± 6.54.9 ± 2.651.9 ± 4.856.5 ± 51203 ± 621525 ± 381364 ± 40
Fischer324 ± 15322 ± 125.6 ± 2.438.7 ± 6.844.5 ± 7.85.8 ± 2.640.3 ± 846.4 ± 9.21193 ± 671393 ± 661293 ± 59
6 months
Lister516 ± 12505 ± 2412.7 ± 3.749.8 ± 14.659.2 ± 9.28.4 ± 2.732.5 ± 8.739.0 ± 6.0NANANA
Wistar646 ± 50637 ± 486.7 ± 4.765.1 ± 15.871.5 ± 15.23.3 ± 2.234.2 ± 1037.5 ± 9.5NANANA
Lewis474 ± 25475 ± 244.3 ± 2.353.4 ± 5.957.6 ± 5.33 ± 1.637.8 ± 4.740.7 ± 4.2NANANA
Fischer396 ± 15387 ± 146.0 ± 2.832.3 ± 5.938.2 ± 5.25.1 ± 2.427.4 ± 5.332.4 ± 4.9NANANA
 Weight gain (g/3d)Food intake (g)Relative food intake (g/kg)RQ (ml O2/kg/h)
 Before testDuring testLight phaseDark phaseTotalLight phaseDark phaseTotalLight phaseDark phaseTotal
  1. Group mean and standard deviation for rat strains in body weight before and after testing, body weight gain over 3 days before and after testing as well as metabolic parameters during the three-day observation period are given for 2, 4 and 6 months of age.
2 months
Lister19 ± 217 ± 330.5 ± 5.138.3 ± 7.568.7 ± 4.433.2 ± 881.5 ± 7.7114.7 ± 11.50.85 ± 0.010.86 ± 0.010.86 ± 0.01
Wistar21 ± 317 ± 718.8 ± 5.460.4 ± 9.879.2 ± 11.025.5 ± 5.767.2 ± 8.992.7 ± 9.80.80 ± 0.020.84 ± 0.010.82 ± 0.01
Lewis18 ± 116 ± 321.9 ± 5.345.9 ± 8.367.8 ± 4.727.1 ± 7.289.4 ± 15.1116.5 ± 13.80.83 ± 0.030.85 ± 0.040.84 ± 0.03
Fischer14 ± 110 ± 412.8 ± 2.037.6 ± 4.050.8 ± 2.321.3 ± 4.356.7 ± 977.5 ± 8.80.83 ± 0.010.86 ± 0.010.84 ± 0.01
4 months
Lister10 ± 13 ± 420.7 ± 4.151.3 ± 4.672.0 ± 4.616.7 ± 3.641.3 ± 3.458 ± 4.10.81 ± 0.010.83 ± 0.010.82 ± 0.01
Wistar11 ± 21 ± 622.5 ± 5.259.0 ± 5.881.4 ± 6.514.2 ± 2.737.7 ± 4.651.9 ± 3.60.74 ± 0.020.79 ± 0.020.77 ± 0.02
Lewis9 ± 14 ± 416.8 ± 4.754.5 ± 5.071.4 ± 3.714.1 ± 3.846 ± 4.660.1 ± 2.80.80 ± 0.020.83 ± 0.020.81 ± 0.02
Fischer5 ± 1−2 ± 412.5 ± 3.132.8 ± 3.144.8 ± 3.212.8 ± 3.133.8 ± 3.646.2 ± 3.70.80 ± 0.020.83 ± 0.020.82 ± 0.01
6 months
Lister0 ± 1−1 ± 319.3 ± 3.051.9 ± 5.271.1 ± 5.112.7 ± 2.134.1 ± 3.346.7 ± 3.5NANANA
Wistar3 ± 2−10 ± 619.1 ± 6.553.1 ± 7.272.2 ± 8.59.8 ± 327.7 ± 4.937.5 ± 5.1NANANA
Lewis2 ± 12 ± 618.4 ± 4.253.6 ± 5.671.9 ± 5.813 ± 338.1 ± 5.551.1 ± 6.2NANANA
Fischer2 ± 1−9 ± 411.5 ± 1.327.8 ± 2.439.5 ± 2.69.7 ± 123.4 ± 2.133.2 ± 2.2NANANA
Table 3. Post-test results for activity-related parameters
 Ambulation (counts)Rearing (counts)Per/cen (ratio)Ambulation (counts)Fine movements (counts)Rearing (counts)
 2 months4 months6 months2 months4 months6 months2 months4 months6 months2 months4 months6 months
