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Effect of rotation preference on spontaneous alternation behavior on Y maze and introduction of a new analytical method, entropy of spontaneous alternation
Behavioural Brain Research 320 (2017) 219–224
Contents lists available at ScienceDirect
Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr
Short communication
Effect of rotation preference on spontaneous alternation behavior on Y maze and introduction of a new analytical method, entropy of spontaneous alternation Jia Bak, Hae-In Pyeon, Jin-I Seok, Yun-Sik Choi ∗ Department of Pharmaceutical Science and Technology, College of Biomedical Science, Catholic University of Daegu, 38430 GyeongSangBukDo, Republic of Korea
h i g h l i g h t s • Rotation preference is associated with turning preference on Y maze. • Linear correlation between rotation preference and percent of spontaneous alternation. • Entropy of spontaneous alternation offsets the effect of rotation preference.
a r t i c l e
i n f o
Article history: Received 20 June 2016 Received in revised form 6 December 2016 Accepted 10 December 2016 Available online 12 December 2016 Keywords: Entropy of spontaneous alternation Y maze Spatial working memory Spontaneous alternation Rotation preference
a b s t r a c t Y maze has been used to test spatial working memory in rodents. To this end, the percentage of spontaneous alternation has been employed. Alternation indicates sequential entries into all three arms; e.g., when an animal visits all three arms clockwise or counterclockwise sequentially, alternation is achieved. Interestingly, animals have a tendency to rotate or turn to a preferred side. Thus, when an animal has a high rotation preference, this may influence their alternation behavior. Here, we have generated a new analytical method, termed entropy of spontaneous alternation, to offset the effect of rotation preference on Y maze. To validate the entropy of spontaneous alternation, we employed a free rotation test using a cylinder and a spatial working memory test on Y maze. We identified that mice showed 65.1% rotation preference on average. Importantly, the percentage of spontaneous alternation in the high preference group (more than 70% rotation to a preferred side) was significantly higher than that in the no preference group (<55%). In addition, there was a clear correlation between rotation preference on cylinder and turning preference on Y maze. On the other hand, this potential leverage effect that arose from rotation preference disappeared when the animal behavior on Y maze was analyzed with the entropy of spontaneous alternation. Further, entropy of spontaneous alternation significantly determined the loss of spatial working memory by scopolamine administration. Combined, these data indicate that the entropy of spontaneous alternation provides higher credibility when spatial working memory is evaluated using Y maze. © 2016 Elsevier B.V. All rights reserved.
1. Introduction A Y maze is comprised of three arms that are at 120◦ angles from each other. It has been widely used to assess spatial working memory in rodents by allowing continuous spontaneous alternation [1–3]. Spontaneous alternation is achieved when an animal
∗ Corresponding author at: Department of Pharmaceutical Science and Technology, Catholic University of Daegu, 13-13 Hayang-ro, Gyeongsan-si Gyeongsangbukdo 38430, Republic of Korea. E-mail address: [email protected] (Y.-S. Choi). http://dx.doi.org/10.1016/j.bbr.2016.12.011 0166-4328/© 2016 Elsevier B.V. All rights reserved.
enters a new arm rather than returning to one visited previously. Since rodents typically prefer to investigate new environments, over the course of multiple arm entries, they usually show a tendency to enter a less recently visited arm. Therefore, the percentage of spontaneous alternation on Y maze is accepted as mirroring the ability of the rodent’s spatial working memory [3–5]. Alternation on Y maze is achieved when an animal visits three consecutive arms clockwise or counterclockwise. Along this line, when an animal has a tendency to turn to one direction, the total number of alternations increases. Of note is that alternation guided by turning preference is less likely to be associated with spatial working memory. Interestingly, there is accumulating
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evidence showing that animals have a structural and functional hemispheric asymmetry. For example, the asymmetry of structural organizations in both global and local connection patterns has been observed [6]. In addition, hemispheric asymmetries have been reported in neurotransmitter release, excitability, and cell proliferation and survival in the adult brain [7–10]. Further, lateralizations of a number of behaviors have been reported in mammals including rodents. For example, a behavioral lateral preference has been noted in rodents in diverse experimental tests, including T maze and circling tests [11–16]. Importantly, there is increasing evidence supporting the idea that behavioral lateralization is related to anatomic and functional brain lateralization [17,18]. That being said, one may presume that in animals that have a high preference to one direction, the percentage of spontaneous alternation may reflect not only its spatial working memory, but also its turning preference. Therefore, it is needed to verify the potential leverage effect of turning preference on spatial working memory test and develop new analytical method to compensate for the behavioral preference. In this study, we investigated the involvement of rotation preference behavior in evaluating spatial working memory on Y maze and compared the percentage of spontaneous alternation with entropy of spontaneous alternation. Finally, we verified the usefulness of our new analytical method in mouse model of scopolamine-induced memory deficit by clarifying whether our new analytical method effectively detects significant difference in conditions where spatial working memory function is affected. 2. Materials and methods 2.1. Animals Male C57BL/6 mice (7 weeks old; Koatech, Kyungki-do, South Korea) were housed at a standard temperature (22 ± 1 ◦ C), humidity (50 ± 5%) conditions with light controlled from 8:00 a.m. to 8:00 p.m. (12 h interval) and access to food and water ad libitum. Animals were allowed at least 7 days-acclimatization to the laboratories before the behavioral test and mice were handled by the researchers for at least 1hr a day. The animal experimental protocol was approved by the Institutional Animal Care and Use Committee of the Catholic University of Daegu (IACUC-2015-040).
