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ORIGINAL ARTICLE |
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Year : 2020 | Volume
: 63
| Issue : 1 | Page : 27-34 |
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Formosan wood mice (Apodemus semotus) exhibit more exploratory behaviors and central dopaminergic activities than C57BL/6 mice in the open field test
Kun-Ruey Shieh1, Shu-Chuan Yang2
1 Department of Physiology, Tzu Chi University, Hualien, Taiwan 2 Holistic Education Center, Tzu Chi University of Science and Technology, Hualien, Taiwan
Date of Submission | 04-Jun-2019 |
Date of Acceptance | 19-Dec-2019 |
Date of Web Publication | 7-Feb-2020 |
Correspondence Address: Prof. Shu-Chuan Yang Holistic Education Center, Tzu Chi University of Science and Technology, No. 880 Chien-Kuo Road, Section 2, Hualien 970 Taiwan
 Source of Support: None, Conflict of Interest: None  | 11 |
DOI: 10.4103/CJP.CJP_47_19
Three-quarters of the lands in Taiwan are over 1000 m above sea level. Formosan wood mice (Apodemus semotus), also called Taiwanese field mice, are largely found at altitudes of 1400 ~ 3700 m and are the dominant rodents in these areas. Notably, Formosan wood mice show high levels of exploratory behaviors, not only in the wild but also in laboratory situations. Therefore, in this study, we examined the behavioral responses and central dopaminergic activities of male C57BL/6J mice and Formosan wood mice in the open field test. Dopamine and its major metabolite 3,4-dihydroxyphenylacetic acid were used as indices of dopaminergic activities. Formosan wood mice showed higher levels of exploration and locomotor activity than C57BL/6J mice in the open field test. Higher central dopaminergic activities in the nucleus accumbens, striatum, and medial prefrontal cortex were found in Formosan wood mice than in C57BL/6J mice in the open field test. Higher levels of locomotion and central dopaminergic activities in Formosan wood mice were consistent after two exposures to the open field test; however, dramatic decreases in levels of locomotion and central dopaminergic activities in C57BL/6J mice were found after two exposures to the open field test. The present study found that Formosan wood mice exhibited higher levels of locomotor activity and exploration and central dopaminergic activities than C57BL/6J mice after one or two exposures to the open field test.
Keywords: Agile, agility, exploration, Taiwan field mice
How to cite this article: Shieh KR, Yang SC. Formosan wood mice (Apodemus semotus) exhibit more exploratory behaviors and central dopaminergic activities than C57BL/6 mice in the open field test. Chin J Physiol 2020;63:27-34 |
How to cite this URL: Shieh KR, Yang SC. Formosan wood mice (Apodemus semotus) exhibit more exploratory behaviors and central dopaminergic activities than C57BL/6 mice in the open field test. Chin J Physiol [serial online] 2020 [cited 2023 Sep 28];63:27-34. Available from: https://www.cjphysiology.org/text.asp?2020/63/1/27/277952 |
Introduction | |  |
Approximately 75% of the district of Taiwan is more than 1000 m above sea level. Apodemus semotus is commonly found in habitats between 1400 m and 3700 m above sea level and is one of nearly 50 endemic species and subspecies of mammals in Taiwan. Formosan wood mice, A. semotus, are also called Taiwanese field mice and are very abundant in the central and southern areas of Taiwan.[1],[2] Previous studies on Formosan wood mice/Taiwanese wood mice have mainly addressed their ecobiology, reproductive characteristics and growth curves,[3],[4] and our previous studies have concentrated on their behavioral responses in the laboratory.[5] Using the elevated plus-maze, our recent study found that Formosan wood mice show high levels of exploratory behaviors in laboratory conditions.[5] However, this result was confounded by the fact that Formosan wood mice jumped off the platform of the apparatus, in contrast to behaviors displayed by common laboratory mice.[6],[7],[8],[9],[10],[11],[12],[13] It is likely that Formosan wood mice exhibit higher levels of exploratory behaviors or locomotor activity in the wild to avoid being killed by predators, such as wild cats, dogs or foxes, as suggested by a previous study.[14]
The open field test and elevated plus maze are commonly used as behavioral tests of anxiety-like responses in mice and rats.[15],[16],[17] These behavioral tests reflect animals' responses to a possible threat and/or fear and their passive avoidance reactions. Although Formosan wood mice showed higher levels of exploratory and locomotor activities than common C57BL/6J laboratory mice, in the elevated plus maze,[5] one-third of the tested Formosan wood mice also jumped down from the platform of the apparatus, as mentioned above. Thus, the results of our previous study cannot rule out that higher levels of exploration in Formosan wood mice were simply an artifact of this increased propensity to jump off the platform. The open field test might be a better alternative to test anxiety-like responses and to examine locomotor activity in Formosan wood mice.[15],[16],[17] In the present study, we tested whether we could replicate our previous findings using an open field test apparatus from which wood mice could not escape. Furthermore, a higher level of novelty in a stimulus an animal approaches is normally associated with locomotor activities or seeking behaviors.[18],[19] When animals encounter the same circumstances recurrently, their behavioral responses and reactions, including locomotion or exploration, are attenuated.[20] Therefore, the behavioral responses, especially locomotor activity, after two exposures to the open field tests in Formosan wood mice were examined in this study.
