Selection of Mice for Object Permanence Cognitive Task Solution

The selection of mice for high (“plus”) and low (“minus”) scores in the puzzle-box test was performed over five generations. This test evaluates the success (or failure) in finding the underpass, leading to the dark part of the box, when it is blocked. This means that the mouse is either able or unable to operate the “object permanence rule” (one of the index’s cognitive abilities). For the “+” strain, animals were bred who solved the test when the underpass test blocked with a plug; the “−” strain comprised those who were unable to solve this task. In mice of the “+” strain, the proportion of animals that was able to solve “plug” stages of the test was higher than in the “−” strain and in the non-selected genetically heterogeneous population. The “+” mice ate significantly more new food in the hyponeophagia test. Animals of both strains demonstrated the ability to “manipulate” the plug blocking the underpass, touching the plug with their paws and muzzle, although the majority of “−” mice were unable to open the underpass effectively. Thus, mice of both selected strains demonstrated that they were able to understand that the underpass does exist, but only “+”-strain animals (at least the majority of them) were able to realize the solution. The selection for plug-stage solution success affected the mouse’s ability to open the hidden underpass.


Introduction
As stated in one behavior genetics historical article, "validation of behavioral constructs and the ways to test them is urgently needed in both animal and human behavioral and psychiatric genetics" [1]. The term "cognitive" behavior, or the ability to solve a test which requires an adaptive response, is usually addressed to behavioral reactions which require classical and/or instrumental conditioning (sometimes of a very complicated structure). Genetic studies of learning abilities have been performed for many years using rodent selection in food-reinforced learning and aversive learning paradigms. Famous rat selection experiments include those by Tryon [2] and Korochkin et al. [3,4] involving rat selection for instrumental learning in the Novosibirsk Institute for Genetics and Selection. Selection experiments for and against successful aversive learning were also performed in Roman, Syracuse and Hatano strains of rat [5][6][7][8]. Experimental evidence concerning genetic differences in mouse learning performance is widespread (among others [9][10][11]), especially in hippocampus-dependent spatial orientation and memory tests. These results describe interstrain differences in mouse learning using different strains, as well as differences in animals with genetically engineered genotypes. The specific properties of respective neuronal networks were also indicated [12][13][14][15][16][17][18].
The term "cognitive behavior", used here, refers to the animal's ability to solve an elementary logic task which is presented to the subject for the first time, i.e., when the subject has no analogous previous experience, as in case of any learning paradigm. So it is not the learned response per se to certain environmental signals. The solution of such a task requires "understanding" the elementary logic of the used paradigm. According to Krushinsky's definition [19], the animal's "cognitive ability" (or elementary reasoning) is = 97, 63 ♂♂, 34 ♀♀, in total), though F1-F3 control animals were not tested due to a technical problem.
Mice were housed in plastic cages (size 35 × 56 × 20 cm) with food (Firm Laboratorkorm) and water ad libitum with a natural light-dark schedule.
Statement on the welfare of animals. The experimental protocol was accepted by the Bioethical Commission of Moscow State University, session no. 49 of 18 June 2014.
Puzzle-box test. An animal was placed into the brightly lit part of the experimental box, from which it could easily go into the dark part of the box, avoiding the light (see Figure 1). The underpass leading to the dark part of the box was submerged below the floor level and the animal could use it to easily penetrate into a more comfortable, dark compartment.
During test stage 1 of the test, this underpass stayed unobstructed, and the animal could freely enter the dark. At test stage 2, the underpass was masked (covered up to the floor level by fresh wood shavings). At stages 3 and 4, the underpass was blocked by means of a plug (made from carton and plastic), which animals could easily remove by using their teeth, or move aside using a muzzle and paws. The animal was given 180 s to solve stages 1 and 2, whereas for stages 3 and 4 (which required more effort), it was given 240 s. After animal entered the dark part of the box, it was left there for 15-20 s, Neurol. Int. 2022, 14, FOR PEER REVIEW 3 = 97, 63 ♂♂, 34 ♀♀, in total), though F1-F3 control animals were not tested due to a technical problem. Mice were housed in plastic cages (size 35 × 56 × 20 cm) with food (Firm Laboratorkorm) and water ad libitum with a natural light-dark schedule.
Statement on the welfare of animals. The experimental protocol was accepted by the Bioethical Commission of Moscow State University, session no. 49 of 18 June 2014.
Puzzle-box test. An animal was placed into the brightly lit part of the experimental box, from which it could easily go into the dark part of the box, avoiding the light (see Figure 1). The underpass leading to the dark part of the box was submerged below the floor level and the animal could use it to easily penetrate into a more comfortable, dark compartment.
During test stage 1 of the test, this underpass stayed unobstructed, and the animal could freely enter the dark. At test stage 2, the underpass was masked (covered up to the floor level by fresh wood shavings). At stages 3 and 4, the underpass was blocked by means of a plug (made from carton and plastic), which animals could easily remove by using their teeth, or move aside using a muzzle and paws. The animal was given 180 s to solve stages 1 and 2, whereas for stages 3 and 4 (which required more effort), it was given 240 s. After animal entered the dark part of the box, it was left there for 15-20 s, , F1 ("−") 22 subject has no analogous previous experience, as in case of any learning paradigm is not the learned response per se to certain environmental signals. The solution of task requires "understanding" the elementary logic of the used paradigm. Accord Krushinsky's definition [19], the animal's "cognitive ability" (or elementary reason the ability to "grasp" the empirical laws which connect objects and events in the ex world and to develop further adaptive behavioral reactions using such information The present paper describes the results of an experiment in which mice we lected over five generations for high and low scores in the puzzle-box test solutio applied to mouse experiments by Galsworthy et al. [20]. The puzzle-box experim design requires an animal to understand that the object (an underpass, which the m is eager to use, as it leads into the safe box compartment) is hidden, being mask wood shavings or a plug, but can be discovered [21]. The ability to solve the puzz test requires that the animal apply the rule of "object permanence" (according to see [22]). The animals in the initial population for this selection experiment were m the EX strain. This strain was selected earlier for high scores in the extrapolatio [23]. The correct extrapolation task solution is also based (at least partly) on an an ability to understand the rule of "object permanence". In the mental operation o trapolation", the animal has to find the new location of food bait on the basis of mation perceived, although the food bait is no longer seen, as it has been moved from view to the right or to the left of the animal, which perceives the food bai small opening at the base of the box's front wall.
In order to make the selection routine more feasible, we simplified the puzz procedure so that the experimental testing could be performed over one day, in co ison to the longer test schedules used initially [20,21].
The selection criterion for the "plus" strain was short latency of test solutions stages when the underpass was masked by a plug, and the lack of these solutions arbitrary time interval of 240 s) in the "plug" stages was the criterion for the stra lected as "minus". Previously published data [24] demonstrated success in this sel process for F1-F3. The behavior of mice from "plus" and "minus" strains was also pared in a hyponeophagia test [25]; the amount of new food eaten during the tes was persistently higher in "plus" mice. In the hyponeophagia test, the new food cubes of cheese) was presented to the hungry mouse in a new, but not frightenin vironment. Animal behavior in this test is affected by both the necessity of handli "novelty" (new food) and the "concurrent" anxiety reaction, which is aroused new environment (and the novelty of cheese as a food) and could obscure the reac this novelty. This test has been successfully used to evaluate the effects of antid sants [26], and was earlier regarded as a test for anxiety [27].
Thus, the results of the five-generation selection for this cognitive trait in m presented below. The working memory indices, which could be drawn from the col of the puzzle-box solution, were also analyzed.

Material and Methods
Experimental animals. Mice were bred selectively starting from F20 of the previously selected for high scores in the extrapolation test (see above). All an born in each generation (males and females), were tested with a simplified puzz test (for details, see below). Animal numbers (for "+" and "-" strains, respectively as follows: F1 ("+") 31 ♂♂, 29 ♀♀, F1 ("-") 22 ♂♂,17 ♀♀, F2 ("+"), 28 ♂♂, 3 F2 ("-") 22♂♂, 27 ♀♀, F3 ("+") 41 ♂♂, 42 ♀♀, F3 ("-") 28♂♂, 18 ♀♀, F4 (" ♂♂, 33 ♀♀, F4 ("-") 26♂♂, 18 ♀♀, F5 ("+") 27 ♂♂, 39 ♀♀, F5 ("-") 44 ♂ ♀♀. Some animals in these animal groups from each generation were tested hyponeophagia test (the limited number of animals in this test was due to tec problems). Mice in the control, non-selected, heterogeneous populations from t spective generations were also tested in parallel with F4 and F5 of the selected stra subject has no analogous previous experience, as in case of any learning paradi is not the learned response per se to certain environmental signals. The solution task requires "understanding" the elementary logic of the used paradigm. Acc Krushinsky's definition [19], the animal's "cognitive ability" (or elementary reas the ability to "grasp" the empirical laws which connect objects and events in the world and to develop further adaptive behavioral reactions using such informat The present paper describes the results of an experiment in which mice lected over five generations for high and low scores in the puzzle-box test solu applied to mouse experiments by Galsworthy et al. [20]. The puzzle-box expe design requires an animal to understand that the object (an underpass, which th is eager to use, as it leads into the safe box compartment) is hidden, being m wood shavings or a plug, but can be discovered [21]. The ability to solve the pu test requires that the animal apply the rule of "object permanence" (according see [22]). The animals in the initial population for this selection experiment wer the EX strain. This strain was selected earlier for high scores in the extrapola [23]. The correct extrapolation task solution is also based (at least partly) on an ability to understand the rule of "object permanence". In the mental operatio trapolation", the animal has to find the new location of food bait on the basis mation perceived, although the food bait is no longer seen, as it has been mov from view to the right or to the left of the animal, which perceives the food b small opening at the base of the box's front wall.
In order to make the selection routine more feasible, we simplified the pu procedure so that the experimental testing could be performed over one day, in ison to the longer test schedules used initially [20,21].
The selection criterion for the "plus" strain was short latency of test solutio stages when the underpass was masked by a plug, and the lack of these solutio arbitrary time interval of 240 s) in the "plug" stages was the criterion for the lected as "minus". Previously published data [24] demonstrated success in this process for F1-F3. The behavior of mice from "plus" and "minus" strains was a pared in a hyponeophagia test [25]; the amount of new food eaten during the was persistently higher in "plus" mice. In the hyponeophagia test, the new foo cubes of cheese) was presented to the hungry mouse in a new, but not frighte vironment. Animal behavior in this test is affected by both the necessity of han "novelty" (new food) and the "concurrent" anxiety reaction, which is arouse new environment (and the novelty of cheese as a food) and could obscure the re this novelty. This test has been successfully used to evaluate the effects of an sants [26], and was earlier regarded as a test for anxiety [27].