on the floor level). Animals were placed on the same arm facing the wall of the arm and this arm was designated as a start arm. The inside of Y maze was cleaned with 70% ethanol between trials and allowed to dry. Arm entry was defined as entry of the whole body into an arm [20,21]. 2.4. Spontaneous alternation score Spontaneous alternation consists of sequential entries into all three arms. Thus, the percentage of spontaneous alternation was calculated by dividing the number of alternations by the number of possible alternations [number of alternation/(number of total arm entries-2)]. 2.5. Evaluation of spatial working memory with entropy of spontaneous alternation (ESA ) On Y maze, six alternation scenarios are possible (clockwise: ABC, BCA, and CAB; counterclockwise: ACB, CBA, and BAC). First, we calculated the percentage of alternation in each alternation case [e.g., number of alternation of ABC/(number of total arm entries-2)] and designated the value as a probability (Pi ). Next, we determined the logarithm of each Pi and multiplied it with Pi . Finally, we summarized all the scores. The equation is as follows: E SA = −˙[Pi (n) × logPi (n)]. n indicates all six alternation cases. 2.6. Y maze test in scopolamine-induced memory deficit model Scopolamine hydrobromide (Sigma, St. Louis, MO, USA) was dissolved in 0.9% saline and administered intraperitoneally (1 mg/kg) to mice. One hour later, mice were allowed to freely navigate three arms of Y maze for 6 min and spontaneous alternation was analyzed as described above. 2.7. Statistical analysis Data are presented as mean ± SEM, and significance was assessed with the two-tailed student’s t-test from GraphPad Prism (version 3.0). Statistical significance was accepted when p < 0.05.
2.2. Rotation preference test on cylinder 3. Results To measure rotation preference, we used a cylinder made of Plexiglas (25 cm height, 30 cm diameter). Each mouse was placed at the center of the cylinder and animal behavior was recorded with a camcorder for 5 min. Rotation was counted when the mouse turned to left or right direction over 90◦ and the percentage of rotations to either direction was calculated. For the repetitive rotation preference test, animal behavior was tested once a week for three weeks (day 1, 8 and 15). In this study first two tests (day 1 and 8) were performed under normal condition and the last test (day 15) was performed one hour after scopolamine administration.
3.1. Rotation preference on round cylinder First, we tested whether each animal had a rotation preference on round cylinder. For this purpose, a total of 20 mice were tested. Each animal showed a preference to left or right rotation, and the mean number and percentage of rotation to preferred direction was 22 and 65.1%, respectively. The lowest percentage was 50% and the highest percentage was 87.5%. 3.2. Effect of rotation preference on spontaneous alternation score
2.3. Spatial working memory test on Y maze The Y maze made by black Plexiglas (5 cm width, 35 cm length, 10 cm height) consisted of identical three arms with 120◦ . There were no visual cues inside the maze, but different extra-maze cues were visible from all three arms to enable spatial orientation. Spatial working memory test on Y maze was performed as described by Choi and Choi [19]. Briefly, each animal was allowed to freely navigate all three arms for 6 min and its behavior was recorded with a camcorder. Behavioral test (spontaneous alternation and rotation test) was started at 8:30 p.m. under dim lightening condition (8 lx
One hour after the rotation preference test, we examined spatial working memory on Y maze for 6 min. In this experiment, 18 out of 20 mice showed total arm entries of 20 or higher, and we included the data from these 18 mice for further study. First, we divided the mice into two groups-a higher rotation preference group and a lower rotation preference group-and compared their spontaneous alternation scores. As shown in Fig. 1A, although the spontaneous alternation score of the higher rotation preference group was higher than that of the lower rotation preference group, there was no significant difference (p = 0.1479). Next, we compared the
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Fig 1. Effect of rotation preference on spontaneous alternation on Y maze. (A) Mice were divided into two groups depending on the percentage of rotation preference on the cylinder and the percentage of spontaneous alternation was compared. Although the higher group (50% of mice showed higher percentage of rotation preference) showed higher percentage of spontaneous alternation, there was no significant difference between two groups (n = 9 for each group). n.s.: not significant. (B) The percentage of spontaneous alternation was compared between no preference group (lower than 55% of rotation preference) and high preference group (higher than 70% of rotation preference) and there was significant difference between two groups (n = 3 for no preference group and 4 for high preference group, respectively). (C) Scattered plot was generated with rotation preference on the cylinder and spontaneous alternation on Y maze. Animals having higher rotation preference showed higher performance of spontaneous alternation. However, there was no statistical significance (p = 0.0593). (D) Scattered plot was generated with rotation preference on the cylinder and turning preference on Y maze and there was significant correlation between two animal parameters (p = 0.0095). Data were represented as mean ± SEM.