  1. Significance levels from group comparisons for rat strains in novelty-induced behavior as well as activity-related parameters during the three-day observation period are given for 2, 4 and 6 months of age (*P < .05, **P < .01, ***P < .001, ***P < .0001).
Light
Lister–Wistar*nsns***********nsns***nsns
Lister–Lewisns**ns*****ns****nsns****nsns
Lister–Fischernsnsns****nsns****nsns****nsns
Wistar–Lewisnsnsnsnsnsnsnsnsnsnsnsns
Wistar–Fischernsnsnsnsns*nsnsns*nsns
Lewis–Fischernsnsnsnsnsnsnsns*nsnsns
Dark
Lister–Wistarnsnsns****nsns****nsns****nsns
Lister–Lewisns*ns***nsns***nsnsnsnsns
Lister–Fischer***ns****nsns****ns*nsnsns
Wistar–Lewisnsnsns**nsns*nsns****nsns
Wistar–Fischernsnsnsnsnsnsnsnsns****nsns
Lewis–Fischernsnsnsnsnsnsnsnsnsnsnsns
Total
Lister–Wistarnsnsns**nsnsnsnsns****nsns
Lister–Lewisnsnsnsnsnsnsnsnsnsnsnsns
Lister–Fischernsnsns*ns*nsns*nsnsns
Wistar–Lewisnsnsns*nsnsnsnsns****nsns
Wistar–Fischernsnsnsnsns*nsnsns****nsns
Lewis–Fischernsnsnsnsns*nsns*nsnsns
Table 4. Post-test results for metabolic parameters
 Water intake (ml)Relative water intake (ml/kg)Relative VO2 (ml O2/kg/h)Food intake (g)Relative food intake (g/kg)RQ (ml O2/kg/h)
 2 months4 months6 months2 months4 months6 months2 months4 months2 months4 months6 months2 months4 months6 months2 months4 months
  1. Significance levels from group comparisons for rat strains in metabolic parameters during the three-day observation period are given for 2, 4 and 6 months of age (*P < .05, **P < .01, ***P < .001, ***P < .0001).
  2. RQ, respiratory quotient.
Light
Lister–Wistar****ns*****nsns****ns****nsns***nsns********
Lister–Lewis***************nsnsnsns****nsns**nsns*ns
Lister–Fischer*************nsns***ns**************nsns*ns
Wistar–Lewisns**ns**nsns*****ns*nsnsnsns******
Wistar–Fischer***nsnsnsnsnsns*********nsnsns******
Lewis–Fischer***nsns***nsns****ns****ns***nsnsnsns
Dark
Lister–Wistar**************nsnsnsns*****ns****nsnsns****
Lister–Lewis****nsns****nsns****ns*nsns*nsnsnsns
Lister–Fischerns**********nsnsnsnsns************ns**nsns
Wistar–Lewis*******nsnsnsns*********nsns*******ns***
Wistar–Fischer**************nsnsnsns**************nsns*****
Lewis–Fischerns********ns*ns**************************nsns
Total
Lister–Wistar*********nsnsns**ns*****ns****ns*********
Lister–Lewisnsnsns***nsns*nsnsnsnsnsnsnsnsns
Lister–Fischerns********nsnsns*ns*********************nsns
Wistar–Lewis**********nsnsns************ns*************
Wistar–Fischer**************nsnsnsns****************nsns******
Lewis–Fischer***************nsns****ns************************nsns