Central mesocortical, mesostriatal, and mesolimbic DAergic systems have been shown to play important roles in novelty-seeking or exploratory behavior.[21],[22],[23] Previous studies have shown that novelty-seeking behavior is disrupted by the destruction or blockade of mesolimbic and mesostriatal DAergic systems.[21],[22] Locomotor activity has also been shown to be related to mesocortical and mesolimbic DAergic systems in a study of N-methyl-D-aspartate antagonist-induced locomotion.[24] Our previous study also showed that locomotor activity in the elevated plus maze is associated with central mesocortical, mesostriatal, and mesolimbic DAergic systems in Formosan wood mice.[5] Since the major projections of the mesocortical, mesolimbic, and mesostriatal DAergic systems include the medial prefrontal cortex (MPFC), nucleus accumbens (NA), and striatum (ST), respectively, herein, we tested both common C57BL/6J laboratory mice and Formosan wood mice in the open field test and tested for associations between behaviors displayed in the open field test and DAergic activity.
Materials and Methods | |  |
Animals
Male 4-month-old C57BL/6J mice and Formosan wood mice/Taiwanese field mice (A. semotus) (n = 24 in each group; weighing 30–35 g; from the National Laboratory Animal Center, Taipei, Taiwan, and the Laboratory Animal Center of Tzu Chi University, Hualien, Taiwan, respectively) were used in this study. Animals were maintained with a 12-h light/dark cycle (lights-on from 6 AM to 6 PM) with temperature- (22°C ± 1°C) controlled room, and had free access to tap water and rodent chow. Groups of three male Formosan wood mice or three male C57BL/6J mice were maintained together in a common cage (45 cm in length, 30 cm in width, and 30 cm in height). In the first experiment, male Formosan wood mice and C57BL/6J mice (n = 9 in each group) were challenged in the open field test. In the second experiment, male Formosan wood mice and C57BL/6J mice (n = 9 in each group) were subjected to the open field test once. The other male Formosan wood mice and C57BL/6J mice (n = 6 in each group) were subjected to the open field test twice. The interval between two trials of the open field test was 3 days. All experimental procedures were approved by the Institutional Animal Care and Use Committee at the Tzu Chi University (TCU, #96080 and #108082). The care and use for each mouse followed the institutional guidelines in accordance with 3Rs principles. The institutional guidelines were followed for the care and use of animals and conducted in accordance with the European Community Council Directive of November 24, 1986 (86/609/EEC).
Open field test
The apparatus for the open field test was a box (50 cm × 50 cm × 50 cm), and a 300 lux light intensity was applied to the central area. The central area was defined as a central 20 cm × 20 cm square, and the other region was defined as the peripheral area. Mice were placed in the center of the open field box, and the distance travelled and the time spent in the center of the open field box in 30 min were recorded. Entry into the central area was defined as the placement of all four paws in the central zone of the open field apparatus. The EthoVision system (Noldus Information Technology, Wageningen, the Netherlands) recorded the behavioral responses in the open field test, as performed in previous studies.[5],[25],[26],[27] Finally, the number of fecal boli was manually recorded after the open field test. Seventy percent alcohol was used to remove odors in the open field test apparatus between all sessions.