spontaneous alternation score between the no-preference group (rotation preference <55%; n = 3) and the high-preference group (rotation preference >70%; n = 4). In this analysis, we found that the spontaneous alternation score of the high-preference group was significantly higher compared to that of the no-preference group (Fig. 1B). 3.3. Correlation between rotation preference and spontaneous alternation behavior To identify the relationship between the rotation preference on the cylinder and the spontaneous alternation behavior on Y maze, we generated a scatter plot and conducted a linear regression analysis. As shown in Fig. 1C, the correlation was marginally out of significance (p = 0.059). Spontaneous alternation on Y maze comprised two consecutive turns to one direction. In other words, if the animal has a preference to one direction (i.e., a turning preference) on Y maze, a high alternation score could be achieved. To analyze the effect of rotation preference on spatial working memory, we generated a scatter plot between the rotation preferences on cylinder and the turning preferences on Y maze during the spontaneous alternation test. As shown in Fig. 1D, the correlation was statistically significant (p = 0.009). Taken together, these data indicate that although a statistical significance in the correlation between rotation preference on cylinder and spontaneous alternation score was not observed, rotation preference may have had a leverage effect on the spontaneous alternation score on Y maze. 3.4. Analysis of the spatial working memory with entropy of spontaneous alternation Again, we divided the mice into two groups, higher and lower rotation preference groups, and compared their entropy of spon-
taneous alternation. As shown in Fig. 2A, there was no statistically significant difference between the two groups. Of note is that the difference of the mean average was markedly reduced between the two groups, which means that entropy of spontaneous alternation minimized the leverage effect of rotation preference on the spatial working memory test. In addition, there was no significant difference in the entropy of spontaneous alternation between the no-rotation preference group and the high-rotation preference group (Fig. 2B). These data may indicate that entropy of spontaneous alternation offsets the leverage effect of rotation preference on spatial working memory test on Y maze. 3.5. Correlation between rotation preference and entropy of spontaneous alternation Next, we tested the correlation between rotation preference and entropy of spontaneous alternation. We generated a scatter plot between rotation preference on the cylinder and entropy of spontaneous alternation on Y maze. As shown in Fig. 2C, the entropy of spontaneous alternation was not affected by rotation preference. These data therefore indicate that our new analytical equation with the algorithm is independent from rotation preference; thus, entropy of spontaneous alternation is an advanced analytical tool for evaluating spatial working memory on Y maze. 3.6. Entropy of spontaneous alternation in animal model of scopolamine-induced memory deficit Next, we asked whether the entropy of spontaneous alternation could mirror the change of spatial working memory correctly in rodents. To answer this question, we employed animal model of scopolamine-induced memory deficit. As shown in Fig. 3, the percentage of spontaneous alternation was significantly reduced.
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Fig. 2. Effect of rotation preference on the entropy of spontaneous alternation on Y maze. (A) Mice were divided into two groups depending on the percentage of rotation preference on the cylinder and the entropy of spontaneous alternation was compared. There was no significant difference between two groups (n = 9 for each group). n.s.: not significant. (B) The entropy of spontaneous alternation was compared between no preference group (lower than 55% of rotation preference) and high preference group (higher than 70% of rotation preference) and there was no significant difference between two groups (n = 3 for no preference group and 4 for high preference group, respectively). Data were represented as mean ± SEM. n.s.: not significant. (C) Scattered plot was generated with rotation preference on the cylinder and entropy of spontaneous alternation on Y maze. The entropy of spontaneous alternation was not affected by the rotation preference.
Likewise, the entropy of spontaneous alternation was also significantly reduced. However, there was no significant difference in turning preference between two groups. These data combined with our previous report in animal model of enriched environment indicate that the entropy of spontaneous alternation could reflect the difference of spatial working memory correctly on Y maze [22]. 3.7. Rotation preference by scopolamine administration Next, we asked whether the rotation preference is maintained over a period of time and affected by scopolamine treatment. For this study, we tested rotation preference for three weeks; at day 1 and 8 rotation was tested under normal condition and at day 15 it was tested one hour after scopolamine administration. As shown in Table 1, the dominant rotational direction was
Fig. 3. Analysis of spatial working memory in animal model of scopolamine-induced memory loss. (A) Spatial working memory was analyzed by the percentage of spontaneous alternation between control and scopolamine-administered groups. There was significant difference between two groups (n = 8 for each group). (B) Spatial working memory was analyzed by the entropy of spontaneous alternation between two groups. The entropy of spontaneous alternation from scopolamineadministered group was significantly lower than that from control group. (C) Turning preference was 70.2% and 70.0% for vehicle- and scopolamine-treated groups, respectively and there was no significant difference between two groups. Data were represented as mean ± SEM. * p < 0.05.
maintained under normal condition in 10 out of 13 animals. In addition, after scopolamine administration dominant rotational direction was unchanged in 10 out of 13 mice. Interestingly, 9 of 13 animals showed consistent preferred rotational direction through all three tests. This data indicate that although not all the cases, rotational preference is maintained and it is not affected by scopolamine treatment. In addition, the percentage of rotation preference was slightly increased by scopolamine administration. Further, the number of animals having high rotation preference (≥70%) was increased by scopolamine treatment. These data further support that rotation preference is not associated with spatial working memory. 4. Discussion Here, we showed the linear correlation between rotation preference on cylinder and turning preference on Y maze, and these
J. Bak et al. / Behavioural Brain Research 320 (2017) 219–224
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Table 1 The effect of rotation preference by scopolamine. Condition
Day 1 (normal condition) Day 8 (normal condition) Day 15(scopolamine treated)
% of rotation preference
65.31 ± 2.96 64.00 ± 2.66 67.69 ± 2.14
Dominant direction (Left/Right, No. of animals)
7/6 8/5 7/6
Rotation preference (No. of animals)
50–55%
≥70%
1 2 1
4 4 7
Consistency of dominant rotational direction (No. of animals)
10/13
– 10/13
–
Data are expressed as mean ± SEM.
