Excerpt
Table of Contents
Table of Contents
Table of figures and tables
Abbreviations
1. Introduction
1.1 Definition
1.2 State of the Art
1.3 Standardization
1.4 Hypotheses
2. Animals, Material, and Methods
2.1 Animals, housing, and experimental conditions
2.2 Behavioral tests
2.3 Experimental Design
2.3.1 The Elevated Plus Maze (EPM)
2.3.2 The Open Field Test (OF)
2.3.3 The Object Exploration Test (OBX)
2.3.4 The Social Investigation Test (SI)
2.3.5 The Dark/Light Test (DL)
2.3.6 The Free Exploration Task (FET)
2.4 Videos and computer software
2.4.1 VirtualDub
2.4.2 Optimas
2.4.3 Tracking Analysis
2.4.4 Statistical analysis
3. Results
3.1 Correlations over time
3.1.1 The Elevated Plus Maze (EPM)
3.1.2 The Open Field Test (OF)
3.1.3 The Object Exploration Test (OBX)
3.1.4 The Social Investigation Test (SI)
3.1.5 The Dark/Light Test (DL)
3.1.6 The Free Exploration Task (FET)
3.2 Correlation across contexts
3.2.1 The Elevated Plus Maze (EPM)
3.2.2 The Open Field Test (OF)
3.2.3 The Object Exploration Test (OBX)
3.2.4 The Social Investigation Test (SI)
3.2.5 The Dark/Light Test (DL)
3.2.6 The Free Exploration Task (FET)
3.3 Differences between individuals
4. Discussion
4.1 Correlation over time
4.2 Correlation across contexts
4.3 Differences between individuals
4.4 Implications
5. Conclusion
6. References
7. Acknowledgements
Table of figures
Figure 1: The open field
Figure 2: The Elevated Plus Maze (EPM)
Figure 3: The Social Investigation Test (SI)
Figure 4: The Dark/Light Test (DL)
Figure 5: Video Recording
Figure 6: The imaging program "Optimas” (Media Cybernetics)
Figure 7: The tracking program "Tracking Analysis” (www.phenotyping.com)
Figure 8: Correlation over time between the time spent in the open arms in the Elevated Plus Maze Test (EPM)
Figure 9: Correlation over time between the covered distance in the Open Field Test (OF) in trial 1 and trial
Figure 10 : Correlation over time for the parameter “path” in the Object Exploration Test (OBX)
Figure 11: Correlation across context between the Open Field Test (OF) and the Object Exploration Test (OBX)
Figure 12: Correlation across context between the Object Exploration Test (OBX) and the Social Investigation female Test (SIf)
Figure 13: Correlation across context between the Social Investigation female (SIf) and the Social Investigation male Test (SIm).
Figure 14: Correlation across context between the Social Investigation female (SIf) and the Dark/Light Test (DL)
Table 1: Experimental Design
Table 2: Support (+) of the hypotheses and contradiction (-) to the hypotheses 1) Individual behavior is correlated over time. 2) Individual behavior is correlated across contexts
Abbreviations
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1. Introduction
It is generally assumed that human beings have personalities, while animals do not. This assumption is not only derived from common sense, but also a basic principle of western philosophy and social theory (Mead, 1934). Still, dog owners will not stop mumbling about their cute puppy's unique personality. And they find support from unexpected source: Biologists argue that humans are nothing more, nothing less than animals. So they, too, begin to take into consideration that animals - including humans - might have a personality. What seems like a solely philosophical question at first glance, has important practical impacts, because the existence of animal personality contradicts the assumption that animals used in biological experiments can be standardized, and have uniform properties. If despite all efforts of standardization a residual variation cannot be eliminated, pharmaceutical and medical experiments might lose part of their validity.
Therefore the hypothesis that laboratory mice possess a personality was tested in an empirical study. In this study a set of standard behavior tests was performed with 40 female mice of the strain C57BL/6N. The data was then analyzed for correlations over time and across contexts, since this is a requirement for animal personality.
1.1 Definition
As a starting point, it is essential to define the concept of animal personality. As for most scientific concepts, a univocally shared understanding of animal personality does not exist. Definitions range from concepts, which stress the plasticity of personality, to the other extreme, which focuses on its invariability and static nature. As Dingemanse and Réale (2010) put it: „The first, prevalent in behavioural ecology, considers behaviours as highly plastic traits with individuals being capable of rapidly changing the expression of behaviour in response to changes in the surrounding environmental conditions […] Alternatively, each individual might be limited in its expression of a behavioural trait relative to the overall expression of that trait in the population”. One argument for the second, relatively stable understanding of personality is also the mutual reinforcement of different personality traits. Dingemanse and Réale (2010) put this as follows: „Selection often also acts on the correlation between seemingly unrelated traits, such that focusing on a single trait might result in a mismatch with the predictions of adaptive models“.