Novelty-induced behavior

The first hour of measurement

Ambulatory and rearing activity as well as the activity in the periphery relative to the activity in the center of the cage (per/cen) were analyzed during the first hour in the PM. Rats showed a similar change in behavior during the first hour at all ages (Fig. S1). For convenience, only the results from 2-month-old rats are displayed and discussed here (Fig. 4, Table 1).

Figure 4.

Habituation to the PhenoMaster cages. Ambulation, rearing and per/cen activity are shown for Listar, Wistar, Lewis and Fischer rats during the first, second and third 20 min time interval in the PhenoMaster. Values are group mean and standard deviation of the sum of beam break counts over 20 min. Statistics: rmanova2, Dunnett's post-test, P-values for the comparison of the second and third 20-min time interval with the first 20-min time interval are given as lowercase letters (a = Lister rats, b = Wistar rats, c = Lewis rats and d = Fischer rats) with one letter = P < 0.05, two letters = P < 0.01, three letters = P < 0.001 and four letters = P < 0.0001.

Ambulation decreased significantly from the first to the second 20-min interval and remained at a low level during the third 20-min interval in most animals (F2,88 = 47.95, P < 0.0001, Fig. 4).

Rearing activity also decreased within the first hour of observation (F2,88 = 15.14, P < 0.0001). However, this only reached statistical significance for Lewis and Fischer rats, although Lister and Wistar rats also showed a gradual decline in rearing (Fig. 4). Per/cen activity did not significantly change during the first hour (Fig. 4).

It appeared that some animals decreased their ambulatory and rearing activity to a very low level or even to zero within the first hour. Observations by the experimenter revealed that a part of the animals went to sleep after an initial exploration of the cage. While no animals were found with a particularly low activity during the first 20 min, the number of animals with a low level of activity differed between strains during the following 40 min. The frequency was highest for Lewis rats (9/12), followed by Fischer (6/12) and Wistar rats (4/12 animals), and did not occur at all in Lister rats.

Strain differences during the first 20 min of testing

Because of the decrease in activity, which can be regarded as habituation effect, only the behavior during the initial 20 min was used to assess differences in novelty-induced behavior between the different rat strains.

Novelty-induced ambulation and rearing decreased with age (ambulation: F2,88 = 35.72, P < 0.0001; rearing: F2,88 = 39.60, P < 0.0001, Fig. 5). Age also influenced per/cen activity, with older rats showing a lower per/cen ratio (F2,86 = 25.59, P < 0.0001, Fig. 5). Significant strain differences were observed in all three parameters (ambulation: F3,44 = 4.571, P = 0.0072; rearing: F3,44 = 5.407, P = 0.0030; per/cen: F3,43 = 7.224, P = 0.0005). Post hoc analysis indicated that these were mainly based on Lister rats differing from the other strains in ambulatory and rearing activity, while Lewis rats differed in per/cen activity. Lister rats showed higher mean values for ambulatory activity compared to the other strains at all ages. Significant differences were found between Lister and Wistar rats at 2 months (P < 0.05) and between Lister and Fischer rats at 6 months of age (P < 0.01, Fig. 5). Lister rats also appeared to rear more than the other strains at 4 and 6 months of age, reaching statistical significance compared to Fischer rats at 4 months (P < 0.01) and compared to Lewis (P < 0.05) and Fischer rats (P < 0.05) at 6 months of age (Fig. 5). Significant differences in per/cen activity occurred between Lister and Lewis rats at 2 months (P < 0.05) and 4 months of age (P < 0.05), whereat Lewis rats showed highest mean values for per/cen activity of all strains. However, we detected a very high individual variability in this parameter (Fig. 5).

Figure 5.

Novelty-induced behavior. Ambulation, rearing and per/cen activity are shown for Listar, Wistar, Lewis and Fischer rats at the age of 2, 4 and 6 months. Values are group mean and standard deviation of the sum of beam break counts over 20 min. Statistics: rmanova2, Dunnett's post-test, P-values for the comparison of activity at 4 and 6 months with the activity at 2 months of age are given as lowercase letters (a = Lister rats, b = Wistar rats, c = Lewis rats and d = Fischer rats) with one letter = P < 0.05, two letters = P < 0.01, three letters = P < 0.001 and four letters = P < 0.0001.

3-Day observation of activity-related and metabolic parameters

Following the first hour of observation, the animals were monitored for an additional 69-h period. Ambulation, fine movements, rearing, water intake, food intake, VO2 and RQ were assessed and analyzed for the overall three-day observation as well as for light and dark phase separately.

Circadian rhythm

Time series analysis indicated that all rats showed a circadian rhythm entrained by the 12/12 h dark/light cycle. In line with this, all four rat strains showed higher levels of ambulation, rearing, drinking, feeding, VO2 and RQ during the dark phase compared to the light phase. Since the results were similar for all parameters at all ages, only the data for ambulation are displayed as an example (Figs. 6,S2).