Thus, the results of the five-generation selection for this cognitive trait in presented below. The working memory indices, which could be drawn from t col of the puzzle-box solution, were also analyzed.
During test stage 1 of the test, this underpass stayed unobstruc could freely enter the dark. At test stage 2, the underpass was maske floor level by fresh wood shavings). At stages 3 and 4, the underp means of a plug (made from carton and plastic), which animals cou using their teeth, or move aside using a muzzle and paws. The anima solve stages 1 and 2, whereas for stages 3 and 4 (which required given 240 s. After animal entered the dark part of the box, it was le Neurol Puzzle-box test. An animal was placed into the brightly lit pa box, from which it could easily go into the dark part of the box, a Figure 1). The underpass leading to the dark part of the box was s floor level and the animal could use it to easily penetrate into a m compartment.
During test stage 1 of the test, this underpass stayed unobstru could freely enter the dark. At test stage 2, the underpass was mask floor level by fresh wood shavings). At stages 3 and 4, the under means of a plug (made from carton and plastic), which animals co using their teeth, or move aside using a muzzle and paws. The anim solve stages 1 and 2, whereas for stages 3 and 4 (which required given 240 s. After animal entered the dark part of the box, it was , F2 ("+"), 28 subject has no analogous previous experience, as in case is not the learned response per se to certain environmenta task requires "understanding" the elementary logic of th Krushinsky's definition [19], the animal's "cognitive abil the ability to "grasp" the empirical laws which connect o world and to develop further adaptive behavioral reactio The present paper describes the results of an expe lected over five generations for high and low scores in t applied to mouse experiments by Galsworthy et al. [20 design requires an animal to understand that the object ( is eager to use, as it leads into the safe box compartme wood shavings or a plug, but can be discovered [21]. Th test requires that the animal apply the rule of "object pe see [22]). The animals in the initial population for this se the EX strain. This strain was selected earlier for high [23]. The correct extrapolation task solution is also based ability to understand the rule of "object permanence". trapolation", the animal has to find the new location of mation perceived, although the food bait is no longer s from view to the right or to the left of the animal, whi small opening at the base of the box's front wall.
In order to make the selection routine more feasibl procedure so that the experimental testing could be perf ison to the longer test schedules used initially [20,21].
The selection criterion for the "plus" strain was sho stages when the underpass was masked by a plug, and arbitrary time interval of 240 s) in the "plug" stages wa lected as "minus". Previously published data [24] demo process for F1-F3. The behavior of mice from "plus" and pared in a hyponeophagia test [25]; the amount of new was persistently higher in "plus" mice.
In the hyponeop cubes of cheese) was presented to the hungry mouse in vironment. Animal behavior in this test is affected by bo "novelty" (new food) and the "concurrent" anxiety rea new environment (and the novelty of cheese as a food) a this novelty. This test has been successfully used to ev sants [26], and was earlier regarded as a test for anxiety [ Thus, the results of the five-generation selection fo presented below. The working memory indices, which col of the puzzle-box solution, were also analyzed.

Material and Methods
Experimental animals. Mice were bred selectively previously selected for high scores in the extrapolatio born in each generation (males and females), were teste test (for details, see below). Animal numbers (for "+" an as follows: F1 ("+") 31 ♂♂, 29 ♀♀, F1 ("-") 22 ♂♂,17 F2 ("-") 22♂♂, 27 ♀♀, F3 ("+") 41 ♂♂, 42 ♀♀, F3 (" ♂♂, 33 ♀♀, F4 ("-") 26♂♂, 18 ♀♀, F5 ("+") 27 ♂♂ ♀♀. Some animals in these animal groups from each hyponeophagia test (the limited number of animals in problems). Mice in the control, non-selected, heterogen spective generations were also tested in parallel with F4 subject has no analogous previous experience, as in c is not the learned response per se to certain environme task requires "understanding" the elementary logic o Krushinsky's definition [19], the animal's "cognitive a the ability to "grasp" the empirical laws which connec world and to develop further adaptive behavioral rea The present paper describes the results of an e lected over five generations for high and low scores i applied to mouse experiments by Galsworthy et al. design requires an animal to understand that the obje is eager to use, as it leads into the safe box compart wood shavings or a plug, but can be discovered [21]. test requires that the animal apply the rule of "object see [22]). The animals in the initial population for this the EX strain. This strain was selected earlier for hig [23]. The correct extrapolation task solution is also ba ability to understand the rule of "object permanence trapolation", the animal has to find the new location mation perceived, although the food bait is no longe from view to the right or to the left of the animal, w small opening at the base of the box's front wall.
In order to make the selection routine more feas procedure so that the experimental testing could be p ison to the longer test schedules used initially [20,21].
The selection criterion for the "plus" strain was s stages when the underpass was masked by a plug, an arbitrary time interval of 240 s) in the "plug" stages lected as "minus". Previously published data [24] dem process for F1-F3. The behavior of mice from "plus" pared in a hyponeophagia test [25]; the amount of ne was persistently higher in "plus" mice.
In the hypon cubes of cheese) was presented to the hungry mouse vironment. Animal behavior in this test is affected by "novelty" (new food) and the "concurrent" anxiety new environment (and the novelty of cheese as a food this novelty. This test has been successfully used to sants [26], and was earlier regarded as a test for anxiet Thus, the results of the five-generation selection presented below. The working memory indices, whic col of the puzzle-box solution, were also analyzed.
During test stage 1 of the test, this un could freely enter the dark. At test stage 2, floor level by fresh wood shavings). At st means of a plug (made from carton and p using their teeth, or move aside using a mu solve stages 1 and 2, whereas for stages given 240 s. After animal entered the dark Mice were housed in plastic cages ( torkorm) and water ad libitum with a nat Statement on the welfare of animal the Bioethical Commission of Moscow Sta Puzzle-box test. An animal was plac box, from which it could easily go into t Figure 1). The underpass leading to the d floor level and the animal could use it to compartment.
During test stage 1 of the test, this u could freely enter the dark. At test stage 2 floor level by fresh wood shavings). At means of a plug (made from carton and using their teeth, or move aside using a m solve stages 1 and 2, whereas for stages given 240 s. After animal entered the da , F2 ("−") 22 subject has no analogous previous experience, as in case of any learning paradigm. So it is not the learned response per se to certain environmental signals. The solution of such a task requires "understanding" the elementary logic of the used paradigm. According to Krushinsky's definition [19], the animal's "cognitive ability" (or elementary reasoning) is the ability to "grasp" the empirical laws which connect objects and events in the external world and to develop further adaptive behavioral reactions using such information.
The present paper describes the results of an experiment in which mice were selected over five generations for high and low scores in the puzzle-box test solution, first applied to mouse experiments by Galsworthy et al. [20]. The puzzle-box experimental design requires an animal to understand that the object (an underpass, which the mouse is eager to use, as it leads into the safe box compartment) is hidden, being masked by wood shavings or a plug, but can be discovered [21]. The ability to solve the puzzle-box test requires that the animal apply the rule of "object permanence" (according to Piajet, see [22]). The animals in the initial population for this selection experiment were mice of the EX strain. This strain was selected earlier for high scores in the extrapolation task [23]. The correct extrapolation task solution is also based (at least partly) on an animal's ability to understand the rule of "object permanence". In the mental operation of "extrapolation", the animal has to find the new location of food bait on the basis of information perceived, although the food bait is no longer seen, as it has been moved away from view to the right or to the left of the animal, which perceives the food bait via a small opening at the base of the box's front wall.
In order to make the selection routine more feasible, we simplified the puzzle-box procedure so that the experimental testing could be performed over one day, in comparison to the longer test schedules used initially [20,21].
The selection criterion for the "plus" strain was short latency of test solutions at test stages when the underpass was masked by a plug, and the lack of these solutions (in an arbitrary time interval of 240 s) in the "plug" stages was the criterion for the strain selected as "minus". Previously published data [24] demonstrated success in this selection process for F1-F3. The behavior of mice from "plus" and "minus" strains was also compared in a hyponeophagia test [25]; the amount of new food eaten during the test time was persistently higher in "plus" mice. In the hyponeophagia test, the new food (small cubes of cheese) was presented to the hungry mouse in a new, but not frightening, environment. Animal behavior in this test is affected by both the necessity of handling the "novelty" (new food) and the "concurrent" anxiety reaction, which is aroused by the new environment (and the novelty of cheese as a food) and could obscure the reaction to this novelty. This test has been successfully used to evaluate the effects of antidepressants [26], and was earlier regarded as a test for anxiety [27].
Thus, the results of the five-generation selection for this cognitive trait in mice are presented below. The working memory indices, which could be drawn from the protocol of the puzzle-box solution, were also analyzed.
The present paper describes the results of an experiment in which mice were selected over five generations for high and low scores in the puzzle-box test solution, first applied to mouse experiments by Galsworthy et al. [20]. The puzzle-box experimental design requires an animal to understand that the object (an underpass, which the mouse is eager to use, as it leads into the safe box compartment) is hidden, being masked by wood shavings or a plug, but can be discovered [21]. The ability to solve the puzzle-box test requires that the animal apply the rule of "object permanence" (according to Piajet, see [22]). The animals in the initial population for this selection experiment were mice of the EX strain. This strain was selected earlier for high scores in the extrapolation task [23]. The correct extrapolation task solution is also based (at least partly) on an animal's ability to understand the rule of "object permanence". In the mental operation of "extrapolation", the animal has to find the new location of food bait on the basis of information perceived, although the food bait is no longer seen, as it has been moved away from view to the right or to the left of the animal, which perceives the food bait via a small opening at the base of the box's front wall.
In order to make the selection routine more feasible, we simplified the puzzle-box procedure so that the experimental testing could be performed over one day, in comparison to the longer test schedules used initially [20,21].
The selection criterion for the "plus" strain was short latency of test solutions at test stages when the underpass was masked by a plug, and the lack of these solutions (in an arbitrary time interval of 240 s) in the "plug" stages was the criterion for the strain selected as "minus". Previously published data [24] demonstrated success in this selection process for F1-F3. The behavior of mice from "plus" and "minus" strains was also compared in a hyponeophagia test [25]; the amount of new food eaten during the test time was persistently higher in "plus" mice. In the hyponeophagia test, the new food (small cubes of cheese) was presented to the hungry mouse in a new, but not frightening, environment. Animal behavior in this test is affected by both the necessity of handling the "novelty" (new food) and the "concurrent" anxiety reaction, which is aroused by the new environment (and the novelty of cheese as a food) and could obscure the reaction to this novelty. This test has been successfully used to evaluate the effects of antidepressants [26], and was earlier regarded as a test for anxiety [27].