data, along other data presented in this paper, indicate that rotation preference potentially affects spontaneous alternation score on Y maze. Of note is that the entropy of spontaneous alternation offset the leverage effect of rotation preference in evaluating the ability of spatial working memory on Y maze. Further, the entropy of spontaneous alternation successfully detected the impaired spatial working memory by scopolamine administration in mice. Taken together, it is inferred that the entropy of spontaneous alternation could be an advanced and trustworthy analytical tool evaluating spatial working memory on Y maze. We provided evidence showing that mice with a high rotation preference have a tendency to demonstrate a high percentage of spontaneous alternation. In addition, we verified the correlation between rotation preference on cylinder and turning preference on Y maze, and the percentage of spontaneous alternation from the high-rotation preference group (>70% to the preferred direction) was significantly higher than that of the no-rotation preference group (50% to 55% to the preferred direction). These data indicate that in animal having high rotation preference, the percentage of spontaneous alternation on Y maze seems not to reflect the ability of spatial working memory properly. To overcome the shortcomings of the current analytical method of spatial working memory with spontaneous alternation, we generated a new function, entropy of spontaneous alternation. Compared with the percentage of spontaneous alternation, the most notable merit of our new function is that it counts not only the percentage of alternation, but also the direction as well as location of the alternation. Importantly, when an animal shows alternation in all direction as well as location, the entropy of spontaneous alternation increases. To validate the benefits of our new analytical method it is critical to elucidate whether rotation preference and turning preference are associated with spatial working memory. To answer this question, we administrated scopolamine to mice and tested spatial working memory on Y maze. Scopolamine, a muscarinic receptor antagonist, has been widely used to impair spatial working memory in animals [23–25]. In this experiment we identified that although spatial working memory was significantly impaired by scopolamine administration, turning preference was almost identical between scopolamine- and saline-treated groups. This data indicate that mice have spontaneous rotation to the preferred direction and this behavior is not associated with spatial working memory. Supporting this idea, accumulating evidence indicates that biochemical asymmetries are associated with behavioral preference. For example, under normal conditions, 68% of rats showed a strong preference for using a preferred paw to reach for food and a significantly higher percentage of rotation preference was observed [18]. In addition, in a free-swimming rotatory test, 45% of mice showed a preference to one side over the other more than 70%, but in mice rendered acallosal by prenatal gamma irradiation, the percentage of mice showed preference was increased up to 70% [24]. Therefore, when spatial working memory is tested on Y maze, it would be reasonable to take turning preference into consideration. Next, we asked whether rotational behavior is intrinsic, thus preferred rotational direction is consistent from week to week and
affected by scopolamine. For this study, we tested spontaneous alternation three times with a week interval. For the first two trials, we tested spontaneous rotation under normal condition and for the third trial mice were administered scopolamine. In this experiment, 9 out of 13 animals showed the same dominant direction preference through all three trials. In addition, the mean percentage of rotational preference was slightly increased by scopolamine treatment (64.0% and 67.7% for normal and scopolamine-treated condition). These data combined with data presented above indicate that mice mostly have intrinsic preference in rotational behavior and this rotational preference is not associated with spatial working memory on Y maze. We have to admit that the percentage of spontaneous alternation on Y maze is a good tool to evaluate spatial working memory in rodents. Spontaneous alternation on Y maze was first applied to the study of drug effects about 40 years ago [5]. Along this line, the percentage of alternation has been widely used as a parameter of spatial working memory in studies of pharmacology as well as pathophysiology [26–28]. The difference of our new analytical method is that it also considers the location and direction of spontaneous alternation occurred. We compared these two analytical methods in scopolamine-administered memory impairing model and environmental enrichment-induced memory inducing model [22]. As shown above, in case of scopolamine-induced memory impairing experiment the p values were 0.0313 and 0.0194 for the percentage and entropy of spontaneous alternation, respectively. On the other hand, in environmental enrichment-induced memory enhancing experiment the p values were 0.0044 and 0.0199 for the percentage and entropy of spontaneous alternation, respectively. Therefore, it is important to note that our new analytical equation does not make it difficult to acquire statistical significance. Instead, we are providing a new and deliberate tool for spatial working memory test on Y maze by minimizing the involvement of rotational preference. In summary, in the current study we generated entropy of spontaneous alternation rendering the ability of spatial working memory on Y maze. We provided evidence showing that entropy of spontaneous alternation offset the demerits arising from turning preference on Y maze, and thus it is highly expected that the entropy of spontaneous alternation will increase the credibility of spatial working memory test on Y maze. Conflict of interest The authors declare no conflict of interest with any person or any organization. References [1] M. Ukai, Y. Watanabe, T. Kameyama, Effects of endomorphins-1 and −2 endogenous mu-opioid receptor agonists, on spontaneous alternation performance in mice, Eur. J. Pharmacol. 395 (2000) 211–215. [2] A.T. Mugwagwa, L.L. Gadaga, W. Pote, D. Tagwireyi, Antiamnesic effects of a hydroethanolic extract of crinum macowanii on scopolamine-induced memory impairment in mice, J. Neurodegener Dis. 2015 (2015) 242505.