Figure 6.

Ambulatory activity over the three observation days. Ambulatory activity is displayed as sum of counts per hour over the 70-h observation period at 2 months of age. Each point represents the group mean of Lister, Wistar, Lewis and Fischer rats. Error bars are not shown in order to enable a better visual comparison. The dark phase is indicated by the black bars below the x-axis.

Ambulatory activity

Total values for ambulation revealed moderate differences between the rat strains (F3,43 = 3.680, P = 0.0191), but showed a clear age effect, with older animals having a lower ambulatory activity (F2,86 = 352.6, P < 0.0001) as well as a significant strain difference in the influence of age on ambulation (F6,86 = 3.226, P = 0.0066, Fig. 7). Total ambulatory activity was highest in Wistar and Fischer rats at 2 months of age. No significant differences between strains were detected at 4 months of age, while Fischer rats were again most active at 6 months of age.

Figure 7.

Light phase, dark phase and total amount of activity-related parameters. Ambulatory activity, fine movements and rearing activity are given per light phase, dark phase and total 70-h observation period for Lister, Wistar, Lewis and Fischer rats. Each data point represents group mean and standard deviation at 2 months, 4 months and 6 months of age.

Separate analysis of light and dark phases revealed similar significant age effects as seen for the total observation period (F2,88 = 172.1, light phase: P < 0.0001; dark phase: F2,86 = 147.5, P < 0.0001) but showed highly significant strain effects (light phase: F3,44 = 24.37, P < 0.0001; dark phase: F3,43 = 9.793, P < 0.0001) as well as strain*age interactions (light phase: F6,88 = 9.486, P < 0.0001; dark phase: F6,86 = 8.032, P < 0.0001, Fig. 7). Strain differences were based to a large degree on Lister rats, being significantly more active during the light phase and in turn less active during the dark phase at 2 months and 4 months of age. Wistar rats on the other hand were least active during the light phase, becoming significantly different from Lister and Fischer rats at 6 months of age.

Fine movements

Analysis of the total amount of fine movements revealed minor differences between strains (F3,43 = 3.401, P = 0.0260, Fig. 7). Fine movements were also found to decrease with age in all strains (F2,86 = 180.5, P < 0.0001). The development over time did not differ between strains. Strain differences in fine movements resulted from an increased amount in Fischer rats at 6 months of age.

Analysis of the distribution of fine movements during the light and dark phase revealed more pronounced strain differences (light phase: F3,44 = 13.89, P < 0.0001; dark phase: F3,43 = 10.85, P < 0.0001) and a significant age effect (light phase: F2,88 = 56.68, P < 0.0001; dark phase: F2,86 = 146.4, P < 0.0001) as well as strain*age interaction (light phase: F6,88 = 8.242, P < 0.0001; dark phase: F6,86 = 6.015, P < 0.0001, Fig. 7). Lister rats showed significantly more fine movements during the light phase and less fine movements during the dark phase at 2 months of age. The increased total amount of fine movements found for Fischer rats at 6 months of age was based on a moderately higher number of counts during the dark phase and a more prominently higher number of counts during the light phase.

Rearing activity

Total rearing activity decreased with age (F2,84 = 178.7, P < 0.0001) and the strains developed differently over time (F6,84 = 4.780, P < 0.0001) (Fig. 7). Strain differences were present (F3,42 = 4.239, P = 0.0028), but appeared only at 2 months of age according to post-test results. At that age, Wistar rats reared in total significantly more than the other rats.

Looking at light and dark phase also revealed significant differences between strains at 2 months of age (light phase: F3,42 = 12.09, P < 0.0001; dark phase: F3,43 = 5.626, P = 0.0002) as well as a significant age effect (light phase: F2,84 = 131.7, P < 0.0001; dark phase: F2,86 = 148.2, P < 0.0001) as well as strain*age interaction (light phase: F6,84 = 3.848, P = 0.0019; dark phase: F6,86 = 8.497, P < 0.0001, Fig. 7). The higher total amount of rearing found for Wistar rats at 2 months of age, was based on a significantly higher rearing activity during the dark phase. Similar as it was found for ambulation and fine movements, Lister rats reared more than the other strains during the light phase at 2 months of age. Fischer rats reared least during the light phase.