Thus, the results of the five-generation selection for this cognitive trait in mice are presented below. The working memory indices, which could be drawn from the protocol of the puzzle-box solution, were also analyzed.
During test stage 1 of the test, this underpass stayed unobstructed, and the animal could freely enter the dark. At test stage 2, the underpass was masked (covered up to the floor level by fresh wood shavings). At stages 3 and 4, the underpass was blocked by means of a plug (made from carton and plastic), which animals could easily remove by using their teeth, or move aside using a muzzle and paws. The animal was given 180 s to solve stages 1 and 2, whereas for stages 3 and 4 (which required more effort), it was Puzzle-box test. An animal was placed into the brightly lit part of the experimental box, from which it could easily go into the dark part of the box, avoiding the light (see Figure 1). The underpass leading to the dark part of the box was submerged below the floor level and the animal could use it to easily penetrate into a more comfortable, dark compartment.
During test stage 1 of the test, this underpass stayed unobstructed, and the animal could freely enter the dark. At test stage 2, the underpass was masked (covered up to the floor level by fresh wood shavings). At stages 3 and 4, the underpass was blocked by means of a plug (made from carton and plastic), which animals could easily remove by using their teeth, or move aside using a muzzle and paws. The animal was given 180 s to solve stages 1 and 2, whereas for stages 3 and 4 (which required more effort), it was , F3 ("+") 41 subject has no analogous previous experience, as in case of any learning paradigm. So it is not the learned response per se to certain environmental signals. The solution of such a task requires "understanding" the elementary logic of the used paradigm. According to Krushinsky's definition [19], the animal's "cognitive ability" (or elementary reasoning) is the ability to "grasp" the empirical laws which connect objects and events in the external world and to develop further adaptive behavioral reactions using such information.
The present paper describes the results of an experiment in which mice were selected over five generations for high and low scores in the puzzle-box test solution, first applied to mouse experiments by Galsworthy et al. [20]. The puzzle-box experimental design requires an animal to understand that the object (an underpass, which the mouse is eager to use, as it leads into the safe box compartment) is hidden, being masked by wood shavings or a plug, but can be discovered [21]. The ability to solve the puzzle-box test requires that the animal apply the rule of "object permanence" (according to Piajet, see [22]). The animals in the initial population for this selection experiment were mice of the EX strain. This strain was selected earlier for high scores in the extrapolation task [23]. The correct extrapolation task solution is also based (at least partly) on an animal's ability to understand the rule of "object permanence". In the mental operation of "extrapolation", the animal has to find the new location of food bait on the basis of information perceived, although the food bait is no longer seen, as it has been moved away from view to the right or to the left of the animal, which perceives the food bait via a small opening at the base of the box's front wall.
In order to make the selection routine more feasible, we simplified the puzzle-box procedure so that the experimental testing could be performed over one day, in comparison to the longer test schedules used initially [20,21].
The selection criterion for the "plus" strain was short latency of test solutions at test stages when the underpass was masked by a plug, and the lack of these solutions (in an arbitrary time interval of 240 s) in the "plug" stages was the criterion for the strain selected as "minus". Previously published data [24] demonstrated success in this selection process for F1-F3. The behavior of mice from "plus" and "minus" strains was also compared in a hyponeophagia test [25]; the amount of new food eaten during the test time was persistently higher in "plus" mice. In the hyponeophagia test, the new food (small cubes of cheese) was presented to the hungry mouse in a new, but not frightening, environment. Animal behavior in this test is affected by both the necessity of handling the "novelty" (new food) and the "concurrent" anxiety reaction, which is aroused by the new environment (and the novelty of cheese as a food) and could obscure the reaction to this novelty. This test has been successfully used to evaluate the effects of antidepressants [26], and was earlier regarded as a test for anxiety [27].
Thus, the results of the five-generation selection for this cognitive trait in mice are presented below. The working memory indices, which could be drawn from the protocol of the puzzle-box solution, were also analyzed.
The present paper describes the results of an experiment in which mice were selected over five generations for high and low scores in the puzzle-box test solution, first applied to mouse experiments by Galsworthy et al. [20]. The puzzle-box experimental design requires an animal to understand that the object (an underpass, which the mouse is eager to use, as it leads into the safe box compartment) is hidden, being masked by wood shavings or a plug, but can be discovered [21]. The ability to solve the puzzle-box test requires that the animal apply the rule of "object permanence" (according to Piajet, see [22]). The animals in the initial population for this selection experiment were mice of the EX strain. This strain was selected earlier for high scores in the extrapolation task [23]. The correct extrapolation task solution is also based (at least partly) on an animal's ability to understand the rule of "object permanence". In the mental operation of "extrapolation", the animal has to find the new location of food bait on the basis of information perceived, although the food bait is no longer seen, as it has been moved away from view to the right or to the left of the animal, which perceives the food bait via a small opening at the base of the box's front wall.
In order to make the selection routine more feasible, we simplified the puzzle-box procedure so that the experimental testing could be performed over one day, in comparison to the longer test schedules used initially [20,21].
The selection criterion for the "plus" strain was short latency of test solutions at test stages when the underpass was masked by a plug, and the lack of these solutions (in an arbitrary time interval of 240 s) in the "plug" stages was the criterion for the strain selected as "minus". Previously published data [24] demonstrated success in this selection process for F1-F3. The behavior of mice from "plus" and "minus" strains was also compared in a hyponeophagia test [25]; the amount of new food eaten during the test time was persistently higher in "plus" mice. In the hyponeophagia test, the new food (small cubes of cheese) was presented to the hungry mouse in a new, but not frightening, environment. Animal behavior in this test is affected by both the necessity of handling the "novelty" (new food) and the "concurrent" anxiety reaction, which is aroused by the new environment (and the novelty of cheese as a food) and could obscure the reaction to this novelty. This test has been successfully used to evaluate the effects of antidepressants [26], and was earlier regarded as a test for anxiety [27].
Thus, the results of the five-generation selection for this cognitive trait in mice are presented below. The working memory indices, which could be drawn from the protocol of the puzzle-box solution, were also analyzed.
During test stage 1 of the test, this underpass stayed unobstructed, and the could freely enter the dark. At test stage 2, the underpass was masked (covered u floor level by fresh wood shavings). At stages 3 and 4, the underpass was bloc means of a plug (made from carton and plastic), which animals could easily rem using their teeth, or move aside using a muzzle and paws. The animal was given solve stages 1 and 2, whereas for stages 3 and 4 (which required more effort) Neurol Puzzle-box test. An animal was placed into the brightly lit part of the exp box, from which it could easily go into the dark part of the box, avoiding the Figure 1). The underpass leading to the dark part of the box was submerged b floor level and the animal could use it to easily penetrate into a more comforta compartment.
During test stage 1 of the test, this underpass stayed unobstructed, and th could freely enter the dark. At test stage 2, the underpass was masked (covered floor level by fresh wood shavings). At stages 3 and 4, the underpass was bl means of a plug (made from carton and plastic), which animals could easily re using their teeth, or move aside using a muzzle and paws. The animal was give solve stages 1 and 2, whereas for stages 3 and 4 (which required more effor , F3 ("−") 28 subject has no analogous previous experience, as in case of any learn is not the learned response per se to certain environmental signals. Th task requires "understanding" the elementary logic of the used para Krushinsky's definition [19], the animal's "cognitive ability" (or elem the ability to "grasp" the empirical laws which connect objects and ev world and to develop further adaptive behavioral reactions using suc The present paper describes the results of an experiment in w lected over five generations for high and low scores in the puzzle-bo applied to mouse experiments by Galsworthy et al. [20]. The puzzl design requires an animal to understand that the object (an underpas is eager to use, as it leads into the safe box compartment) is hidden wood shavings or a plug, but can be discovered [21]. The ability to s test requires that the animal apply the rule of "object permanence" ( see [22]). The animals in the initial population for this selection exper the EX strain. This strain was selected earlier for high scores in the [23]. The correct extrapolation task solution is also based (at least pa ability to understand the rule of "object permanence". In the menta trapolation", the animal has to find the new location of food bait on mation perceived, although the food bait is no longer seen, as it has from view to the right or to the left of the animal, which perceives small opening at the base of the box's front wall.
In order to make the selection routine more feasible, we simpli procedure so that the experimental testing could be performed over o ison to the longer test schedules used initially [20,21].
The selection criterion for the "plus" strain was short latency of stages when the underpass was masked by a plug, and the lack of th arbitrary time interval of 240 s) in the "plug" stages was the criterio lected as "minus". Previously published data [24] demonstrated succ process for F1-F3. The behavior of mice from "plus" and "minus" str pared in a hyponeophagia test [25]; the amount of new food eaten d was persistently higher in "plus" mice. In the hyponeophagia test, t cubes of cheese) was presented to the hungry mouse in a new, but vironment. Animal behavior in this test is affected by both the neces "novelty" (new food) and the "concurrent" anxiety reaction, which new environment (and the novelty of cheese as a food) and could obs this novelty. This test has been successfully used to evaluate the ef sants [26], and was earlier regarded as a test for anxiety [27].
Thus, the results of the five-generation selection for this cogniti presented below. The working memory indices, which could be dra col of the puzzle-box solution, were also analyzed.

Material and Methods
Experimental animals. Mice were bred selectively starting from previously selected for high scores in the extrapolation test (see a born in each generation (males and females), were tested with a sim test (for details, see below). Animal numbers (for "+" and "-" strains as follows: F1 ("+") 31 ♂♂, 29 ♀♀, F1 ("-") 22 ♂♂,17 ♀♀, F2 ("+" F2 ("-") 22♂♂, 27 ♀♀, F3 ("+") 41 ♂♂, 42 ♀♀, F3 ("-") 28♂♂, 1 ♂♂, 33 ♀♀, F4 ("-") 26♂♂, 18 ♀♀, F5 ("+") 27 ♂♂, 39 ♀♀, F ♀♀. Some animals in these animal groups from each generation hyponeophagia test (the limited number of animals in this test w problems). Mice in the control, non-selected, heterogeneous popul spective generations were also tested in parallel with F4 and F5 of th subject has no analogous previous experience, as in case of any lea is not the learned response per se to certain environmental signals. task requires "understanding" the elementary logic of the used pa Krushinsky's definition [19], the animal's "cognitive ability" (or ele the ability to "grasp" the empirical laws which connect objects and world and to develop further adaptive behavioral reactions using s The present paper describes the results of an experiment in lected over five generations for high and low scores in the puzzleapplied to mouse experiments by Galsworthy et al. [20]. The pu design requires an animal to understand that the object (an underp is eager to use, as it leads into the safe box compartment) is hidd wood shavings or a plug, but can be discovered [21]. The ability to test requires that the animal apply the rule of "object permanence see [22]). The animals in the initial population for this selection exp the EX strain. This strain was selected earlier for high scores in [23]. The correct extrapolation task solution is also based (at least ability to understand the rule of "object permanence". In the me trapolation", the animal has to find the new location of food bait mation perceived, although the food bait is no longer seen, as it h from view to the right or to the left of the animal, which perceiv small opening at the base of the box's front wall.