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Contents lists available at ScienceDirect
Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr
Short communication
Effect of rotation preference on spontaneous alternation behavior on Y maze and introduction of a new analytical method, entropy of spontaneous alternation Jia Bak, Hae-In Pyeon, Jin-I Seok, Yun-Sik Choi ∗ Department of Pharmaceutical Science and Technology, College of Biomedical Science, Catholic University of Daegu, 38430 GyeongSangBukDo, Republic of Korea
h i g h l i g h t s • Rotation preference is associated with turning preference on Y maze. • Linear correlation between rotation preference and percent of spontaneous alternation. • Entropy of spontaneous alternation offsets the effect of rotation preference.
a r t i c l e
i n f o
Article history: Received 20 June 2016 Received in revised form 6 December 2016 Accepted 10 December 2016 Available online 12 December 2016 Keywords: Entropy of spontaneous alternation Y maze Spatial working memory Spontaneous alternation Rotation preference
a b s t r a c t Y maze has been used to test spatial working memory in rodents. To this end, the percentage of spontaneous alternation has been employed. Alternation indicates sequential entries into all three arms; e.g., when an animal visits all three arms clockwise or counterclockwise sequentially, alternation is achieved. Interestingly, animals have a tendency to rotate or turn to a preferred side. Thus, when an animal has a high rotation preference, this may influence their alternation behavior. Here, we have generated a new analytical method, termed entropy of spontaneous alternation, to offset the effect of rotation preference on Y maze. To validate the entropy of spontaneous alternation, we employed a free rotation test using a cylinder and a spatial working memory test on Y maze. We identified that mice showed 65.1% rotation preference on average. Importantly, the percentage of spontaneous alternation in the high preference group (more than 70% rotation to a preferred side) was significantly higher than that in the no preference group (<55%). In addition, there was a clear correlation between rotation preference on cylinder and turning preference on Y maze. On the other hand, this potential leverage effect that arose from rotation preference disappeared when the animal behavior on Y maze was analyzed with the entropy of spontaneous alternation. Further, entropy of spontaneous alternation significantly determined the loss of spatial working memory by scopolamine administration. Combined, these data indicate that the entropy of spontaneous alternation provides higher credibility when spatial working memory is evaluated using Y maze. © 2016 Elsevier B.V. All rights reserved.
1. Introduction A Y maze is comprised of three arms that are at 120◦ angles from each other. It has been widely used to assess spatial working memory in rodents by allowing continuous spontaneous alternation [1–3]. Spontaneous alternation is achieved when an animal
∗ Corresponding author at: Department of Pharmaceutical Science and Technology, Catholic University of Daegu, 13-13 Hayang-ro, Gyeongsan-si Gyeongsangbukdo 38430, Republic of Korea. E-mail address: [email protected] (Y.-S. Choi). http://dx.doi.org/10.1016/j.bbr.2016.12.011 0166-4328/© 2016 Elsevier B.V. All rights reserved.
enters a new arm rather than returning to one visited previously. Since rodents typically prefer to investigate new environments, over the course of multiple arm entries, they usually show a tendency to enter a less recently visited arm. Therefore, the percentage of spontaneous alternation on Y maze is accepted as mirroring the ability of the rodent’s spatial working memory [3–5]. Alternation on Y maze is achieved when an animal visits three consecutive arms clockwise or counterclockwise. Along this line, when an animal has a tendency to turn to one direction, the total number of alternations increases. Of note is that alternation guided by turning preference is less likely to be associated with spatial working memory. Interestingly, there is accumulating
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evidence showing that animals have a structural and functional hemispheric asymmetry. For example, the asymmetry of structural organizations in both global and local connection patterns has been observed [6]. In addition, hemispheric asymmetries have been reported in neurotransmitter release, excitability, and cell proliferation and survival in the adult brain [7–10]. Further, lateralizations of a number of behaviors have been reported in mammals including rodents. For example, a behavioral lateral preference has been noted in rodents in diverse experimental tests, including T maze and circling tests [11–16]. Importantly, there is increasing evidence supporting the idea that behavioral lateralization is related to anatomic and functional brain lateralization [17,18]. That being said, one may presume that in animals that have a high preference to one direction, the percentage of spontaneous alternation may reflect not only its spatial working memory, but also its turning preference. Therefore, it is needed to verify the potential leverage effect of turning preference on spatial working memory test and develop new analytical method to compensate for the behavioral preference. In this study, we investigated the involvement of rotation preference behavior in evaluating spatial working memory on Y maze and compared the percentage of spontaneous alternation with entropy of spontaneous alternation. Finally, we verified the usefulness of our new analytical method in mouse model of scopolamine-induced memory deficit by clarifying whether our new analytical method effectively detects significant difference in conditions where spatial working memory function is affected. 2. Materials and methods 2.1. Animals Male C57BL/6 mice (7 weeks old; Koatech, Kyungki-do, South Korea) were housed at a standard temperature (22 ± 1 ◦ C), humidity (50 ± 5%) conditions with light controlled from 8:00 a.m. to 8:00 p.m. (12 h interval) and access to food and water ad libitum. Animals were allowed at least 7 days-acclimatization to the laboratories before the behavioral test and mice were handled by the researchers for at least 1hr a day. The animal experimental protocol was approved by the Institutional Animal Care and Use Committee of the Catholic University of Daegu (IACUC-2015-040).