Water intake

Total water intake differed significantly between strains at each age (F3,37 = 48.83, P < 0.0001) (Fig. 8). It further developed differently over time in the three rat strains (F6,74 = 5.626, P < 0.0001) with Wistar, Lewis and Fischer rats showing a decrease in water consumption from 2 to 6 months of age (mean difference in water intake between 2 and 6 months Wistar: 22.4 ml, P < 0.0001; Lewis: 11.9 ml, P > 0.001; Fischer 13.9 ml, P < 0.0001), while Lister rats increased their water consumption from 2 to 4 months of age (mean difference in water intake between 2 and 4 months: −9.1 ml, P < 0.01). Strain differences in the overall amount of water consumed during the 3-day experiment reflected the differences in body size of the animals. In total, Wistar rats drank significantly more than the other strains at all ages. Lister and Lewis rats did not differ in their overall water intake and Fischer rats drank least at any time.

Figure 8.

Light phase, dark phase and total amount of metabolic parameters. Absolute water intake in ml, relative water intake in ml per kg of body weight, absolute food intake in g, relative food intake in g per kg of body weight, relative oxygen consumption in ml per kg of body weight per hour and respiratory quotient are given per light phase, dark phase and total 70-h observation period for Lister, Wistar, Lewis and Fischer rats. Each data point represents group mean and standard deviation at 2 months, 4 months and 6 months of age.

The amount of water consumed during the light and dark phase was also found to differ significantly between strains (light phase: F3,37 = 33.10, P < 0.0001; dark phase: F3,37 = 33.86, P < 0.0001) with a significant effect of age (light phase: F2,74 = 45.78, P < 0.0001; dark phase: F2,74 = 15.46, P < 0.0001) and a significant strain*age interaction (light phase: F6,74 = 9.520, P < 0.0001; dark phase: F6,74 = 11.94, P < 0.0001, Fig. 8). The strain differences in total water intake were mostly recapitulated in the results for dark phase water intake. During the light phase, Lister rats had the highest water intake at all ages and Wistar rats drank more than Lewis and Fischer rats at 4 months of age.

Total water intake relative to body weight [ml/kg] decreased with age in all strains (F2,74 = 653.4, P < 0.0001), but also showed differences in the exact development over time between the strains (F6,74 = 3.475, P = 0.0044, Fig. 8). Strain differences in relative water intake resulted from differences at 2 months of age (F3,37 = 6.165, P = 0.0017). At that age, Lewis and Wistar rats consumed most, while Fischer rats consumed least.

Relative water intake during light and dark phase both decreased with age in all strains (light phase: F2,82 = 111.5, P < 0.0001; dark phase: F2,76 = 363.8, P < 0.0001), but not in a similar manner (light phase: F6,82 = 13.78, P < 0.0001; dark phase: F6,76 = 16.91, P < 0.0001, Fig. 8). Strain differences were found at 2 months only (light phase: F3,41 = 22.48, P < 0.0001; dark phase: F3,38 = 10.66, P < 0.0001) with Lister and Lewis rats consuming significantly more water relative to body weight than Wistar rats, which in turn consumed more than Fischer rats during the light phase. During the dark phase, Lister rats drank the least amount of water per kg body weight compared to the other strains.

Food intake

The three rat strains differed significantly in their total food consumption (F3,40 = 194.6, P < 0.0001, Fig. 8). The strains further developed differently with age (F6,80 = 5.396, P < 0.0001), with Wistar and Fischer rats decreasing their food consumption from 2 to 6 months of age (mean difference in food intake between 2 and 6 months Wistar: 7.1 g, P < 0.01; Fischer 12.0 g, P < 0.0001), while Lister and Lewis rats kept a constant consumption level (age effect: F2,80 = 5.502, P = 0.0036). Strain differences in food intake resembled body weight to a lesser degree than water intake: At 2 months of age, the results for food intake were in accordance with body weight, with Wistar rats eating most, Fischer rats eating least and Lister and Lewis rats eating the same amount of food during the 3-day observation period. At 4 and 6 months of age though, the difference between Wistar, Lister and Lewis rats disappeared due to the drop in food intake in Wistar rats.