The first, rather fluid concept grasps personality as a phenomenon which can change at any time in any situation, and which is only shaped and influenced by environmental conditions. The theoretical value of the concept of personality consists in the fact that the action or reaction of an individual is not completely determined by environmental conditions. Hence, the explanatory value of a concept that focuses on the plasticity of personality is rather limited for the discussion at hand.
This study is based on the second, more stable understanding of personality, which focuses on the intrinsic dimensions of personality. The stable character of the concept of personality is tested in the study at hand.
Therefore, the following understanding of the concept is proposed: Personality refers to the intra-individual consistency in behavior, even a) when an individual is faced with different environmental challenges (across-context consistency) and b) at different points of time (inter temporal consistency). In addition, the assumption of an individual personality explains inter individual variety in behavior even when individuals are faced with the same environmental challenge (inter-individual variety even in similar situations).
1.2 State of the Art
This definition reflects three major insights of the existing literature on animal personalities: First, one has to consider the different situations in which behavior is consistent. Cross-contextual consistency is sometimes understood as behavioral consistency under different environmental challenges. Herborn and colleagues (2010), for example, observed that individual birds show similar exploration activity in the wild and in a cage. Even the hermit crab (Pagurus bernhardus) was found to show cross-context consistency (Briffa et al, 2008). The researchers studied the crabs' startle responses in the laboratory and at other locations and observed individual consistency in behavior. Similar observations were made in great tits (Parus major, V erbeek et al, 1994). Birds that approached a novel object more quickly were also quicker to explore a novel environment. In a similar vein, consistent behavior under varying risk of predation was observed in noble crayfish (Astacus astacus, Vainikka et al, 2011).
Context might also refer to “behavioral categories of different quality and function” (Uher 2011). A possible explanation for this understanding of across-context consistency is what some authors describe as behavioral syndrome (Bell, 2007, Sih et al, 2004). This concept refers to the correlation of different character traits. An example for such a correlation has been reported by Vainikka and colleagues (2011), who have studied boldness in the noble crayfish and propose a correlation between aggressiveness and boldness. They refer to this correlation as the „aggressive- bold behavioral syndrome“.
Other behavioral correlations have been observed by Hollander and colleagues (2007) who detected a link between exploratory behavior and nest defense in great tits (Parus major), and Adriaenssens and Johnsson (2011) who observed that less explorative brown trouts (Salmo trutta) were also less aggressive. These examples show that consistency across different dimensions of behavior, which is considered as an essential indicator of animal personality, is present in some species.
Second, inter-temporal consistency seems to be a major trait of animal personality, which has been empirically observed in a vast number of studies. The diagnosis of inter-temporal consistency, however, requires that the time period, over which a behavior is consistent, must be considered. Several authors report that there is short-term consistency in some species, for instance, in streamside salamanders (Ambystoma barbouri, Sih et al, 2003) and in sticklebacks (Gasterosteus aculeatus, Bell and Stamps, 2004), while other data indicate that even a long-term consistency can be observed in some species as the bighorn sheep (Ovis canadensis, Réale et al. 2000). It is evident that long-term and short-term are relative terms, therefore it is necessary for future research, to explicitly indicate over which time span consistency is analyzed.
Third, different individuals act differently in the same situation. A commonly used example for this phenomenon is the distinction between fast and slow explorers. (Exnerová et al, 2010, Dingemanse et al, 2004) Fast explorers are usually described as bold and aggressive, while slow explorers are shy and less aggressive. The former are also characterized as more proactive, thus tend to shape their environment, while the latter tend to be responsive to changes in their environment (Exnerovà et al, 2010).
A wide range of studies has detected animal personality in almost every existing higher developed animal taxon (Gosling 2001). The focus of research is on vertebrate species, but the existence of personality is even affirmed for firebugs (Pyrrhocoris apterus, Gyuris et al. 2010).
Nevertheless, it must be considered that the underlying understanding of animal personality differs immensely in the scientific community.
1.3 Standardization
In the beginning of behavioral research test animals were scarcely standardized. Ever since, scientists have tried to reduce the variance of experiments involving animals to a minimum. Only recently, studies were performed, in which it was questioned, whether it is truly possible to equalize animals and completely eliminate variability from animal tests. This new approach emerged, when Crabbe and colleagues (1999) conducted a study with a set of highly standardized tests, which were performed in laboratories all over the world. They found out that even with very precise experimental instructions and highly standardized experimental conditions, the results still showed considerable variability. Lewejohann and colleagues (2006) conducted a similar multi-laboratory study, which showed that a residual variance escapes all efforts of standardization. Therefore, some researchers propose to intentionally integrate a certain variance, which makes the experimental design less vulnerable to environmental influences (Richter et al, 2010). This variance might be different housing of the animals (e. g. an enriched environment for some subjects), or different characteristics of the test animals (e. g. different age or different sex). Results which can still be observed in all different test designs, will be reproducible with the utmost probability (or, as Richter put and colleagues put it, “systematic variation of only two factors was sufficient to mimic the range of differences between the replicate experiments, which guaranteed virtually perfect reproducibility”.)