on the floor level). Animals were placed on the same arm facing the wall of the arm and this arm was designated as a start arm. The inside of Y maze was cleaned with 70% ethanol between trials and allowed to dry. Arm entry was defined as entry of the whole body into an arm [20,21]. 2.4. Spontaneous alternation score Spontaneous alternation consists of sequential entries into all three arms. Thus, the percentage of spontaneous alternation was calculated by dividing the number of alternations by the number of possible alternations [number of alternation/(number of total arm entries-2)]. 2.5. Evaluation of spatial working memory with entropy of spontaneous alternation (ESA ) On Y maze, six alternation scenarios are possible (clockwise: ABC, BCA, and CAB; counterclockwise: ACB, CBA, and BAC). First, we calculated the percentage of alternation in each alternation case [e.g., number of alternation of ABC/(number of total arm entries-2)] and designated the value as a probability (Pi ). Next, we determined the logarithm of each Pi and multiplied it with Pi . Finally, we summarized all the scores. The equation is as follows: E SA = −˙[Pi (n) × logPi (n)]. n indicates all six alternation cases. 2.6. Y maze test in scopolamine-induced memory deficit model Scopolamine hydrobromide (Sigma, St. Louis, MO, USA) was dissolved in 0.9% saline and administered intraperitoneally (1 mg/kg) to mice. One hour later, mice were allowed to freely navigate three arms of Y maze for 6 min and spontaneous alternation was analyzed as described above. 2.7. Statistical analysis Data are presented as mean ± SEM, and significance was assessed with the two-tailed student’s t-test from GraphPad Prism (version 3.0). Statistical significance was accepted when p < 0.05.
2.2. Rotation preference test on cylinder 3. Results To measure rotation preference, we used a cylinder made of Plexiglas (25 cm height, 30 cm diameter). Each mouse was placed at the center of the cylinder and animal behavior was recorded with a camcorder for 5 min. Rotation was counted when the mouse turned to left or right direction over 90◦ and the percentage of rotations to either direction was calculated. For the repetitive rotation preference test, animal behavior was tested once a week for three weeks (day 1, 8 and 15). In this study first two tests (day 1 and 8) were performed under normal condition and the last test (day 15) was performed one hour after scopolamine administration.
3.1. Rotation preference on round cylinder First, we tested whether each animal had a rotation preference on round cylinder. For this purpose, a total of 20 mice were tested. Each animal showed a preference to left or right rotation, and the mean number and percentage of rotation to preferred direction was 22 and 65.1%, respectively. The lowest percentage was 50% and the highest percentage was 87.5%. 3.2. Effect of rotation preference on spontaneous alternation score
2.3. Spatial working memory test on Y maze The Y maze made by black Plexiglas (5 cm width, 35 cm length, 10 cm height) consisted of identical three arms with 120◦ . There were no visual cues inside the maze, but different extra-maze cues were visible from all three arms to enable spatial orientation. Spatial working memory test on Y maze was performed as described by Choi and Choi [19]. Briefly, each animal was allowed to freely navigate all three arms for 6 min and its behavior was recorded with a camcorder. Behavioral test (spontaneous alternation and rotation test) was started at 8:30 p.m. under dim lightening condition (8 lx
One hour after the rotation preference test, we examined spatial working memory on Y maze for 6 min. In this experiment, 18 out of 20 mice showed total arm entries of 20 or higher, and we included the data from these 18 mice for further study. First, we divided the mice into two groups-a higher rotation preference group and a lower rotation preference group-and compared their spontaneous alternation scores. As shown in Fig. 1A, although the spontaneous alternation score of the higher rotation preference group was higher than that of the lower rotation preference group, there was no significant difference (p = 0.1479). Next, we compared the
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Fig 1. Effect of rotation preference on spontaneous alternation on Y maze. (A) Mice were divided into two groups depending on the percentage of rotation preference on the cylinder and the percentage of spontaneous alternation was compared. Although the higher group (50% of mice showed higher percentage of rotation preference) showed higher percentage of spontaneous alternation, there was no significant difference between two groups (n = 9 for each group). n.s.: not significant. (B) The percentage of spontaneous alternation was compared between no preference group (lower than 55% of rotation preference) and high preference group (higher than 70% of rotation preference) and there was significant difference between two groups (n = 3 for no preference group and 4 for high preference group, respectively). (C) Scattered plot was generated with rotation preference on the cylinder and spontaneous alternation on Y maze. Animals having higher rotation preference showed higher performance of spontaneous alternation. However, there was no statistical significance (p = 0.0593). (D) Scattered plot was generated with rotation preference on the cylinder and turning preference on Y maze and there was significant correlation between two animal parameters (p = 0.0095). Data were represented as mean ± SEM.