In order to make the selection routine more feasible, we sim procedure so that the experimental testing could be performed ove ison to the longer test schedules used initially [20,21].
The selection criterion for the "plus" strain was short latency stages when the underpass was masked by a plug, and the lack of arbitrary time interval of 240 s) in the "plug" stages was the crite lected as "minus". Previously published data [24] demonstrated su process for F1-F3. The behavior of mice from "plus" and "minus" pared in a hyponeophagia test [25]; the amount of new food eate was persistently higher in "plus" mice. In the hyponeophagia tes cubes of cheese) was presented to the hungry mouse in a new, b vironment. Animal behavior in this test is affected by both the nec "novelty" (new food) and the "concurrent" anxiety reaction, wh new environment (and the novelty of cheese as a food) and could o this novelty. This test has been successfully used to evaluate the sants [26], and was earlier regarded as a test for anxiety [27].
Thus, the results of the five-generation selection for this cogn presented below. The working memory indices, which could be d col of the puzzle-box solution, were also analyzed.

Material and Methods
Experimental animals. Mice were bred selectively starting f previously selected for high scores in the extrapolation test (see born in each generation (males and females), were tested with a test (for details, see below). Animal numbers (for "+" and "-" stra as follows: Puzzle-box test. An animal was placed into the bri box, from which it could easily go into the dark part o Figure 1). The underpass leading to the dark part of th floor level and the animal could use it to easily penetra compartment.
During test stage 1 of the test, this underpass stay could freely enter the dark. At test stage 2, the underpas floor level by fresh wood shavings). At stages 3 and 4 means of a plug (made from carton and plastic), which using their teeth, or move aside using a muzzle and paw solve stages 1 and 2, whereas for stages 3 and 4 (whi Puzzle-box test. An animal was placed into the b box, from which it could easily go into the dark part Figure 1). The underpass leading to the dark part of floor level and the animal could use it to easily penet compartment.
During test stage 1 of the test, this underpass sta could freely enter the dark. At test stage 2, the underp floor level by fresh wood shavings). At stages 3 and means of a plug (made from carton and plastic), whic using their teeth, or move aside using a muzzle and pa solve stages 1 and 2, whereas for stages 3 and 4 (w , F4 ("+"), 39 subject has no analogous previous experien is not the learned response per se to certain e task requires "understanding" the elementa Krushinsky's definition [19], the animal's "c the ability to "grasp" the empirical laws wh world and to develop further adaptive beha The present paper describes the resul lected over five generations for high and lo applied to mouse experiments by Galswor design requires an animal to understand tha is eager to use, as it leads into the safe box wood shavings or a plug, but can be discov test requires that the animal apply the rule see [22]). The animals in the initial populati the EX strain. This strain was selected earl [23]. The correct extrapolation task solution ability to understand the rule of "object pe trapolation", the animal has to find the new mation perceived, although the food bait is from view to the right or to the left of the small opening at the base of the box's front w In order to make the selection routine procedure so that the experimental testing c ison to the longer test schedules used initial The selection criterion for the "plus" st stages when the underpass was masked by arbitrary time interval of 240 s) in the "plu lected as "minus". Previously published da process for F1-F3. The behavior of mice from pared in a hyponeophagia test [25]; the am was persistently higher in "plus" mice. In t cubes of cheese) was presented to the hung vironment. Animal behavior in this test is a "novelty" (new food) and the "concurrent new environment (and the novelty of cheese this novelty. This test has been successfull sants [26], and was earlier regarded as a test Thus, the results of the five-generation presented below. The working memory ind col of the puzzle-box solution, were also ana

Material and Methods
Experimental animals. Mice were bred previously selected for high scores in the born in each generation (males and female test (for details, see below). Animal number as follows: F1 ("+") 31 ♂♂, 29 ♀♀, F1 ("-" F2 ("-") 22♂♂, 27 ♀♀, F3 ("+") 41 ♂♂, 4 ♂♂, 33 ♀♀, F4 ("-") 26♂♂, 18 ♀♀, F5 ♀♀. Some animals in these animal group hyponeophagia test (the limited number o problems). Mice in the control, non-selecte spective generations were also tested in par subject has no analogous previous exper is not the learned response per se to certai task requires "understanding" the eleme Krushinsky's definition [19], the animal's the ability to "grasp" the empirical laws w world and to develop further adaptive be The present paper describes the res lected over five generations for high and applied to mouse experiments by Galsw design requires an animal to understand is eager to use, as it leads into the safe wood shavings or a plug, but can be disc test requires that the animal apply the ru see [22]). The animals in the initial popul the EX strain. This strain was selected e [23]. The correct extrapolation task soluti ability to understand the rule of "object trapolation", the animal has to find the mation perceived, although the food bai from view to the right or to the left of t small opening at the base of the box's fro In order to make the selection routi procedure so that the experimental testin ison to the longer test schedules used init The selection criterion for the "plus" stages when the underpass was masked arbitrary time interval of 240 s) in the "p lected as "minus". Previously published process for F1-F3. The behavior of mice f pared in a hyponeophagia test [25]; the was persistently higher in "plus" mice. I cubes of cheese) was presented to the hu vironment. Animal behavior in this test i "novelty" (new food) and the "concurre new environment (and the novelty of che this novelty. This test has been successf sants [26], and was earlier regarded as a t Thus, the results of the five-generat presented below. The working memory col of the puzzle-box solution, were also
During test stage 1 of the could freely enter the dark. At floor level by fresh wood sha means of a plug (made from c using their teeth, or move asid solve stages 1 and 2, whereas Puzzle-box test. An anim box, from which it could eas Figure 1). The underpass lea floor level and the animal co compartment.
During test stage 1 of th could freely enter the dark. A floor level by fresh wood sh means of a plug (made from using their teeth, or move as solve stages 1 and 2, where subject has no analogous previous experience, as in case of any learning paradigm. So it is not the learned response per se to certain environmental signals. The solution of such a task requires "understanding" the elementary logic of the used paradigm. According to Krushinsky's definition [19], the animal's "cognitive ability" (or elementary reasoning) is the ability to "grasp" the empirical laws which connect objects and events in the external world and to develop further adaptive behavioral reactions using such information. The present paper describes the results of an experiment in which mice were selected over five generations for high and low scores in the puzzle-box test solution, first applied to mouse experiments by Galsworthy et al. [20]. The puzzle-box experimental design requires an animal to understand that the object (an underpass, which the mouse is eager to use, as it leads into the safe box compartment) is hidden, being masked by wood shavings or a plug, but can be discovered [21]. The ability to solve the puzzle-box test requires that the animal apply the rule of "object permanence" (according to Piajet, see [22]). The animals in the initial population for this selection experiment were mice of the EX strain. This strain was selected earlier for high scores in the extrapolation task [23]. The correct extrapolation task solution is also based (at least partly) on an animal's ability to understand the rule of "object permanence". In the mental operation of "extrapolation", the animal has to find the new location of food bait on the basis of information perceived, although the food bait is no longer seen, as it has been moved away from view to the right or to the left of the animal, which perceives the food bait via a small opening at the base of the box's front wall.
In order to make the selection routine more feasible, we simplified the puzzle-box procedure so that the experimental testing could be performed over one day, in comparison to the longer test schedules used initially [20,21].
The selection criterion for the "plus" strain was short latency of test solutions at test stages when the underpass was masked by a plug, and the lack of these solutions (in an arbitrary time interval of 240 s) in the "plug" stages was the criterion for the strain selected as "minus". Previously published data [24] demonstrated success in this selection process for F1-F3. The behavior of mice from "plus" and "minus" strains was also compared in a hyponeophagia test [25]; the amount of new food eaten during the test time was persistently higher in "plus" mice. In the hyponeophagia test, the new food (small cubes of cheese) was presented to the hungry mouse in a new, but not frightening, environment. Animal behavior in this test is affected by both the necessity of handling the "novelty" (new food) and the "concurrent" anxiety reaction, which is aroused by the new environment (and the novelty of cheese as a food) and could obscure the reaction to this novelty. This test has been successfully used to evaluate the effects of antidepressants [26], and was earlier regarded as a test for anxiety [27].
Thus, the results of the five-generation selection for this cognitive trait in mice are presented below. The working memory indices, which could be drawn from the protocol of the puzzle-box solution, were also analyzed.

Material and Methods
Experimental animals. Mice were bred selectively starting from F20 of the strain, previously selected for high scores in the extrapolation test (see above). All animals, born in each generation (males and females), were tested with a simplified puzzle-box test (for details, see below). Animal numbers (for "+" and "-" strains, respectively) were as follows: subject has no analogous previous experience, as in case of any learning paradigm. So it is not the learned response per se to certain environmental signals. The solution of such a task requires "understanding" the elementary logic of the used paradigm. According to Krushinsky's definition [19], the animal's "cognitive ability" (or elementary reasoning) is the ability to "grasp" the empirical laws which connect objects and events in the external world and to develop further adaptive behavioral reactions using such information. The present paper describes the results of an experiment in which mice were selected over five generations for high and low scores in the puzzle-box test solution, first applied to mouse experiments by Galsworthy et al. [20]. The puzzle-box experimental design requires an animal to understand that the object (an underpass, which the mouse is eager to use, as it leads into the safe box compartment) is hidden, being masked by wood shavings or a plug, but can be discovered [21]. The ability to solve the puzzle-box test requires that the animal apply the rule of "object permanence" (according to Piajet, see [22]). The animals in the initial population for this selection experiment were mice of the EX strain. This strain was selected earlier for high scores in the extrapolation task [23]. The correct extrapolation task solution is also based (at least partly) on an animal's ability to understand the rule of "object permanence". In the mental operation of "extrapolation", the animal has to find the new location of food bait on the basis of information perceived, although the food bait is no longer seen, as it has been moved away from view to the right or to the left of the animal, which perceives the food bait via a small opening at the base of the box's front wall.
In order to make the selection routine more feasible, we simplified the puzzle-box procedure so that the experimental testing could be performed over one day, in comparison to the longer test schedules used initially [20,21].