Significant strain differences appeared as well when looking at light and dark phases separately (light phase: F3,40 = 33.73, P < 0.0001; dark phase: F3,41 = 77.85, P < 0.0001) with a significant effect of age on food intake (light phase: F2,80 = 10.05, P = 0.0001; dark phase: F2,82 = 6.121, P = 0.0033) and a significantly different development of the strains with age (light phase: F6,80 = 6.431, P < 0.0001; dark phase: F6,82 = 13.85, P < 0.0001, Fig. 8). As it was found for water intake, food intake during the dark phase was reflecting differences in total food consumption, while during the light phase differences between groups were smaller and were based on Lister rats eating most at 2 months of age and Fischer rats eating least at all ages.

Food intake relative to body weight (g/kg) decreased with age in all strains (F2,80 = 1077, P < 0.0001) showing a significant strain*age interaction (F6,80 = 13.20, P < 0.0001, Fig. 8). Lister and Lewis rats had a higher food intake relative to body weight compared to Wistar rats at all ages (F3,40 = 49.98, P < 0.0001) and Wistar rats ate more than Fischer rats at 2 months of age.

The decrease in the relative amount of food intake (light phase: F2,80 = 259.2, P < 0.0001; dark phase: F2,82 = 812.5, P < 0.0001) as well as the difference in strain*age interaction was also found for both light and dark phase (light phase: F6,80 = 4.425, P = 0.0007, dark phase: F2,82 = 10.63, P < 0.0001, Fig. 8). At 2 months of age, Lister rats showed the highest relative food consumption during the light phase, in accordance with their absolute amount of food intake; at the same age, Lewis rats consumed more food than all other strains, followed by Lister rats, while Fischer rats ate least. Differences were less pronounced at older ages, but mean values of Lister and particularly Lewis rats remained above Wistar and Fischer rats.

VO2

The relative hourly consumption of oxygen (ml O2/kg/h) showed significant strain differences (F3,44 = 16.72, P < 0.0001; Fig. 8), a significant decrease of VO2 relative to body weight with age (F1,44 = 1277, P < 0.0001) and a significant strain* age interaction (F3,44 = 3.876, P < 0.0152). Strain differences comprised a higher metabolic rate of Lewis over Lister rats and in turn Lister over Wistar and Fischer rats at 2 months of age. At 4 months of age, metabolic rates converged, leaving only Lewis rats at a slightly higher level.

Strain differences (light phase: F3,44 = 20.16, P < 0.0001; dark phase: F3,44 = 12.68, P < 0.0001), age effect (light phase: F1,44 = 831.9, P < 0.0001; dark phase: F1,44 = 1752, P < 0.0001) and strain*age interaction (light phase: F3,44 = 6.73, P < 0.0008; dark phase: F3,44 = 2.304, P = 0.0900) were also found for light and dark phase separately and were mainly based on differences at 2 months of age (Fig. 8). The higher VO2 of Lister rats resulted from an increase during the light phase, while Lewis rats showed high VO2 levels during both light and dark phase.

Respiratory quotient

Strains differed significantly in their mean RQ during the experiment (F3,44 = 23.82, P < 0.0001), based on Wistar rats having a significantly lower RQ compared to all other strains (Fig. 8). Respiratory quotient further decreased with age in all strains (F1,44 = 172.1, P < 0.0001), but in a moderately different manner (F3,44 = 4.8914, P < 0.0051).

Strain differences in RQ were also found during the light (F3,44 = 34.13, P < 0.0001) and dark phase (F3,44 = 10.46, P < 0.0001) and were consistent with the differences observed for food intake (Fig. 8). Lister rats had a significantly higher RQ during the light phase, while the RQ was significantly reduced in Wistar rats during both light and dark phase compared to the other strains. The decrease of RQ with age was also present for light (F1,44 = 144.3, P < 0.0001) and dark phase (F1,44 = 98.61, P < 0.0001) with a moderate difference in strain*age interaction (light phase: F3,44 = 4.214, P < 0.0105; dark phase F3,44 = 2.984, P < 0.0413).