spontaneous alternation score between the no-preference group (rotation preference <55%; n = 3) and the high-preference group (rotation preference >70%; n = 4). In this analysis, we found that the spontaneous alternation score of the high-preference group was significantly higher compared to that of the no-preference group (Fig. 1B). 3.3. Correlation between rotation preference and spontaneous alternation behavior To identify the relationship between the rotation preference on the cylinder and the spontaneous alternation behavior on Y maze, we generated a scatter plot and conducted a linear regression analysis. As shown in Fig. 1C, the correlation was marginally out of significance (p = 0.059). Spontaneous alternation on Y maze comprised two consecutive turns to one direction. In other words, if the animal has a preference to one direction (i.e., a turning preference) on Y maze, a high alternation score could be achieved. To analyze the effect of rotation preference on spatial working memory, we generated a scatter plot between the rotation preferences on cylinder and the turning preferences on Y maze during the spontaneous alternation test. As shown in Fig. 1D, the correlation was statistically significant (p = 0.009). Taken together, these data indicate that although a statistical significance in the correlation between rotation preference on cylinder and spontaneous alternation score was not observed, rotation preference may have had a leverage effect on the spontaneous alternation score on Y maze. 3.4. Analysis of the spatial working memory with entropy of spontaneous alternation Again, we divided the mice into two groups, higher and lower rotation preference groups, and compared their entropy of spon-
taneous alternation. As shown in Fig. 2A, there was no statistically significant difference between the two groups. Of note is that the difference of the mean average was markedly reduced between the two groups, which means that entropy of spontaneous alternation minimized the leverage effect of rotation preference on the spatial working memory test. In addition, there was no significant difference in the entropy of spontaneous alternation between the no-rotation preference group and the high-rotation preference group (Fig. 2B). These data may indicate that entropy of spontaneous alternation offsets the leverage effect of rotation preference on spatial working memory test on Y maze. 3.5. Correlation between rotation preference and entropy of spontaneous alternation Next, we tested the correlation between rotation preference and entropy of spontaneous alternation. We generated a scatter plot between rotation preference on the cylinder and entropy of spontaneous alternation on Y maze. As shown in Fig. 2C, the entropy of spontaneous alternation was not affected by rotation preference. These data therefore indicate that our new analytical equation with the algorithm is independent from rotation preference; thus, entropy of spontaneous alternation is an advanced analytical tool for evaluating spatial working memory on Y maze. 3.6. Entropy of spontaneous alternation in animal model of scopolamine-induced memory deficit Next, we asked whether the entropy of spontaneous alternation could mirror the change of spatial working memory correctly in rodents. To answer this question, we employed animal model of scopolamine-induced memory deficit. As shown in Fig. 3, the percentage of spontaneous alternation was significantly reduced.
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Fig. 2. Effect of rotation preference on the entropy of spontaneous alternation on Y maze. (A) Mice were divided into two groups depending on the percentage of rotation preference on the cylinder and the entropy of spontaneous alternation was compared. There was no significant difference between two groups (n = 9 for each group). n.s.: not significant. (B) The entropy of spontaneous alternation was compared between no preference group (lower than 55% of rotation preference) and high preference group (higher than 70% of rotation preference) and there was no significant difference between two groups (n = 3 for no preference group and 4 for high preference group, respectively). Data were represented as mean ± SEM. n.s.: not significant. (C) Scattered plot was generated with rotation preference on the cylinder and entropy of spontaneous alternation on Y maze. The entropy of spontaneous alternation was not affected by the rotation preference.
Likewise, the entropy of spontaneous alternation was also significantly reduced. However, there was no significant difference in turning preference between two groups. These data combined with our previous report in animal model of enriched environment indicate that the entropy of spontaneous alternation could reflect the difference of spatial working memory correctly on Y maze [22]. 3.7. Rotation preference by scopolamine administration Next, we asked whether the rotation preference is maintained over a period of time and affected by scopolamine treatment. For this study, we tested rotation preference for three weeks; at day 1 and 8 rotation was tested under normal condition and at day 15 it was tested one hour after scopolamine administration. As shown in Table 1, the dominant rotational direction was
Fig. 3. Analysis of spatial working memory in animal model of scopolamine-induced memory loss. (A) Spatial working memory was analyzed by the percentage of spontaneous alternation between control and scopolamine-administered groups. There was significant difference between two groups (n = 8 for each group). (B) Spatial working memory was analyzed by the entropy of spontaneous alternation between two groups. The entropy of spontaneous alternation from scopolamineadministered group was significantly lower than that from control group. (C) Turning preference was 70.2% and 70.0% for vehicle- and scopolamine-treated groups, respectively and there was no significant difference between two groups. Data were represented as mean ± SEM. * p < 0.05.
maintained under normal condition in 10 out of 13 animals. In addition, after scopolamine administration dominant rotational direction was unchanged in 10 out of 13 mice. Interestingly, 9 of 13 animals showed consistent preferred rotational direction through all three tests. This data indicate that although not all the cases, rotational preference is maintained and it is not affected by scopolamine treatment. In addition, the percentage of rotation preference was slightly increased by scopolamine administration. Further, the number of animals having high rotation preference (≥70%) was increased by scopolamine treatment. These data further support that rotation preference is not associated with spatial working memory. 4. Discussion Here, we showed the linear correlation between rotation preference on cylinder and turning preference on Y maze, and these
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Table 1 The effect of rotation preference by scopolamine. Condition
Day 1 (normal condition) Day 8 (normal condition) Day 15(scopolamine treated)
% of rotation preference
65.31 ± 2.96 64.00 ± 2.66 67.69 ± 2.14
Dominant direction (Left/Right, No. of animals)
7/6 8/5 7/6
Rotation preference (No. of animals)
50–55%
≥70%
1 2 1
4 4 7
Consistency of dominant rotational direction (No. of animals)
10/13
– 10/13
–
Data are expressed as mean ± SEM.