The selection criterion for the "plus" strain was short latency of test solutions at test stages when the underpass was masked by a plug, and the lack of these solutions (in an arbitrary time interval of 240 s) in the "plug" stages was the criterion for the strain selected as "minus". Previously published data [24] demonstrated success in this selection process for F1-F3. The behavior of mice from "plus" and "minus" strains was also compared in a hyponeophagia test [25]; the amount of new food eaten during the test time was persistently higher in "plus" mice. In the hyponeophagia test, the new food (small cubes of cheese) was presented to the hungry mouse in a new, but not frightening, environment. Animal behavior in this test is affected by both the necessity of handling the "novelty" (new food) and the "concurrent" anxiety reaction, which is aroused by the new environment (and the novelty of cheese as a food) and could obscure the reaction to this novelty. This test has been successfully used to evaluate the effects of antidepressants [26], and was earlier regarded as a test for anxiety [27].
Thus, the results of the five-generation selection for this cognitive trait in mice are presented below. The working memory indices, which could be drawn from the protocol of the puzzle-box solution, were also analyzed.
During test stage 1 of the test, this underpass stayed unobstructed, and the animal could freely enter the dark. At test stage 2, the underpass was masked (covered up to the floor level by fresh wood shavings). At stages 3 and 4, the underpass was blocked by means of a plug (made from carton and plastic), which animals could easily remove by using their teeth, or move aside using a muzzle and paws. The animal was given 180 s to Puzzle-box test. An animal was placed into the brightly lit part of the experimental box, from which it could easily go into the dark part of the box, avoiding the light (see Figure 1). The underpass leading to the dark part of the box was submerged below the floor level and the animal could use it to easily penetrate into a more comfortable, dark compartment.
During test stage 1 of the test, this underpass stayed unobstructed, and the animal could freely enter the dark. At test stage 2, the underpass was masked (covered up to the floor level by fresh wood shavings). At stages 3 and 4, the underpass was blocked by means of a plug (made from carton and plastic), which animals could easily remove by using their teeth, or move aside using a muzzle and paws. The animal was given 180 s to , F5 ("+") 27 Neurol. Int. 2022, 14, FOR PEER REVIEW subject has no analogous previous experience, as in case of any learning paradigm. is not the learned response per se to certain environmental signals. The solution of s task requires "understanding" the elementary logic of the used paradigm. Accordi Krushinsky's definition [19], the animal's "cognitive ability" (or elementary reasoni the ability to "grasp" the empirical laws which connect objects and events in the ext world and to develop further adaptive behavioral reactions using such information. The present paper describes the results of an experiment in which mice wer lected over five generations for high and low scores in the puzzle-box test solution applied to mouse experiments by Galsworthy et al. [20]. The puzzle-box experim design requires an animal to understand that the object (an underpass, which the m is eager to use, as it leads into the safe box compartment) is hidden, being maske wood shavings or a plug, but can be discovered [21]. The ability to solve the puzzl test requires that the animal apply the rule of "object permanence" (according to P see [22]). The animals in the initial population for this selection experiment were m the EX strain. This strain was selected earlier for high scores in the extrapolation [23]. The correct extrapolation task solution is also based (at least partly) on an ani ability to understand the rule of "object permanence". In the mental operation of trapolation", the animal has to find the new location of food bait on the basis of i mation perceived, although the food bait is no longer seen, as it has been moved from view to the right or to the left of the animal, which perceives the food bait small opening at the base of the box's front wall.
In order to make the selection routine more feasible, we simplified the puzzl procedure so that the experimental testing could be performed over one day, in com ison to the longer test schedules used initially [20,21].
The selection criterion for the "plus" strain was short latency of test solutions a stages when the underpass was masked by a plug, and the lack of these solutions ( arbitrary time interval of 240 s) in the "plug" stages was the criterion for the strai lected as "minus". Previously published data [24] demonstrated success in this sele process for F1-F3. The behavior of mice from "plus" and "minus" strains was also pared in a hyponeophagia test [25]; the amount of new food eaten during the test was persistently higher in "plus" mice. In the hyponeophagia test, the new food ( cubes of cheese) was presented to the hungry mouse in a new, but not frightening vironment. Animal behavior in this test is affected by both the necessity of handlin "novelty" (new food) and the "concurrent" anxiety reaction, which is aroused b new environment (and the novelty of cheese as a food) and could obscure the reacti this novelty. This test has been successfully used to evaluate the effects of antide sants [26], and was earlier regarded as a test for anxiety [27].
Thus, the results of the five-generation selection for this cognitive trait in mic presented below. The working memory indices, which could be drawn from the p col of the puzzle-box solution, were also analyzed.

Material and Methods
Experimental animals. Mice were bred selectively starting from F20 of the s previously selected for high scores in the extrapolation test (see above). All ani born in each generation (males and females), were tested with a simplified puzzl test (for details, see below). Animal numbers (for "+" and "-" strains, respectively) as follows: F1 ("+") 31 ♂♂, 29 ♀♀, F1 ("-") 22 ♂♂,17 ♀♀, F2 ("+"), 28 ♂♂, 30 F2 ("-") 22♂♂, 27 ♀♀, F3 ("+") 41 ♂♂, 42 ♀♀, F3 ("-") 28♂♂, 18 ♀♀, F4 ("+ ♂♂, 33 ♀♀, F4 ("-") 26♂♂, 18 ♀♀, F5 ("+") 27 ♂♂, 39 ♀♀, F5 ("-") 44 ♂♂ ♀♀. Some animals in these animal groups from each generation were tested i hyponeophagia test (the limited number of animals in this test was due to tech problems). Mice in the control, non-selected, heterogeneous populations from th spective generations were also tested in parallel with F4 and F5 of the selected strai Neurol. Int. 2022, 14, FOR PEER REVIEW subject has no analogous previous experience, as in case of any learning paradig is not the learned response per se to certain environmental signals. The solution o task requires "understanding" the elementary logic of the used paradigm. Acco Krushinsky's definition [19], the animal's "cognitive ability" (or elementary reaso the ability to "grasp" the empirical laws which connect objects and events in the world and to develop further adaptive behavioral reactions using such informati The present paper describes the results of an experiment in which mice w lected over five generations for high and low scores in the puzzle-box test soluti applied to mouse experiments by Galsworthy et al. [20]. The puzzle-box exper design requires an animal to understand that the object (an underpass, which the is eager to use, as it leads into the safe box compartment) is hidden, being ma wood shavings or a plug, but can be discovered [21]. The ability to solve the puz test requires that the animal apply the rule of "object permanence" (according t see [22]). The animals in the initial population for this selection experiment were the EX strain. This strain was selected earlier for high scores in the extrapolat [23]. The correct extrapolation task solution is also based (at least partly) on an a ability to understand the rule of "object permanence". In the mental operation trapolation", the animal has to find the new location of food bait on the basis o mation perceived, although the food bait is no longer seen, as it has been move from view to the right or to the left of the animal, which perceives the food ba small opening at the base of the box's front wall.
In order to make the selection routine more feasible, we simplified the puz procedure so that the experimental testing could be performed over one day, in c ison to the longer test schedules used initially [20,21].
The selection criterion for the "plus" strain was short latency of test solution stages when the underpass was masked by a plug, and the lack of these solution arbitrary time interval of 240 s) in the "plug" stages was the criterion for the st lected as "minus". Previously published data [24] demonstrated success in this s process for F1-F3. The behavior of mice from "plus" and "minus" strains was al pared in a hyponeophagia test [25]; the amount of new food eaten during the t was persistently higher in "plus" mice. In the hyponeophagia test, the new foo cubes of cheese) was presented to the hungry mouse in a new, but not frighten vironment. Animal behavior in this test is affected by both the necessity of hand "novelty" (new food) and the "concurrent" anxiety reaction, which is aroused new environment (and the novelty of cheese as a food) and could obscure the rea this novelty. This test has been successfully used to evaluate the effects of ant sants [26], and was earlier regarded as a test for anxiety [27].
Thus, the results of the five-generation selection for this cognitive trait in m presented below. The working memory indices, which could be drawn from th col of the puzzle-box solution, were also analyzed.
Statement on the welfare of animals. The experimental protoco the Bioethical Commission of Moscow State University, session no. 49 Puzzle-box test. An animal was placed into the brightly lit part o box, from which it could easily go into the dark part of the box, avoi Figure 1). The underpass leading to the dark part of the box was sub floor level and the animal could use it to easily penetrate into a more compartment.
During test stage 1 of the test, this underpass stayed unobstructe could freely enter the dark. At test stage 2, the underpass was masked floor level by fresh wood shavings). At stages 3 and 4, the underpa means of a plug (made from carton and plastic), which animals could using their teeth, or move aside using a muzzle and paws. The animal Puzzle-box test. An animal was placed into the brightly lit part box, from which it could easily go into the dark part of the box, av Figure 1). The underpass leading to the dark part of the box was su floor level and the animal could use it to easily penetrate into a mo compartment.
During test stage 1 of the test, this underpass stayed unobstruc could freely enter the dark. At test stage 2, the underpass was maske floor level by fresh wood shavings). At stages 3 and 4, the underp means of a plug (made from carton and plastic), which animals cou using their teeth, or move aside using a muzzle and paws. The anim , F5 ("−") 44 Neurol. Int. 2022, 14, FOR PEER REVIEW subject has no analogous previous experience, as in case o is not the learned response per se to certain environmental task requires "understanding" the elementary logic of the Krushinsky's definition [19], the animal's "cognitive ability the ability to "grasp" the empirical laws which connect obj world and to develop further adaptive behavioral reaction The present paper describes the results of an experi lected over five generations for high and low scores in the applied to mouse experiments by Galsworthy et al. [20]. design requires an animal to understand that the object (an is eager to use, as it leads into the safe box compartment wood shavings or a plug, but can be discovered [21]. The test requires that the animal apply the rule of "object perm see [22]). The animals in the initial population for this sele the EX strain. This strain was selected earlier for high sc [23]. The correct extrapolation task solution is also based ( ability to understand the rule of "object permanence". In trapolation", the animal has to find the new location of f mation perceived, although the food bait is no longer see from view to the right or to the left of the animal, which small opening at the base of the box's front wall.
In order to make the selection routine more feasible, procedure so that the experimental testing could be perfor ison to the longer test schedules used initially [20,21].
The selection criterion for the "plus" strain was short stages when the underpass was masked by a plug, and th arbitrary time interval of 240 s) in the "plug" stages was lected as "minus". Previously published data [24] demons process for F1-F3. The behavior of mice from "plus" and " pared in a hyponeophagia test [25]; the amount of new fo was persistently higher in "plus" mice.
In the hyponeoph cubes of cheese) was presented to the hungry mouse in a vironment. Animal behavior in this test is affected by both "novelty" (new food) and the "concurrent" anxiety reac new environment (and the novelty of cheese as a food) and this novelty. This test has been successfully used to eval sants [26], and was earlier regarded as a test for anxiety [27 Thus, the results of the five-generation selection for presented below. The working memory indices, which co col of the puzzle-box solution, were also analyzed.