Animal Personality in standardized animals
Lewejohann and colleagues (in press) analyzed existing data of tests with laboratory mice in order to find out whether constant and cross-contextual differences among individuals - also referred to as personality - underlie the variability that can usually be detected in studies with standardized mice. They indeed identified inter-temporal correlations of some parameters, e. g. the number of open/closed arm entries and the amount of time spent on the open/closed arms of an Elevated Plus Maze (EPM), as well as cross-contextual correlations, e. g. time spent in the center of the arena in the Open field Test (OF) and time spent in the center of the open field in the Free Exploration Task (FET). They concluded that nonrandom behavioral consistency accounts for part of the variability in biomedical studies.
Their conclusion that residual individual differences possibly escape all standardization is to be tested in the present study. On that account a set of standard behavioral tests, widely used in biomedical animal research, was performed in two trials, aiming to find out whether individual behavioral differences would be consistent across time and context.
1.4 Hypotheses
As mentioned above, the overall aim of this study was to test whether or not laboratory inbred mice show personality. According to the previously mentioned definition of animal personality, the following three hypotheses can be formulated:
1. Individual behavior is correlated over time.
2. Individual behavior is correlated across contexts.
3. Individuals show differences in behavior.
The first hypothesis implies that mice show consistent behavior over several weeks. Thus, it was expected that an individual that showed bold and proactive behavior and little anxiety-like behavior in the first trial, would also be fast-exploring and less anxious in the second trial. The second hypothesis imports that an individual shows a similar behavioral pattern in different situations. In the present study it was tested, whether an individual's behavior is correlated across different tests which assess different properties (state anxiety/ trait anxiety/ social exploration/ object exploration). The third hypothesis states that even standardized animals behave differently.
2. Animals, Material, and Methods
2.1 Animals, housing, and experimental conditions
In this study mice of the inbred strain C57BL/6N were scanned for personality under standardized laboratory conditions. This strain was chosen, because C57BL/6N is a very widely used mouse strain in animal research. The tests were conducted with 40 female mice that were twelve weeks old on the first test day and 17 weeks on the first day of the second trial. None of the mice experienced previous testing and all experiments were performed by the same experimenter (the author performed all of the tests herself) The mice were marked with ear cuts in order to distinguish between the four mice that shared a cage; possible combinations were one cut at the left ear, one cut at the right ear, two cuts at the left ear, and two cuts at the right ear. The home cages were standard Makrolon cages Type III (39 cm (length) x 22 cm (width) x 16 cm (height)) of transparent polypropylene with a fitted steel wire lid. Food (Altromin GmbH, Lage, Germany) and tab water were provided ad libitum in the lid. Additionally, the mice were fed with oat flakes once a week. The cage bottom was covered with wood shavings as bedding material and a paper towel as nesting material. Every Friday afternoon, the cages were cleaned and replaced. The mice were then transferred to the new cage along with a handful of old wood shavings to ease familiarization with the new home cage.
The cages were stored in a room with artificial lighting from 8.00 am. to 8.00 pm, a temperature of 22 °C and a highly fluctuating relative humidity between 30 and 50 %.
2.2 Behavioral tests
The experiments were performed in a 12 m² room, which was darkened, thus no daylight entered. All tests took place in an open field (figure 1, an 80 cm x 80 cm gray square arena surrounded by walls 40 cm high), which was illuminated by a lamp suspended 1.5 meter above the open field (255 lux). In case of the Dark Light Test (DL) additional neon tubes were switched on to make the light compartment more aversive (271 lux vs. 2.8 lux in the dark compartment). All test equipment was wiped out with 70% ethanol after each individual in order to eliminate scent trails. All mice were tested individually in five-minute-sessions for tests 2.3.1. to 2.3.5.
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Figure 1: The open field. All tests were performed in this 80 cm x 80 cm gray square arena surrounded by walls 40 cm high. The light intensity in the open field is 255 lux.
2.3 Experimental Design
In the present study behavior coding was used to measure the animals' behavior. Behavior coding comprises the instantaneous behavior of an individual in a specific context, whereas another kind of empirical studies, called trait rating, involves a human observer who evaluates an animal's behavioral tendencies along various behavioral dimensions (Highfill et al, 2010). The mice were tested in a range of tests for anxiety-like, exploratory, and social behavior in order to investigate their behavioral profile across different contexts. The animals' unconditioned anxiety-related and exploratory behavior was assessed in six different behavioral tests: The Elevated Plus Maze (EPM), the Open Field Test (OF), the Object Exploration Test (OBX), the Social Investigation Test (SI), the Dark/Light Test (DL), and the Free Exploration Task (FET). The behavioral testing was performed two times at an interval of five weeks in order to test for across-time consistency (table 1). All tests took place on weekdays, giving the mice time to acclimatize to the new home cage on weekends. The tests were conducted between 9 am. and 13 pm. Usually, only one test per day was performed, only the OF and the OBX were conducted back-to-back.
