Highlights: Selection for cognitive trait induced changes in the intermale aggression
Intermale aggression is presumably related to anxiety level
Less aggressive mice are not afraid of novel food
Keywords: Intermale Aggression; Cognitive Abilities; Artificial Selection; Anxiety; Novelty; Mice
The data obtained demonstrated, that in rodents, selected for different levels of aggressiveness, the respective phenotypic correlations [i.e. the plausible correspondence of aggressiveness, anxiety and general reactivity levels] are only partly based on common physiological mechanisms. The same seems to be true when animals, selected for other behavioral traits, were compared. No clear correlations could be seen as well when aggression levels are compared with cognitive tests performance [19]. Although the knocking out of one BDNF allele, or the forebrain-restricted deletion of both these alleles in mice induced not only increased aggression but also the increase in anxiety and deficits in cognition. Thus, rather complicated pattern of correlations between aggression and other behavioral traits emerges, presumably due to [i] heterogeneity of aggression traits as different aggressive genotypes could be created in technically different selection experiments, while the “aggressive” traits in selected lines look rather similar by their phenotype [20,21] and [ii] the heterogeneity of anxiety phenotypes. The complexity of genetic bases of cognitive traits could make its own impact in the complicated patterns of phenotype correlations discovered.
The objective of the present work was to determine the intermale aggression level [in standard opponent test] in EX mice, i.e. line selected for high scores of elementary logic task solution [extrapolation test], in comparison to these indices in control unselected mice - population CoEX [22-24]. The data from elevated plus maze test [as anxiety indices] for EX and CoEX mice are also presented which were obtained previously during several selection generations [starting from F4]. Light-dark box test data for mice of both groups from F16 are also included.
The animals which were chosen for breeding in each generation [for EX strain] should conform two criteria – i] the animal should display the correct solution of extrapolation task at its first presentation [when an animal had no previous analogous experience] and demonstrate the 5-6 correct solutions during 6 subsequent task presentations and ii] apart from extrapolation test success they should reveal no “refusals to solve the task”, when the mouse did not approach the central feeding opening during 3 min [arbitrary interval]. Such refusals are the distinct signs of anxiety, aroused in animal by the new environment, as well as the cases when the solution took more than 120 s [also arbitrary chosen interval].
The behavior of EX mice in all tests was compared with that of mice from the unselected population [CoEX], which were bred in parallel with EX line. The numbers of mice tested in dyadic encounters are presented in table 1.
Generation |
Trait |
First attack latency, seconds |
Number of Attacks |
||||
Days of Test |
1 |
2 |
3 |
1 |
2 |
3 |
|
F5 |
EX n=8 |
606.3 ± 122.6* |
608.6±139.8* |
421.3±122.4** |
5.0±1.6 |
11.8±2.7** |
10.1±2.4* |
CoEX n=8 |
1113.8 ± 122.6 |
1069.7±139 |
1082.7±121.6 |
1.8±1.5 |
1.5±2.7 |
1.4±2.3 |
|
F10 |
EX n=20 |
1171.3 ±66.9*** |
1096.9±66.5*** |
1051.8±98.7** |
0.4±0.3***5 |
1.5 ±0.7** |
1.3±0.7* |
CoEX С n=19 |
782.4±68.6 |
623.8±68.2 |
556.4±101.2 |
6.7±1.8 |
9.3±2.9 |
8.9±2.9 |
|
F12 |
EX n=19 |
1200±65.6** |
1149.9±70.5* |
1147.8±85.9* |
0* |
0.3±0.7* |
0.4±0.8* |
CoEX n=20 |
913.7±63.9 |
902.4±68.7 |
860.4±83.7 |
1.8±0.5* |
2.5±0.7 |
2.8±0.8 |
|
F15 |
EX n=18 |
1178.1±48.9** |
1131.1±66.0* |
1094.9±73.5** |
0.1±0.8 |
0.7±1.2* |
3.1±2.2 |
CoEX n=18 |
960.3±48.9 |
923.8±66.0 |
791.7±73.5 |
2.7±0.8 |
4.8±1.2 |
6.7±2.2 |
All animals were kept in plastic cages [ 26.7 x 20.7 x 14 cm] with natural light-dark schedule and food [Chara firm] and water ad lib. The experimental procedures were performed in accordance with ES 2010 Directive.
Standard opponent test procedure. Before the start of tests on intermale aggression EX and CoEX males were kept isolated in individual cages [26.7 х 20.7 х 14 cm] in mouse colony room for 10 days.
At the start of the test two males [experimental one and the “standard opponent”] were placed simultaneously in the plastic cage [42.5 х 26.6 х 15 cm] with fresh bedding [wood shaving] and were watched for 20 min. Latency of the first attack, total number of attacks, the number of aggressive “forced” grooming and tail rattling episodes as well as sniffing of the partner [data not presented] were manually recorded. The proportions of animals from each group which demonstrated the directed aggression toward the rival were also estimated. When animal aggression was scored relying on the data from the single encounter there is the risk to get the biased data due to chance variability [26]. Thus the repetitive testing was performed - each pair of males was tested during three successive days.