Material and Methods
Experimental animals. Mice were bred selectively st previously selected for high scores in the extrapolation born in each generation (males and females), were tested test (for details, see below). Animal numbers (for "+" and as follows: F1 ("+") 31 ♂♂, 29 ♀♀, F1 ("-") 22 ♂♂,17 ♀ F2 ("-") 22♂♂, 27 ♀♀, F3 ("+") 41 ♂♂, 42 ♀♀, F3 ("-" ♂♂, 33 ♀♀, F4 ("-") 26♂♂, 18 ♀♀, F5 ("+") 27 ♂♂ ♀♀. Some animals in these animal groups from each g hyponeophagia test (the limited number of animals in t problems). Mice in the control, non-selected, heterogene spective generations were also tested in parallel with F4 an Neurol. Int. 2022, 14, FOR PEER REVIEW subject has no analogous previous experience, as in cas is not the learned response per se to certain environmen task requires "understanding" the elementary logic of t Krushinsky's definition [19], the animal's "cognitive abi the ability to "grasp" the empirical laws which connect world and to develop further adaptive behavioral reacti The present paper describes the results of an exp lected over five generations for high and low scores in applied to mouse experiments by Galsworthy et al. [2 design requires an animal to understand that the object is eager to use, as it leads into the safe box compartm wood shavings or a plug, but can be discovered [21]. T test requires that the animal apply the rule of "object p see [22]). The animals in the initial population for this s the EX strain. This strain was selected earlier for high [23]. The correct extrapolation task solution is also base ability to understand the rule of "object permanence". trapolation", the animal has to find the new location o mation perceived, although the food bait is no longer s from view to the right or to the left of the animal, wh small opening at the base of the box's front wall.
In order to make the selection routine more feasib procedure so that the experimental testing could be per ison to the longer test schedules used initially [20,21].
The selection criterion for the "plus" strain was sho stages when the underpass was masked by a plug, and arbitrary time interval of 240 s) in the "plug" stages w lected as "minus". Previously published data [24] demo process for F1-F3. The behavior of mice from "plus" an pared in a hyponeophagia test [25]; the amount of new was persistently higher in "plus" mice.
In the hyponeo cubes of cheese) was presented to the hungry mouse i vironment. Animal behavior in this test is affected by b "novelty" (new food) and the "concurrent" anxiety re new environment (and the novelty of cheese as a food) this novelty. This test has been successfully used to ev sants [26], and was earlier regarded as a test for anxiety Thus, the results of the five-generation selection fo presented below. The working memory indices, which col of the puzzle-box solution, were also analyzed.
During test stage 1 of the test, this unde could freely enter the dark. At test stage 2, th floor level by fresh wood shavings). At stag means of a plug (made from carton and plas using their teeth, or move aside using a muzz Puzzle-box test. An animal was placed box, from which it could easily go into the Figure 1). The underpass leading to the da floor level and the animal could use it to e compartment.
During test stage 1 of the test, this un could freely enter the dark. At test stage 2, t floor level by fresh wood shavings). At st means of a plug (made from carton and pl using their teeth, or move aside using a mu . Some animals in these animal groups from each generation were tested in the hyponeophagia test (the limited number of animals in this test was due to technical problems). Mice in the control, non-selected, heterogeneous populations from the respective generations were also tested in parallel with F4 and F5 of the selected strains (n = 97, 63 Neurol. Int. 2022, 14, FOR PEER REVIEW subject has no analogous previous experience, as in case of a is not the learned response per se to certain environmental si task requires "understanding" the elementary logic of the u Krushinsky's definition [19], the animal's "cognitive ability" the ability to "grasp" the empirical laws which connect objec world and to develop further adaptive behavioral reactions u The present paper describes the results of an experim lected over five generations for high and low scores in the p applied to mouse experiments by Galsworthy et al. [20]. T design requires an animal to understand that the object (an u is eager to use, as it leads into the safe box compartment) wood shavings or a plug, but can be discovered [21]. The ab test requires that the animal apply the rule of "object perma see [22]). The animals in the initial population for this selecti the EX strain. This strain was selected earlier for high scor [23]. The correct extrapolation task solution is also based (at ability to understand the rule of "object permanence". In t trapolation", the animal has to find the new location of foo mation perceived, although the food bait is no longer seen, from view to the right or to the left of the animal, which p small opening at the base of the box's front wall.
In order to make the selection routine more feasible, w procedure so that the experimental testing could be perform ison to the longer test schedules used initially [20,21].
The selection criterion for the "plus" strain was short la stages when the underpass was masked by a plug, and the l arbitrary time interval of 240 s) in the "plug" stages was th lected as "minus". Previously published data [24] demonstra process for F1-F3. The behavior of mice from "plus" and "m pared in a hyponeophagia test [25]; the amount of new foo was persistently higher in "plus" mice.
In the hyponeophag cubes of cheese) was presented to the hungry mouse in a n vironment. Animal behavior in this test is affected by both t "novelty" (new food) and the "concurrent" anxiety reactio new environment (and the novelty of cheese as a food) and c this novelty. This test has been successfully used to evalua sants [26], and was earlier regarded as a test for anxiety [27].
Thus, the results of the five-generation selection for th presented below. The working memory indices, which coul col of the puzzle-box solution, were also analyzed.

Material and Methods
Experimental animals. Mice were bred selectively star previously selected for high scores in the extrapolation te born in each generation (males and females), were tested w test (for details, see below). Animal numbers (for "+" and "as follows: F1 ("+") 31 ♂♂, 29 ♀♀, F1 ("-") 22 ♂♂,17 ♀♀ F2 ("-") 22♂♂, 27 ♀♀, F3 ("+") 41 ♂♂, 42 ♀♀, F3 ("-") 2 ♂♂, 33 ♀♀, F4 ("-") 26♂♂, 18 ♀♀, F5 ("+") 27 ♂♂, 3 ♀♀. Some animals in these animal groups from each ge hyponeophagia test (the limited number of animals in thi problems). Mice in the control, non-selected, heterogeneou spective generations were also tested in parallel with F4 and Neurol. Int. 2022, 14, FOR PEER REVIEW subject has no analogous previous experience, as in case o is not the learned response per se to certain environmental task requires "understanding" the elementary logic of the Krushinsky's definition [19], the animal's "cognitive ability the ability to "grasp" the empirical laws which connect ob world and to develop further adaptive behavioral reaction The present paper describes the results of an experi lected over five generations for high and low scores in the applied to mouse experiments by Galsworthy et al. [20]. design requires an animal to understand that the object (a is eager to use, as it leads into the safe box compartmen wood shavings or a plug, but can be discovered [21]. The test requires that the animal apply the rule of "object perm see [22]). The animals in the initial population for this sele the EX strain. This strain was selected earlier for high sc [23]. The correct extrapolation task solution is also based ability to understand the rule of "object permanence". In trapolation", the animal has to find the new location of f mation perceived, although the food bait is no longer see from view to the right or to the left of the animal, which small opening at the base of the box's front wall.
In order to make the selection routine more feasible, procedure so that the experimental testing could be perfor ison to the longer test schedules used initially [20,21].
The selection criterion for the "plus" strain was short stages when the underpass was masked by a plug, and th arbitrary time interval of 240 s) in the "plug" stages was lected as "minus". Previously published data [24] demons process for F1-F3. The behavior of mice from "plus" and " pared in a hyponeophagia test [25]; the amount of new fo was persistently higher in "plus" mice.
In the hyponeoph cubes of cheese) was presented to the hungry mouse in a vironment. Animal behavior in this test is affected by both "novelty" (new food) and the "concurrent" anxiety reac new environment (and the novelty of cheese as a food) and this novelty. This test has been successfully used to eval sants [26], and was earlier regarded as a test for anxiety [27 Thus, the results of the five-generation selection for presented below. The working memory indices, which co col of the puzzle-box solution, were also analyzed.
During test stage 1 of the test, this underp Puzzle-box test. An animal was placed in box, from which it could easily go into the d Figure 1). The underpass leading to the dark floor level and the animal could use it to easi compartment.
During test stage 1 of the test, this unde , in total), though F1-F3 control animals were not tested due to a technical problem.
Mice were housed in plastic cages (size 35 × 56 × 20 cm) with food (Firm Laboratorkorm) and water ad libitum with a natural light-dark schedule.
Statement on the welfare of animals. The experimental protocol was accepted by the Bioethical Commission of Moscow State University, session no. 49 of 18 June 2014.
Puzzle-box test. An animal was placed into the brightly lit part of the experimental box, from which it could easily go into the dark part of the box, avoiding the light (see Figure 1). The underpass leading to the dark part of the box was submerged below the floor level and the animal could use it to easily penetrate into a more comfortable, dark compartment. using their teeth, or move aside using a muzzle and paws. The animal was given 180 s to solve stages 1 and 2, whereas for stages 3 and 4 (which required more effort), it was given 240 s. After animal entered the dark part of the box, it was left there for 15-20 s, and then placed in a separate clean cage for 45-60 s before the next stage of the test initiated. The latencies of animal reaction (when entering into the dark part of the box) were registered manually. At stages 3 and 4 (i.e., stages with the plug), the animal movements to remove the plug ("manipulations", i.e., the attempts to enter the dark by seizing the plug with their teeth, as well as the attempts to raise it) were also registered. In cases when the animal failed to solve the plug stage (for 240 s), the presence or absence of this type of manipulation was considered to be an important index for evaluation of the interstrain behavioral differences. The proportions of mice from the given group which were able to solve stages 3 and 4 of the puzzle-box test were registered as well. Hyponeophagia test. Animals were food (but not water)-deprived for 10-12 h. The mouse was placed in the dimly lit round arena (diam. 40 cm) surrounded by a plastic wall (height 35 cm), and a small cup with pieces of cheese was placed in the center of this arena. During 5 min of testing, the latency of the first approach to the cheese and the number of approaches and retreats were manually registered, and the amount of cheese consumed during the test was determined. During test stage 1 of the test, this underpass stayed unobstructed, and the animal could freely enter the dark. At test stage 2, the underpass was masked (covered up to the floor level by fresh wood shavings). At stages 3 and 4, the underpass was blocked by means of a plug (made from carton and plastic), which animals could easily remove by using their teeth, or move aside using a muzzle and paws. The animal was given 180 s to solve stages 1 and 2, whereas for stages 3 and 4 (which required more effort), it was given 240 s. After animal entered the dark part of the box, it was left there for 15-20 s, and then placed in a separate clean cage for 45-60 s before the next stage of the test initiated. The latencies of animal reaction (when entering into the dark part of the box) were registered manually. At stages 3 and 4 (i.e., stages with the plug), the animal movements to remove the plug ("manipulations", i.e., the attempts to enter the dark by seizing the plug with their teeth, as well as the attempts to raise it) were also registered. In cases when the animal failed to solve the plug stage (for 240 s), the presence or absence of this type of manipulation was considered to be an important index for evaluation of the interstrain behavioral differences. The proportions of mice from the given group which were able to solve stages 3 and 4 of the puzzle-box test were registered as well.