Dopamine turnover analysis
Instantly (within three min) after the final exposure to the open field test, according to the experimental design, mice were euthanized using an overdose of inhaled CO2, and their brains were removed and frozen with dry ice (−75°C). One hour later, the frozen brains were sectioned (300 μm) with a freezing microtome without any fixative procedures. The brain slices, according to a mouse brain Atlas More Details,[28] corresponding to the regions of the MPFC, NA, and ST were dissected as previously described.[5],[25],[29] The different brain regions were preserved at −20°C until examination.
High-pressure liquid chromatography equipped with electrochemical detection (BAS LC480, with a Phase II ODS column at 3.2 mm × 100 mm with 3-μm spheres, and an LC-4C EC detector; Bioanalytical Systems, West Lafayette, IN, USA) was used to measure the concentrations of dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC), a major metabolite of DA, as in previous studies.[5],[25],[29] DOPAC is the main product of the intracellular metabolism of DA through monoamine oxidases, and DOPAC levels, as well as the DOPAC/DA ratio, can be used to estimate the rate of DA metabolism.[30] Therefore, the ratio of DOPAC/DA and DOPAC alone were determined as measures of DAergic turnover or activity. Each tissue pellet was dissolved in NaOH (1N; Sigma-Aldrich, St. Louis, MO, USA) and assayed for protein content.[31]
Statistical analysis
The data are expressed as the mean ± standard error of the mean (SEM). The data were tested for normality by the Kolmogorov–Smirnov test before further statistical tests were performed. The differences in the open field test results and DAergic activities between Formosan wood mice and C57BL/6J mice were determined by two-tailed unpaired Student's t-test. Two-way ANOVA was used in the experiments of one and two exposures to the open field test. A post hoc Bonferroni test was used to examine the differences between the two groups after two-way ANOVA. Spearman's correlation coefficient was calculated to test correlations between the ratio of DOPAC/DA in the NA, ST and MPFC and the total distance travelled, and the distances travelled in the central and peripheral areas of the open field test. P < 0.05 were considered statistically significant for all statistical analyses.
Results | |  |
Behavioral responses related to locomotor and exploratory activity and central DAergic activities in the open field test
The behavioral responses in the open field test were evaluated in C57BL/6J mice (n = 9) and Formosan wood mice (n = 9) [Figure 1]. Time spent in [Figure 1]a; t16 = 4.334, P < 0.001] and number of entries [Figure 1]b; t16 = 2.415, P < 0.05] into the central area of the open field apparatus were higher in Formosan wood mice than in C57BL/6J mice. Defecation levels were similar in both groups [Figure 1]c; t16 = 0.224, P = 0.826]. The distance travelled by Formosan wood mice, both in the central area [Figure 1]d; t16 = 5.869, P < 0.001] and in the peripheral area [Figure 1]e; t16 = 3.177, P < 0.01] of the open field test, was significantly higher than that by C57BL/6J mice. The total distance travelled in the open field test by Formosan wood mice was higher than that by C57BL/6J mice [Figure 1]f; t16 = 5.869, P < 0.001]. | Figure 1: Higher levels of exploration and locomotion and lower levels of anxiety in the open field test in male Formosan wood mice than in male C57BL/6 mice. (a) Time spent in and (b) number of entries into the central area and (c) the amount of defecation in the open field test. Distance travelled in the (d) central area and (e) peripheral area, and (f) total distance travelled in the open field test. The vertical line above each bar represents the SEM (n = 9 for each group). *P < 0.05, **P < 0.01 and ***P < 0.001, compared with C57BL/6 mice. SEM: Standard error of the mean.