data, along other data presented in this paper, indicate that rotation preference potentially affects spontaneous alternation score on Y maze. Of note is that the entropy of spontaneous alternation offset the leverage effect of rotation preference in evaluating the ability of spatial working memory on Y maze. Further, the entropy of spontaneous alternation successfully detected the impaired spatial working memory by scopolamine administration in mice. Taken together, it is inferred that the entropy of spontaneous alternation could be an advanced and trustworthy analytical tool evaluating spatial working memory on Y maze. We provided evidence showing that mice with a high rotation preference have a tendency to demonstrate a high percentage of spontaneous alternation. In addition, we verified the correlation between rotation preference on cylinder and turning preference on Y maze, and the percentage of spontaneous alternation from the high-rotation preference group (>70% to the preferred direction) was significantly higher than that of the no-rotation preference group (50% to 55% to the preferred direction). These data indicate that in animal having high rotation preference, the percentage of spontaneous alternation on Y maze seems not to reflect the ability of spatial working memory properly. To overcome the shortcomings of the current analytical method of spatial working memory with spontaneous alternation, we generated a new function, entropy of spontaneous alternation. Compared with the percentage of spontaneous alternation, the most notable merit of our new function is that it counts not only the percentage of alternation, but also the direction as well as location of the alternation. Importantly, when an animal shows alternation in all direction as well as location, the entropy of spontaneous alternation increases. To validate the benefits of our new analytical method it is critical to elucidate whether rotation preference and turning preference are associated with spatial working memory. To answer this question, we administrated scopolamine to mice and tested spatial working memory on Y maze. Scopolamine, a muscarinic receptor antagonist, has been widely used to impair spatial working memory in animals [23–25]. In this experiment we identified that although spatial working memory was significantly impaired by scopolamine administration, turning preference was almost identical between scopolamine- and saline-treated groups. This data indicate that mice have spontaneous rotation to the preferred direction and this behavior is not associated with spatial working memory. Supporting this idea, accumulating evidence indicates that biochemical asymmetries are associated with behavioral preference. For example, under normal conditions, 68% of rats showed a strong preference for using a preferred paw to reach for food and a significantly higher percentage of rotation preference was observed [18]. In addition, in a free-swimming rotatory test, 45% of mice showed a preference to one side over the other more than 70%, but in mice rendered acallosal by prenatal gamma irradiation, the percentage of mice showed preference was increased up to 70% [24]. Therefore, when spatial working memory is tested on Y maze, it would be reasonable to take turning preference into consideration. Next, we asked whether rotational behavior is intrinsic, thus preferred rotational direction is consistent from week to week and
affected by scopolamine. For this study, we tested spontaneous alternation three times with a week interval. For the first two trials, we tested spontaneous rotation under normal condition and for the third trial mice were administered scopolamine. In this experiment, 9 out of 13 animals showed the same dominant direction preference through all three trials. In addition, the mean percentage of rotational preference was slightly increased by scopolamine treatment (64.0% and 67.7% for normal and scopolamine-treated condition). These data combined with data presented above indicate that mice mostly have intrinsic preference in rotational behavior and this rotational preference is not associated with spatial working memory on Y maze. We have to admit that the percentage of spontaneous alternation on Y maze is a good tool to evaluate spatial working memory in rodents. Spontaneous alternation on Y maze was first applied to the study of drug effects about 40 years ago [5]. Along this line, the percentage of alternation has been widely used as a parameter of spatial working memory in studies of pharmacology as well as pathophysiology [26–28]. The difference of our new analytical method is that it also considers the location and direction of spontaneous alternation occurred. We compared these two analytical methods in scopolamine-administered memory impairing model and environmental enrichment-induced memory inducing model [22]. As shown above, in case of scopolamine-induced memory impairing experiment the p values were 0.0313 and 0.0194 for the percentage and entropy of spontaneous alternation, respectively. On the other hand, in environmental enrichment-induced memory enhancing experiment the p values were 0.0044 and 0.0199 for the percentage and entropy of spontaneous alternation, respectively. Therefore, it is important to note that our new analytical equation does not make it difficult to acquire statistical significance. Instead, we are providing a new and deliberate tool for spatial working memory test on Y maze by minimizing the involvement of rotational preference. In summary, in the current study we generated entropy of spontaneous alternation rendering the ability of spatial working memory on Y maze. We provided evidence showing that entropy of spontaneous alternation offset the demerits arising from turning preference on Y maze, and thus it is highly expected that the entropy of spontaneous alternation will increase the credibility of spatial working memory test on Y maze. Conflict of interest The authors declare no conflict of interest with any person or any organization. References [1] M. Ukai, Y. Watanabe, T. Kameyama, Effects of endomorphins-1 and −2 endogenous mu-opioid receptor agonists, on spontaneous alternation performance in mice, Eur. J. Pharmacol. 395 (2000) 211–215. [2] A.T. Mugwagwa, L.L. Gadaga, W. Pote, D. Tagwireyi, Antiamnesic effects of a hydroethanolic extract of crinum macowanii on scopolamine-induced memory impairment in mice, J. Neurodegener Dis. 2015 (2015) 242505.
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