Weight gain before and during the experiment

A comparison of the average weight gain one week prior to the experiment with the weight gain during the experiment revealed a significant drop in body weight gain during the experiment in Wistar and Fischer rats (Wistar: F1,22 = 32.76; P < 0.0001; Fischer: F1,22 = 41.74; P < 0.0001; Fig. 9). Lister and Lewis rats seemed to be less affected in their weight gain during the experiment, but showed a significantly decreased weight gain at least at 4 months of age (Lister: F1,18 = 17.25; P = 0.0006; Lewis: F1,22 = 8.671; P < 0.0075).

Figure 9.

Body weight development over 3 days measured before and during the experiment. We calculated the average weight gain over 3 days measured one week before the experiment and compared it to the weight gain during the 3 days while exposed to the automated homecage system in Lister, Wistar, Lewis and Fischer rats. Values represent group mean and standard deviation. Statistics: rmanova2, Sidak's multiple comparison test, P-values are given as asterisks with *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Discussion

The PhenoMaster system

Automated homecage systems are of particular interest for the behavioral phenotyping of laboratory animals, as these systems are believed to provide more robust and valid measurements of behavior compared to classical tests. Our study provides basic data on the behavior of four commonly used rat strains in an automated homecage system and reveals some important aspects to consider when using such a system.

The PhenoMaster system as replacement for other tests

The PM software offers the measurement of activity in specific parts of the cage. In order to investigate, if these measurements could provide a valid replacement for an open field (OF) test, we measured ambulatory, rearing and per/cen activity during the first 60 min of testing. The drop in ambulation and rearing after the first 20 min indicates that the animals initially explored and later habituated to the test cage in a similar fashion as it would be expected in an OF (Daenen et al. 2001; Hoy et al. 1999; Simon et al. 1994; Thiel et al. 1999). Habituation behavior was also found in a characterization study of a mouse model using both OF and PM (Painsipp et al. 2008). In contrast to ambulation and rearing, per/cen activity remained constant throughout the first hour of measurement. Although there are indications of habituation effects on similar parameters in the OF (Fonio et al. 2012; Ossenkopp et al. 1994; Simon et al. 1994; Thiel et al. 1999), it is at the moment unclear how the PM results would relate to those, as the smaller size of the cages and the presence of food and water baskets could affect the rats' tendency for thigmotaxis (Eilam 2003; Russell & Williams 1973).

The PhenoMaster as a homecage

A large part of the promotion of automated behavioral testing systems such as the PM focuses on their homecage-like aspects. It is believed that testing animals in a known environment reduces stress and offers results that better represent natural behavior (de Visser et al. 2006; Lipp et al. 2005; Tecott & Nestler 2004). However, the PM system differs notably from the typical homecage environment in our animal facilities. The most profound difference is the demand of single housing. Housing the animals individually is usually avoided because of ethical considerations, but also because it is known to influence performance in behavioral tests (reviewed in Hall 1998; Lukkes et al. 2009). Other aspects concern the cage setup itself. The airtight lids used in our setup are lower than the lids of the actual homecages and might therefore restrain rearing in old or big rats. Further, the lids likely create different acoustic and olfactory conditions compared to the homecages. Finally, food and water is not provided in the same way as in a typical homecage. It might be tempting to neglect these differences and consider the conditions close enough to the normal homecage. However, our measurements suggest that the rats experience some form of stress during testing as indicated by the drop in weight gain. Since this is a general phenomenon we observe with both mouse and rat PM systems using different setups in different animal facilities, it is an important issue for further evaluation. Indeed, it is known from metabolic cages, where the animals are also transferred to a different cage setup and isolated for the duration of the test, that these conditions are stressful to mice and rats (Gil et al. 1999; Kalliokoski et al. 2013). From the current study, it cannot be concluded whether the stress experienced during the PM experiment is a result of the testing conditions per se or rather due to the change in housing conditions. Thus it is possible that keeping the rats in the PM system constantly, or adapting the homecages to better approximate the PM setup, might avoid a stress response. However, as either option would include long-term social isolation, obtained data would most likely not reflect the natural behavior of a social animal. In this regard, it might be better to work with other automated homecage systems like the commercially available PhenoTyper (Noldus Information Technology, Wageningen, The Netherlands) or IntelliCage (NewBehavior AG, Zurich, Switzerland), which allow the assessment of group-housed animals.