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Next, we examined the DOPAC and DA contents as we had done in our previous studies[5],[25] [Figure 2]. The DOPAC contents in the NA [Figure 2]a; t16 = 8.031, P < 0.001], ST [Figure 2]b; t16 = 7.648, P < 0.001] and MPFC [Figure 2]c; t16 = 13.890, P < 0.001] in Formosan wood mice (n = 9) after the open field test were higher than those in C57BL/6J mice (n = 9). The DA content in the NA (86.25 ± 2.21 vs. 85.27 ± 1.83), ST (108.82 ± 1.57 vs. 110.75 ± 1.84) and MPFC (1.10 ± 0.16 vs. 1.11 ± 0.14) in C57BL/6J mice and Formosan wood mice were similar, so the trends in the DOPAC/DA ratios and the DOPAC content were parallel. The DOPAC/DA ratios in the NA [Figure 2]d; t16 = 4.518, P < 0.001], ST [Figure 2]e; t16 = 4.904, P < 0.001] and MPFC [Figure 2]f; t16 = 8.901, P < 0.001] in Formosan wood mice (n = 9) after the open field test were higher than those in C57BL/6J mice (n = 9). | Figure 2: Increase in central dopaminergic activities, including DOPAC content and DOPAC/DA ratio, in the open field test in male Formosan wood mice compared with those in male C57BL/6 mice. DOPAC content in the (a) NA, (b) ST, and (c) MPFC in male C57BL/6 and Formosan wood mice. DOPAC/DA ratios in the (d) NA, (e) ST, and (f) MPFC in male C57BL/6 and Formosan wood mice. The vertical line above each bar represents the SEM (n = 9 for each group). ***P < 0.001, compared with C57BL/6 mice. DOPAC/DA: 3,4-Dihydroxyphenylacetic acid/Dopamine, MPFC: Medial prefrontal cortex, NA: Nucleus accumbens, ST: Striatum, SEM: Standard error of the mean.
Click here to view |
Distance travelled and central DAergic activities after one and two exposures to the open field test
The differences in locomotor activity [Figure 3]a, [Figure 3]b, [Figure 3]c] and central DAergic turnover [Figure 3]d, [Figure 3]e, [Figure 3]f] were examined after one and two exposures to the open field test. Because the distance travelled was the focus of this experimental design, we only showed the total distance travelled and the distance travelled in the central and peripheral areas in [Figure 3]. For the total distance travelled, a significant decrease after two exposures to the open field test [Figure 3]a; two-way ANOVA; species F1, 26 = 166.8, P < 0.001; exposures F1, 26 = 9.96, P < 0.01; interaction F1,26 = 4.04, P = 0.055] was found in C57BL/6J mice (n = 6) [Figure 3]a; F1, 13 = 3.69, P < 0.01] but not in Formosan wood mice (n = 6) [Figure 3]a; F1, 13 = 0.80, P = 0.44]. For the distance travelled in the central area, a significant decrease after two exposures to the open field test [Figure 3]b; two-way ANOVA; species F1, 26 = 309.0, P < 0.001; exposures F1, 26 = 7.72, P < 0.01; interaction F1, 26 = 1.99, P = 0.17] was found in C57BL/6J mice (n = 6) [Figure 3]b; F1,13 = 5.17, P < 0.001] but not in Formosan wood mice (n = 6) [Figure 3]b; F1, 13 = 0.75, P = 0.47]. For distance travelled in the peripheral area, a significant decrease after two exposures to the open field test [Figure 3]c; two-way ANOVA; species F1, 26 = 106.1, P < 0.001; exposures F1, 26 = 8.03, P < 0.01; interaction F1, 26 = 3.73, P = 0.06] was found in C57BL/6J mice (n = 6) [Figure 3]c; F1, 13 = 5.46, P < 0.001] but not in Formosan wood mice (n = 6) [Figure 3]c; F1, 13 = 0.50, P = 0.62]. The DOPAC/DA ratios were measured to examine the differences between one- and two-exposures to the open field tests on central DA activities [Figure 3]d, [Figure 3]e, [Figure 3]f]. For the DOPAC/DA ratio in the NA, a significant decrease after two exposures to the open field test [Figure 3]d; two-way ANOVA; species F1, 26 = 103.6, P < 0.001; exposures F1, 26 = 19.9, P < 0.001; interaction F1, 26 = 3.72, P = 0.06] was found in C57BL/6J mice (n = 6) [Figure 3]d; F1, 13 = 5.57, P < 0.001] but not in Formosan wood mice (n = 6) [Figure 3]d; F1, 13 = 1.55, P = 0.15]. For the DOPAC/DA ratio in the ST, a significant decrease after two exposures to the open field test [Figure 3]e; two-way ANOVA; species F1, 26 = 52.97, P < 0.001; exposures F1, 26 = 20.12, P < 0.001; interaction F1, 26 = 3.25, P = 0.08] was found in C57BL/6J mice (n = 6) [Figure 3]e; F1, 13 = 7.22, P < 0.001] but not in Formosan wood mice (n = 6) [Figure 3]e; F1, 13 = 1.49, P = 0.16]. For the DOPAC/DA ratio in