Hyponeophagia test. Animals were food (but not water)-deprived for 10-12 h. The mouse was placed in the dimly lit round arena (diam. 40 cm) surrounded by a plastic wall (height 35 cm), and a small cup with pieces of cheese was placed in the center of this arena. During 5 min of testing, the latency of the first approach to the cheese and the number of approaches and retreats were manually registered, and the amount of cheese consumed during the test was determined.
Selection. After the completion of behavioral testing, "plus" and "minus" groups of male and female mice were chosen as parents for the next generation. The candidates for the "plus" strain needed to successfully solve stages 3 and 4 of the test with latencies not longer than 60-90 s. As practically all mice under study solved test stages 1 and 2 with rather short latencies, the criterion for selection in the "minus" strain was the lack of solutions of both the 3rd and 4th test stages. As the time to penetrate the dark part of the box during stages 1 and 2 was short in all animal groups, these data were not used as selection criteria, although the latencies in selected "plus" animals were shorter.
The future parents of the next generation (1 male and 1 or 2 females) were placed for mating in the cages of a smaller size (39 × 20.5 × 9 cm). The pregnant females were placed in the separate cages and stayed with their litters up to weaning at the age of 30-34 days. Pups were ear-marked and put into the larger cages (with approximately 6-8 animals per cage, males and females separately). The behavioral testing started not earlier than at 3-3.5 months of age, with the puzzle-box test being the first one, followed (for some animals) by the hyponeophagia test.
Statistic differences evaluation. The statistical significance of differences in latency of puzzle-box solutions and of hyponeophagia test scores was evaluated by means of 1and 2-factorial ANOVA (factors-strain and sex) with the post hoc Fisher LSD test. The differences in proportions of animals which solved the 3rd and 4th stages of the puzzle-box test were evaluated using the Fisher ϕ-test for alternative proportions difference.

Results
Puzzle-box test. The mean latencies of the puzzle-box test for stages 1 and 2 are presented in Figure 2. The histograms demonstrate that the mean time of entrance into the dark at these (more "simple") stages of the test were shorter in the "plus" group even in animals of the first selected generation in spite of the fact that parental groups ("P" in Figure 2), i.e., parents for the "+"and "−" selections, solved the test with similar mean latencies. As the puzzle-box stage with the unobstructed underpass is more or less similar to the procedure of the light-dark test, these differences (not large, but statistically significant) reveal that the selection process presumably also affected the expression of anxiety (although this issue was not yet analyzed in detail). During stage 2 of the test (with the underpass masked by the wood shavings), the performance of "plus" mice was quicker than that of "minus" animals. male and female mice were chosen as parents for the next generation. The candidates for the "plus" strain needed to successfully solve stages 3 and 4 of the test with latencies not longer than 60-90 s. As practically all mice under study solved test stages 1 and 2 with rather short latencies, the criterion for selection in the "minus" strain was the lack of solutions of both the 3rd and 4th test stages. As the time to penetrate the dark part of the box during stages 1 and 2 was short in all animal groups, these data were not used as selection criteria, although the latencies in selected "plus" animals were shorter.
The future parents of the next generation (1 male and 1 or 2 females) were placed for mating in the cages of a smaller size (39 × 20.5 × 9 cm). The pregnant females were placed in the separate cages and stayed with their litters up to weaning at the age of 30-34 days. Pups were ear-marked and put into the larger cages (with approximately 6-8 animals per cage, males and females separately). The behavioral testing started not earlier than at 3-3.5 months of age, with the puzzle-box test being the first one, followed (for some animals) by the hyponeophagia test.
Statistic differences evaluation. The statistical significance of differences in latency of puzzle-box solutions and of hyponeophagia test scores was evaluated by means of 1and 2-factorial ANOVA (factors-strain and sex) with the post hoc Fisher LSD test. The differences in proportions of animals which solved the 3rd and 4th stages of the puzzle-box test were evaluated using the Fisher φ-test for alternative proportions difference.

Results
Puzzle-box test. The mean latencies of the puzzle-box test for stages 1 and 2 are presented in Figure 2. The histograms demonstrate that the mean time of entrance into the dark at these (more "simple") stages of the test were shorter in the "plus" group even in animals of the first selected generation in spite of the fact that parental groups ("P" in Figure 2), i.e., parents for the "+"and "-" selections, solved the test with similar mean latencies. As the puzzle-box stage with the unobstructed underpass is more or less similar to the procedure of the light-dark test, these differences (not large, but statistically significant) reveal that the selection process presumably also affected the expression of anxiety (although this issue was not yet analyzed in detail). During stage 2 of the test (with the underpass masked by the wood shavings), the performance of "plus" mice was quicker than that of "minus" animals. Horizontal axis-groups of animals. P-animals of parental generation, i.e., summed performance of mice, chosen for further "plus" and "minus" selection as parents. F1-F5-selection generations. Light gray columns-mice of "plus" selection strain, dark gray columns-mice of "minus" selection strain. Black columns-mice of the control, non-selected, heterogeneous population. *, **,***-significantly different from the respective values for "minus" and control mice, p < 0.05, 0.01 and 0.001 respectively (post hoc Fisher LSD test, one-way ANO-VA). Horizontal axis-groups of animals. P-animals of parental generation, i.e., summed performance of mice, chosen for further "plus" and "minus" selection as parents. F1-F5-selection generations. Light gray columns-mice of "plus" selection strain, dark gray columns-mice of "minus" selection strain. Black columns-mice of the control, non-selected, heterogeneous population. *, **, ***-significantly different from the respective values for "minus" and control mice, p < 0.05, 0.01 and 0.001 respectively (post hoc Fisher LSD test, one-way ANOVA).
The mean latencies of "plus" and "minis" mice groups during performance at stages 3 and 4 of this test were significantly different, those of "plus" mice, being shorter (data not presented). The longer latencies of "minus" group mice reflected not only their slower reaction during the test but also the occurrence of cases with 240 s scores for "non-solutions". Thus, the more adequate evaluation of solution success at the "plug" stages of this test was the proportion of animals of each group that was able to solve the task when the underpass was blocked by a plug. These scores illustrate the resulting interstrain differences better ( Figure 3A). Figure 3B presents the summarized proportion scores (for both "plug" stages) for "plus" and "minus" groups that failed to solve the task. "non-solutions". Thus, the more adequate evaluation of solution success at the "plug" stages of this test was the proportion of animals of each group that was able to solve the task when the underpass was blocked by a plug. These scores illustrate the resulting interstrain differences better ( Figure 3A). Figure 3B presents the summarized proportion scores (for both "plug" stages) for "plus" and "minus" groups that failed to solve the task. (B) Summarized data for the proportions (%, ordinate) of animals in F1-F5 that failed to solve both "plug" stages. Designations as in Figure 2. *,**, ***-significant difference from "minus"-strain scores for both stages, p < 0.05, 0.01 and 0.001, respectively (Fisher φ-method for significance evaluation between alternative proportions).
Mice of both newly selected strains were active in the attempts to manipulate the plug, which blocked the underpass, even those animals which failed to remove the plug successfully. This fact indicates that they all were able to "grasp" the object permanence rule, but animals selected for "plus" solutions were able to bring these attempts to realization. One may suggest that animals of the "minus" group and controls were less adapted for the "plug" stage solution, being smaller and thus less "muscular". The data for all F5 mice show that this was not the case ( Figure 4). In "plus", "minus" and control groups, males were heavier than females (with a lack of differences in task solution success, data not presented), but the differences in body weight between the same-gender "plus", "minus" and control mice were absent. Thus, one may conclude that animals of the "plus" groups were actually significantly superior to "minus" mice in the task solution (i.e., incidences of plug removal). (B) Summarized data for the proportions (%, ordinate) of animals in F1-F5 that failed to solve both "plug" stages. Designations as in Figure 2. *, ***-significant difference from "minus"-strain scores for both stages, p < 0.05 and 0.001, respectively (Fisher ϕ-method for significance evaluation between alternative proportions).
Mice of both newly selected strains were active in the attempts to manipulate the plug, which blocked the underpass, even those animals which failed to remove the plug successfully. This fact indicates that they all were able to "grasp" the object permanence rule, but animals selected for "plus" solutions were able to bring these attempts to realization. One may suggest that animals of the "minus" group and controls were less adapted for the "plug" stage solution, being smaller and thus less "muscular". The data for all F5 mice show that this was not the case ( Figure 4). In "plus", "minus" and control groups, males were heavier than females (with a lack of differences in task solution success, data not presented), but the differences in body weight between the same-gender "plus", "minus" and control mice were absent. Thus, one may conclude that animals of the "plus" groups were actually significantly superior to "minus" mice in the task solution (i.e., incidences of plug removal).
"non-solutions". Thus, the more adequate evaluation of solution success at the "plug" stages of this test was the proportion of animals of each group that was able to solve the task when the underpass was blocked by a plug. These scores illustrate the resulting interstrain differences better ( Figure 3A). Figure 3B presents the summarized proportion scores (for both "plug" stages) for "plus" and "minus" groups that failed to solve the task. (B) Summarized data for the proportions (%, ordinate) of animals in F1-F5 that failed to solve both "plug" stages. Designations as in Figure 2. *,**, ***-significant difference from "minus"-strain scores for both stages, p < 0.05, 0.01 and 0.001, respectively (Fisher φ-method for significance evaluation between alternative proportions).
Mice of both newly selected strains were active in the attempts to manipulate the plug, which blocked the underpass, even those animals which failed to remove the plug successfully. This fact indicates that they all were able to "grasp" the object permanence rule, but animals selected for "plus" solutions were able to bring these attempts to realization. One may suggest that animals of the "minus" group and controls were less adapted for the "plug" stage solution, being smaller and thus less "muscular". The data for all F5 mice show that this was not the case ( Figure 4). In "plus", "minus" and control groups, males were heavier than females (with a lack of differences in task solution success, data not presented), but the differences in body weight between the same-gender "plus", "minus" and control mice were absent. Thus, one may conclude that animals of the "plus" groups were actually significantly superior to "minus" mice in the task solution (i.e., incidences of plug removal). The comparison of individual solution latencies at stages 3 and 4 ( Figure 5) shows that the shortening of stage 4 latencies (from those of stage 3) was more frequent in the "plus" than in the "minus" strain. These data (preliminary, as they only noted the fact of shorter latency, but not the scope of such differences) could not be regarded as a specific test for working memory, but still they demonstrate the interstrain differences in the effect of previous experience-the quicker realization of a solution by "plus" mice during the second "plug" presentation.