Light-dark box [LDB] test was performed with F16 males from EX [n=16] and CoEX [n=18] groups [in F15 there were not enough animals to form the groups for this test]. LDB was the opaque plastic box divided by partition in two unequal parts – the dark one [15, 5 х 29,5 х 28 cm] and brightly lit [58 х 29,5 х 28 cm]. The partition contained the opening [5,5 х 4,5 cm] for an animal to pass through. Animal was placed into the brightly lit compartment and the latency of the first entrance into the dark was manually recorded. The time spent in the dark, number of returns into the lit compartment, numbers of peeping reactions in and out the dark compartment and defecation boli numbers were also recorded.
Elevated plus maze test [EPM]. The data on EPM performance in EX and CoEX mice were partly described previously [22-24]. During 3 min test the number of animal’s entries into the open EPM arms, time spent there, closed arm-closed arm transitions, hangings from open arms, stretched-attend postures, as well as the number of rearings, grooming episodes and defecation boli were manually registered. This test was presented to males of F4, F6, F8, F9, F10, F12, F16 [n=175 in total for EX and n=178 - for CoEX mice, see (Table 2).
Generation |
Group |
Time in open arms, secс |
Entries into open arms |
Closedarm-closed arm transitions |
Number of grooming episodes |
F4 |
EX n=16 |
51.3 ± 6.1** |
4.5 ± 0.5 |
2.4 ± 0.8 |
1.88 ± 0.3 |
CoEX n=12 |
30.4 ± 7.3 |
2.92 ± 0.6 |
4.08 ± 0.8 |
2.4 ± 0.77 |
|
F6 |
EX n=36 |
29.8 ± 3.4* |
3.08 ± 0.3* |
2.4 ± 0.36* |
1.36 ± 0.2*** |
CoEX n=32 |
14.2 ± 2.3 |
1.44 ± 0.26 |
1.09 ± 0.28 |
4.06 ± 0.4 |
|
F8 |
EX n=14 |
12.2 ± 1.6 |
1.78 ± 0.28 |
5.6 ± 0.9* |
1.9 ± 0.32 |
CoEX n=14 |
19.0 ± 4.1 |
1.78 ± 0.4 |
2.9 ± 0.7 |
1.6 ± 0.26 |
|
F9 |
EX n=25 |
8.7 ± 1.7 |
0.64 ± 0.2 |
2.4 ± 0.5 |
2.88 ± 0.4* |
CoEX n=17 |
8.9 ± 1.9 |
0.76 ± 0.2 |
2.5 ± 0.6 |
1.59 ± 0.3 |
|
F10 |
EX С n=34 |
8.0 ± 1.4* |
0.56 ± 0.15 |
1.2 ± 0.3 |
2.65 ± 0.26* |
CoEX n=53 |
6.6 ± 0.8 |
0.25 ± 0.08 |
2.0 ± 0.37 |
1.8 ± 0.18 |
|
F12 |
EX n=34 |
5.9 ± 1.0 |
0.3 ± 0.1 |
1.0 ± 0.2 |
2.7 ± 0.2*** |
CoEX n=36 |
7.4 ± 1.2 |
0.38 ± 0.1 |
1.36 ± 0.29 |
1.5 ± 0.18 |
|
F16 |
EX n=16 |
0.3 ± 0.4 |
0.18 ± 0.06* |
2.6 ± 0.4 |
0.9 ± 0.3 |
CoEX n=14 |
0 |
0 |
1.9 ± 0.4 |
0.85 ± 0.3 |
All mice from the “standard opponent” group, when not attacked by rivals, demonstrated “neutral” behavior. They emitted no overt reactions when they were olfactory investigated by the rival, they never initiated the contacts and conflicts and they started to freeze for long periods and to avoid the rival after being attacked [data not presented].
The results of aggressive encounters [attack latencies, number of attacks] are presented in the (table 1). F5 EX males demonstrated higher aggression than CoEX mice. This statement is based on several experimental evidences - first attack latencies of EX’s were significantly shorter and the attack numbers during each day of tests – higher, than in CoEX mice [table 1], and the proportion of animals, which demonstrated aggression towards the opponent, was also significantly higher in EX, than in CoEX.
In F10, F12 и F15 the “sign” of intergroup differences changed into the opposite one – the aggression level in EX males was now lower than in CoEX. This difference concerns not only the intergroup comparison, but is seen in absolute trait magnitudes as well - the proportion of aggressive CoEX males in F5 was 12%, while in F15 – it was significantly higher - 67% [p< 0.01].
It should be noted that the behavior of animals during dyadic encounters also changed in the course of selection. In F5 the attacks of EX mice were longer in duration [>20 s] and very intense, while later, in F10 their attacks were sparse, although still of rather long duration. At the same time EX males from F12 and F15 revealed the tendency to avoid the opponent and demonstrated the overt fear reactions. They tended to stay in the cage corners and did not enter the center of this arena. As for CoEX males the picture was the reverse – in F5 their attacks were rare and short [< 5 s], while in F10 both these indices increased and remained at rather high levels in F12 and F15. The proportions of CoEX aggressive mice in these generations were also high (Figure1).