Table 1: Experimental Design. 40 female mice of the strain C57BL/6N numbered from 118 to 157 were tested individually in two trials (trial 1: 9.5.2011-27.5.2011, trial 2: 13.6.2011-1.7.2011) in a set of standard behavioral tests. The test sessions took place on weekdays from 9 am. to 13 am. The pretests were performed with other test mice for the experimenter to practice the procedure. The home cage was a hole cage during the week before the FET. After the SIm it was examined which of the animals was in estrous.
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2.3.1 The Elevated Plus Maze (EPM)
The Elevated Plus Maze Test (EPM) is widely employed as a measurement of anxiety-like behavior in rodents (Belzung et al, 2001) and offers the choice between two opposite open arms (30 cm (length) x 5 cm (width)) and two opposite enclosed arms (20 cm high walls) in 50 cm height: extending from a central platform (figure 2). Mice tend to stay in a place with close contact to a refuge which touches their vibrissae (thigmotaxis) (Treit, 1993). This generates a standard approach-avoidance conflict situation with mice with low anxiety-like behavior spending more time in the open arms. The central platform is not interpreted as either one. The EPM has been chosen for this study because of its prevalent utilization in anxiolytic drug testing (Pellow, 1985).
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Figure 2: The Elevated Plus Maze (EPM). The EPM offers the choice between two opposite open arms and two opposite enclosed arms extending from a central platform.
EPM testing procedure
The age of the mice was twelve weeks on the first day of the EPM, when mouse 118-137 was tested and twelve weeks and one day at the time mouse 138-157 was tested. In trial 2 they were seventeen weeks respectively seventeen weeks and one day old. Before a test mouse was placed in the EPM it was transferred to an empty small cage (24 cm (length) x 18 cm (width) x 14 cm (height)) for one minute. Then it was put on the center platform of the EPM with its head pointing towards one open arm. In the EPM, the covered distance („path“), the animal's average speed („velocity” - a linear function of “path”: path/300s), and the number of stops were analyzed. A halt was counted as a stop, when the mouse did not cover a distance of more than 1 cm during up to one second. The time spent in the closed arms, the time spent in the open arms, and the number of entries in both was also measured. The mouse was only detected in one of the arms when it had completely left the center platform. In order to prevent biases from differing locomotive health of the animals, the percentage of time spent in the open arms was calculated (quotient time,qt: openarmtime/(openarmtime+closedarmtime)*100) as well as the relative number of entries in the open arms (quotient number, qn: openarmentries/(openarmentries+closedarmentries)*100). The number of falls was observed.
2.3.2 The Open Field Test (OF)
The Open Field Test (OF) is the simplest tool for testing anxiety-like behavior. The open field arena is an 80 cm x 80 cm gray square arena surrounded by walls 40 cm high. The OF exposes a mouse to the approach-avoidance conflict, where it is tested how much a test animal tends to stay in contact with the wall. Individuals with little anxiety-like behavior are proposed to spend more time in the center, since the brightly lit and open area is aversive to the mice. The covered distance is not only an indication for locomotive health, but indicates also exploration activity.
OF testing procedure
On the first day of the OF, when mouse 118 through 129 was tested, the animals were twelve weeks and two days old (seventeen weeks and two days in trial 2). Mouse 130 through 145 was tested on the following day. Mouse 146 through 157 was tested at the age of twelve weeks and three days in trial 1 and at the age of seventeen weeks and three days in trial 2. In the OF the test mouse was placed in a 33 cm high cylinder with a diameter of 12 cm in one corner of the open field. After one minute recording was started and the cylinder was removed from the open field. Every individual had the opportunity to examine the OF for five minutes. Measured were path length, velocity, and stops. In addition, the time spent less than 20 cm away from the wall („wall time“), the time the animal spent in the center area of the OF (center: >20 cm distance to the wall), the time spent in the four corners (square areas of 20 cm x 20 cm in the open field corners), as well as the distance that was covered during minute one to minute five (path per minute 1-5). After five minutes of recording, the cylinder was put back in the corner and the mouse was placed inside again. The region of interest (ROI, section 2.4.2) was defined as the open field ground.
2.3.3 The Object Exploration Test (OBX)
The Object Exploration Test (OBX) also serves as a measurement of anxiety-like behavior and of curiosity towards a novel object (Zipser, 2006).
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