The comparison of individual solution latencies at stages 3 and 4 ( Figure 5) shows that the shortening of stage 4 latencies (from those of stage 3) was more frequent in the "plus" than in the "minus" strain. These data (preliminary, as they only noted the fact of shorter latency, but not the scope of such differences) could not be regarded as a specific test for working memory, but still they demonstrate the interstrain differences in the effect of previous experience-the quicker realization of a solution by "plus" mice during the second "plug" presentation. The intergenerational comparison is worth mentioning. In F5, the proportion of mice that were able to solve the first "plug" stage (stage 3 of the puzzle-box test) in the "plus" strain was significantly higher than in F1 (t = 2.89, p < 0.01, φ test), and the opposite tendency was noted for the respective values in the "minus" strain from F1 to F5, with a decrease in the percentage of animals that solved stage 3 of the test (t = 2.07, p < 0.05, φ test).
Hyponeophagia test. Among several behavioral indices registered in this test, the amount of food eaten during 5 min of this test revealed the stable differences along selection generations ( Figure 6). Figure 5. Light gray columns-mice of "plus" selection strain, dark gray columns-mice of "minus" selection strain. Proportions (%, ordinate) of animals in the successive selection generations, which performed stage 4 with shorter latencies than stage 3. *, ***-significant differences between plus and minus strains (Fisher ϕ-method for significance evaluation between alternative proportions). Designations as in Figure 2.
The intergenerational comparison is worth mentioning. In F5, the proportion of mice that were able to solve the first "plug" stage (stage 3 of the puzzle-box test) in the "plus" strain was significantly higher than in F1 (t = 2.89, p < 0.01, ϕ test), and the opposite tendency was noted for the respective values in the "minus" strain from F1 to F5, with a decrease in the percentage of animals that solved stage 3 of the test (t = 2.07, p < 0.05, ϕ test).
Hyponeophagia test. Among several behavioral indices registered in this test, the amount of food eaten during 5 min of this test revealed the stable differences along selection generations ( Figure 6).
The comparison of individual solution latencies at stages 3 and 4 ( Figure 5) shows that the shortening of stage 4 latencies (from those of stage 3) was more frequent in the "plus" than in the "minus" strain. These data (preliminary, as they only noted the fact of shorter latency, but not the scope of such differences) could not be regarded as a specific test for working memory, but still they demonstrate the interstrain differences in the effect of previous experience-the quicker realization of a solution by "plus" mice during the second "plug" presentation. The intergenerational comparison is worth mentioning. In F5, the proportion of mice that were able to solve the first "plug" stage (stage 3 of the puzzle-box test) in the "plus" strain was significantly higher than in F1 (t = 2.89, p < 0.01, φ test), and the opposite tendency was noted for the respective values in the "minus" strain from F1 to F5, with a decrease in the percentage of animals that solved stage 3 of the test (t = 2.07, p < 0.05, φ test).
Hyponeophagia test. Among several behavioral indices registered in this test, the amount of food eaten during 5 min of this test revealed the stable differences along selection generations ( Figure 6). Figure 6. Light gray columns-mice of "plus" selection strain, dark gray columns-mice of "minus" selection strain, black columns-mice of the control, non-selected, heterogeneous population. The amount (ordinate, mgs) of the new food (cheese) eaten by hungry mice in selection generations (horizontal axis) during 5 min of hyponeophagia test. *, ***-significant differences between males and females p < 0.05 and 0.001 (one-way ANOVA, LSD Fisher post hoc test) Designations as in Figure 2.
Mice of the "plus" strain ate more new food (cheese) than "minus" animals and more cheese than mice of the control non-selected genetically heterogeneous population, although the differences were not statistically significant for F1 and F3. The new foodsmall cubicles of cheese-was eaten by an animal in the test situation not all at once but during several approaches to the food cup. The numbers of such approaches, as well as time occupied by consuming the new food, varied among generations in non-systemic way (data not presented). The same was true for the number of freezing episodes during this test, the index being lower for "plus" mice. This difference was statistically significant for F5 only (p < 0.001, 1.52 ± 0.3 for "plus" strain, 6.7 ± 0.3 for "minus" strain and 5.2 ± 0.7 for mice of the heterogeneous population). These data demonstrate that mice selected for successful solutions of puzzle-box cognitive stages ("plus" strain) were less frightened by the new environment with the new food in comparison to two other groups. These data could be interpreted as showing a positive reaction to novelty, and also higher anxiety in "minus" and control mice (which could be also inferred from longer latencies during stages 1 and 2 of the puzzle-box test; see above).

Discussion
Data on puzzle-box successes and failures in mice of "plus" and "minus" strains show that definite cognitive ability traits (i.e., solution of "object permanence" task) could be selected for high and low values. The interstrain differences in reactions to new food by a hungry animal in a new environment (hyponeophagia test) could also be an indication of real difference in the cognitive capacity between "plus" and "minus" mice as well. The regular quantitative data have not yet been obtained for cases of plug "manipulations" by "minus" mice (when animals were not able to solve the plug stage), although this type of behavior displayed by "minus" mice in cases of solution failure could be cautiously discussed in the following way. Both "plus" and "minus" mice presumably understand the object permanence rule (that the plug covers and masks the underpass), but "plus" mice are significantly more effective in real solution performance. This could mean that the interstrain differences in plug-stage behavior could be attributed to the differences in executive functions, i.e., the ability (and maybe persistence) to achieve a definite solution. The executive function notion, acquired in psychology, is now applied to animal cognitive behavior as well [21,[28][29][30][31][32]. In our case, the executive function behavioral expression was the effective removal of the plug within the arbitrary 240 s time interval.
Data obtained also permit us to suggest that the working memory capacities seem to be affected by this selection. The "plus"-strain populations contained larger proportions of animals that acted more quickly when they were presented with the "plug" stage of the puzzle-box test for the second time than the "minus"-strain mice. These two "plug" presentations were separated from one another by dozens of seconds, and thus, we may attribute these interstrain differences to the differential influence of previous solution success in mice of two selected strains. As was stated by other authors, the working memory could be viewed as a flexible system that both maintains current information and supports the simultaneous execution of higher cognitive functions. The possible variations in the efficacy of working memory [33] could impact individual differences in intelligence scores [34]. Our conclusion concerning working memory differences could be regarded as a preliminary one because it needs confirmation in special experiments (as in, e.g., [35]). Memory genetic differences and mouse reactions to novelty were noted not once in the papers published in the last few decades, e.g., [9,16].
The experimental data obtained during the last few decades provide information concerning the genetic basis of animal behavior, although, apart from data on knock-out and knock-in mice, the problem-solving capacity in genetically different populations has not been analyzed. At the same time, the differences in the ranking of strains in different spatial tasks indicate that no single task can reveal the full richness of spatially guided behavior in a wide range of mouse genotypes [17]. As a whole, the correspondence of scores from different learning tasks in animals of different genotypes is a complicated issue. Animals from strains selected for fear conditioning did not differ in their approaches in conditioning task and in Morris water maze performance, and this probably means that the g-factor [35,36] was not affected by such selection [37], but it may be that the selection affected the motivational basis of such traits. Selection based upon physiological traits, related to the function of the autonomic cholinergic system, was also not accompanied by differences in cognitive function in rats [38]. The hippocampal morphological variability, affecting the mossy fiber synaptic projections in correlation with novelty reactions in Naples selected strains, correlated with processes which modulate strain-characteristic responses to a spatial novelty [39], although these authors indicated that this difference is of a nongenetic origin. At the same time, the genetic correlations of the performance in spatial task with the size of mossy fiber projection was established around the same period of time [40,41]. The QTL technique was also able to identify two loci, on chromosomes 4 and 12, which influenced behavior in a probe trial of the water maze [42]. The extrapolation ability in mice with NCX2 gene knock-out (sodium-calcium exchanger gene 2), tested in our experiments earlier, could demonstrate the participation of cell membrane fine machinery in the expression of such a complicated trait [43]. The relatively simple (i.e., oligo-genic) determination of "learning" genes, inferred from the fact of quick selection response and from differences in genetically defined strains [10,14,44], was not confirmed by further detailed molecular genetic investigations, in which gene expression differences were found to be much more complicated and numerous both in the background state and in situations of spatial learning and fear-based conditioning [44]. Thus, it was not surprising that the attention of neurobiologists shifted to studies in which the role of single genes in complicated behavioral expression of cognitive abilities could be identified. In these works, the improvement in cognitive traits' expression was detected after gene expression manipulations (among others [45,46]). The participation of definite genetic elements in cognitive traits' expression was confirmed in many investigations, and attention has now largely shifted to murine models of human diseases in the hope (rather real) of finding therapy approaches [12,18,28,47].
The response to selection in our experiment indicates that there are at least two important points to be aware with. First is the possibility that selection ability in the "plus" strain could vanish in further generations, as was described earlier for selection for mice with high scores on a extrapolation task solution [23], and the second point is (in the case the "plus"-"minus" difference persists) that the elevated executive function in this mouse population was determined by a rather small set of genetic elements. The selection for high and low learning abilities in rodents has been performed several times on the basis of both food and aversive motivations, which is not analyzed here, as it was presented earlier for learning in simpler animals [48]. The differences in g-factor [36,49], especially when the puzzle-box test was used as one of the "units" in a test battery [20,21,50], is also a point of interest. These data being applied to the definite genetic groups of animals may lead to identifying the genetic elements involved in cognitive task solutions [11]. The prefrontal cortex function in the aspect of cognitive ability, working memory and other complicated brain functions is also the focus of investigations with promising results [29,51]. The ability to understand the object permanence rule was recently analyzed in "Comparative cognition in three understudied" ungulate species-European bison, forest buffalos and giraffes [52]. The authors note that such data are important for understanding the evolution of animal cognition. An interesting development of these ideas was recently described by an analysis of trait inheritance in birds, namely pheasants, which are not generally used to study behavior genetics. The heritability and correlations of learning and inhibitory control traits were analyzed in 450 pheasants, Phasianus colchicus, over four generations [53], with the data clearly demonstrating the necessity of more broad analysis of this issue.
Author Contributions: Experiments, Conceptualization-O.V.P. and I.I.P.; writing,-I.I.P. I.I.P. and O.V.P. made equal contributions to the conception and design of the work and to analysis, the interpretation of data, approved the version to be published, agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors have read and agreed to the published version of the manuscript.