*, **, *** - significantly different from CoEX scores (1-factor ANOVA and post hoc Tukey HSD test). Designations as in Fig.1
The anxiety indices of LDB test, performed by F16 mice, demonstrated that time in dark compartment was significantly [p< 0.05] longer in EX mice [92.13 ± 11,3 s] in comparison with CoEX [61.55±9.4 s], and this could signify the higher level of their overall anxiety.
In the neophagophobia test, when the new food was presented to hungry animal in the new [although not provoking avoidance] environment, the anxiety displayed by an animal, is of the type not identical to anxiety displayed in EPM. In other words the signs of anxiety behavior in the hyponeophagophobia test were consistently lower in EX mice in the same generations in which EPM test revealed no such differences [23]. Several authors [15,16,30] also suggest that the anxiety of the type which reflects the species specific fear of open and brightly lit space [displayed in EPM, LDB and open field tests] is not identical to the state of anxiety-alertness, which animals [including rodents] display in the new environment [i.e., in neophagophobia test]. Our data on EPM [starting from F8] and on LDB [F16] tests indicated the higher “overall” species-specific anxiety in EX mice. And up to the present moment there are no obvious explanations of the dynamic changes of this type of anxiety during selection generations.
The pattern of differences which was shown in this study resemble the correspondence of aggression and general anxiety in SAL-LAL and TA-TNA lines [see above], i.e. in lines which were directly selected for aggression traits. The non-aggressive lines in these pairs of lines demonstrated higher anxiety.
Unfortunately the neophagophobia test was not presented to mice of early generations. Starting from F8 the differences in these indices [23] made it possible to suggest that EX mice were more prone to overcome the specific fear of the new environment, than CoEX control mice.
The neophagophobia test results are usually not easy to interpret, as an animal could reveal both - the positive reaction to novelty as well as the fear of it [31]. This means that animal ability to suppress the fear of new environment [which is not the “full developed” species-specific anxiety] was more clearly expressed in EX mice. Although it should be reminded, that EX mice were less aggressive in the late selection generations. At the same time “reaction to novelty” could be considered as the component of the cognitive behavior [23].
Our selection experiment did not result in clear increase of extrapolation task scores [which were selected for], although the success of EX mice in another cognitive test - the burrowing [puzzle-box] task - was noted in comparison with CoEX animals [24]. These results permit to cautiously suggest, that the prevalence of EX mice scores in burrowing task is the response to selection for cognitive trait. It is also possible that the cognitive component of neophagophobia test performance in EX corresponds their ability to overcome the fear of novelty in puzzle-box, and this ability was less evident in CoEX mice [23-24].
Many experimental evidences indicated the role which brain neurotransmitter systems play in determination of aggression level [8]. At the same time the increased anxiety is associated with the serotonergic system hypofunction [5,33,34]The involvement of noradrenergic and glutamatergic systems in the realization of aggressive behavior was also shown [11,35,36]. The genetic studies permitted to localize the set of genes involved in the expression of this behavior [14,37,26,38] The switch-off of genes, participating several signaling pathways, was shown to increase the intermale aggression as well [3,13,35] The interstrain differences in lines, selected for high and low cholinergic system reactivity [Flinders lines], also affected the aggression traits [4], which could be also the evidence of genetic modulation of the aggression level.
The experimental data on intermale aggression in mice, which were selected for cognitive trait, could be regarded as the so called correlated trait, and this in turn means that genetic variability has the impact in this trait expression. The selection for the cognitive trait was successful during initial selection generations [23], although this selection gain was not maintained during further selection. The same pattern was noted for the selection against anxiety behavior, which mice displayed in the new environment [i.e. against “refusals” to solve the task]. No marked differences were noted in this trait in the later selection generations [in comparison to CoEX]. The results of this study permit us to state the following. These traits [anxiety in the new environment and logic task solution ability] have rather complicated genetic basis, but the anxiety, displayed in the EPM test and in the course of extrapolation test are not similar, i.e. the anxiety is not a “uniform” state. In other words we suggest that the notion “anxiety” encompasses: first, the state of general fear-anxiety which is developed in the uncomfortable frightening environment [open-field, EPM and LDB], and second, the anxiety state, which is aroused when animal faces the need to explore the “novelty” when no real overt danger is present and the environment is more or less “comfortable”. This view coincides with opinion developed by other authors [39].
Analyzing the complicated pattern of EX-CoEX behavioral differences one more consideration is also worth mentioning. It could be that the integrated adaptive behavior of an animal [i.e. displayed during rather complicated logic task as that of extrapolation] requires the definite level of anxiety to be expressed in the behavioral repertoire of an animal [may be even anxieties of both types, hypothesized above]. The decrease of anxiety during artificial selection procedure could evoke the compensatory shifts which would counteract such selection effects. Thus one may suggest that changes in EX mice aggression levels in response to selection for high cognitive test scores with the concomitant selection against anxiety signs are the result of complex changes in mouse adaptive behavior.
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