Comparison of four common background rat strains

Novelty-induced behavior

The first 20 min of exposure to the PM cages could constitute a replacement at least for the activity parameter of an OF. Our results indicate that Lister rats are generally more active in terms of total ambulation and rearing counts. These findings might reflect a general trait among pigmented rats, suggesting that they are exploratory rather than anxious when exposed to a new environment (Broersen & Uylings 1999; Onaivi et al. 1992; Ramos et al. 1997; Rex et al. 1996; van der Staay et al. 1996, 2009; Weiss et al. 2000). Although post hoc analysis did not reveal significant strain differences, per/cen ratio indicated that Lewis rats had a stronger preference for sticking to the periphery of the PM cages. However, Lewis rats also showed the steepest decline in ambulatory activity and the highest number of animals with almost no ambulatory and rearing activity after the initial 20 min. Thus, we conclude that the high per/cen ratio might have resulted rather from a reduced exploratory activity than from increased anxiety, again pointing to difficulties in using the PM for the assessment of anxiety parameters. Accordingly, Lewis rats have previously been found to be less explorative (Rex et al. 1996) and either more or equally anxious compared to Fischer rats (Chaouloff et al. 1995; van der Staay et al. 2009). However, it is important to note that different anxiety tests are known to reveal different phenotypes and comparisons to classical anxiety tests might at this time be premature. A more thorough investigation of the per/cen measurement and potential anxiogenic factors of the PM system must be made to provide better understanding of which studies it can be expected to replicate.

3-Day observation period

The data obtained from each 3-day test period were divided into light phase, dark phase and total activity. The total values for ambulation, fine movement and rearing did not reveal any consistent differences between the rat strains. However, when analyzing light and dark phase separately, it became apparent that Lister rats were more active in all three parameters during the day. Furthermore, they showed the least amount of activity during the night. This finding is in line with a comparative study on behavioral pharmacology, revealing a higher diurnal and lower nocturnal homecage locomotor activity during a 24-h observation in Lister Hooded compared to Sprague Dawley and Wistar rats (McDermott & Kelly 2008). As the total level of activity was largely similar between Lister rats and the other strains, this indicates that Lister rats have a different circadian rhythm of activity.

Drinking and feeding differed significantly between the rat strains, and absolute values seemed to recapitulate differences in body size. However, looking at food and water intake relative to body weight revealed that Wistar and Fischer rats ate less than the others. Comparing weight gain before and during testing further showed that Wistar and Fischer rats did not grow properly during PM exposure. Accordingly, oxygen consumption, and even more meaningful respiratory quotient, was lower in Wistar rats compared to the other strains, pointing to inappropriate food supply. Lewis rats had the highest metabolic rate and showed the least effect on body weight gain during the test, indicating them to be least affected by the testing conditions. This idea is supported by literature on Lewis rats being less susceptible to stress due to physiological alterations (Chaouloff et al. 1995; Dhabhar et al. 1993). Lister rats once again showed higher diurnal activity in food and water intake and consistently in oxygen consumption and RQ, which further reinforces the differences seen in their activity pattern.

Summary notes

Automated homecage observation has been promoted as a powerful tool to study animal behavior. The essential advantage lies in the possibility to assess behavioral parameters over long periods of time, which in turn enables analysis across circadian phases as well as separate analysis of both novelty-induced and baseline homecage behavior.

In this study, data on the behavior of wild type rat strains was collected with the commercially available automated homecage system PhenoMaster. The first hour of observation revealed strain differences in novelty-induced behavior, which recreate common findings in literature. In a following 3-day observation period, further differences in circadian activities were identified, which offer new results that can be a useful reference for further studies.

It has to be noted that although the system allowed for minimal animal handling, a range of other stressors appeared to be present. Thus, further investigation is needed in order to establish optimal protocols for behavioral assessment, and to better judge the potential of the PM system.

Acknowledgments

The study was supported through institutional funds of the Institute of Medical Genetics and Applied Genomics. None of the authors have a conflict of interest. The PhenoMaster system was obtained from TSE as part of the RATstream project (EU-FP6-37846). However, the company was not involved in design or execution of the study and did not participate in data interpretation.

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