the MPFC, a significant decrease after two exposures to the open field test [Figure 3]f; two-way ANOVA; species F1, 26 = 66.22, P < 0.001; exposures F1, 26 = 8.86, P < 0.01; interaction F1, 26 = 0.49, P = 0.49] was found in C57BL/6J mice (n = 6) [Figure 3]f; F1, 13 = 8.04, P < 0.001] but not in Formosan wood mice (n = 6) [Figure 3]f; F1, 13 = 1.86, P = 0.09]. Distance travelled and central DAergic activities decreased after two exposures to the open field test in C57BL/6J mice but not in Formosan wood mice [Figure 3]. | Figure 3: Differences in behavioral responses and central dopaminergic activities between one exposure (n = 9 for each group) and two exposures (n = 6 for each group) to the open field test in male Formosan wood mice and C57BL/6 mice. Distance travelled in (a) total, (b) the central area and (c) the peripheral area in male C57BL/6 and Formosan wood mice after one and two exposures to the open field test. DOPAC/DA ratios in the (d) NA, (e) ST, and (f) MPFC in male C57BL/6 and Formosan wood mice after one and two exposures to the open field test. The vertical line above each bar represents the SEM (n = 9 and 6 for each group). **P < 0.01 and ***P < 0.001, compared with the one exposure to the open field. DOPAC/DA: 3,4-Dihydroxyphenylacetic acid/Dopamine, MPFC: Medial prefrontal cortex, NA: Nucleus accumbens, ST: Striatum, SEM: Standard error of the mean.
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Then, we examined the associations between the total distance travelled and distance travelled in the central and peripheral areas and the NA, ST, and MPFC DOPAC/DA ratios after one and two exposures to the open field test [Table 1]. After one or two exposures to the open field test, the total distance travelled was not associated with the DOPAC/DA ratio in either C57BL/6J mice or Formosan wood mice in the NA, ST or MPFC [Table 1]; all P > 0.05]. The distance travelled in the central area after one or two exposures to the open field test was also not associated with the DOPAC/DA ratio in C57BL/6J mice or Formosan wood mice in the NA, ST or MPFC [Table 1]; all P > 0.05]. Finally, after one or two exposures to the open field test, the DOPAC/DA ratio was not associated with the distance travelled in the peripheral area in C57BL/6J mice or Formosan wood mice in the NA, ST or MPFC [Table 1]; all P > 0.05]. | Table 1: No correlations between distance travelled and central DAergic activities after one and two exposures to the open field test in either C57BL/6 mice (B6) or Formosan wood mice (WM)
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Discussion | |  |
Wood mice are widely spread throughout the world, including in Asia, Africa, and Europe; thus, they are not a protected species. Wood mice are known as a nimble rodent in the wild[32] and exhibit higher levels of locomotor activity even in the laboratory.[5] Previous studies on wood mice focused on parasitology, reproduction and ecobiology. Recent studies on wood mice have focused on stress responses, behavioral responses and environmental pollution in the wild.[33],[34],[35],[36],[37] Because wood mice are genetically close relatives of house mice (Mus musculus), an increasing number of studies have addressed behavioral or physiological differences between common laboratory mice and wood mice.[5],[9],[12]
Formosan wood mice/Taiwanese field mice (A. semotus) are an endemic Taiwanese rodent[1],[2] and display similarly deft movements and behaviors to those of other wood mice.[4],[32],[38],[39] The difference between these studies was that the wood mice were either caught from rural regions[32] or were bred in the laboratory.[5] In our previous study, we found that Formosan wood mice show higher levels of locomotor activity and exploratory behavioral responses in the elevated plus maze.[5] However, these mice regularly jumped off the platform of the elevated plus maze apparatus, which is mentioned in the interpretation of our results. Thus, in the current study, we used a better experimental approach, the open field test, to confirm our previous results using the elevated plus maze. In the present study, we compared behavioral responses and central DAergic activities in the open field test, without escape events, between common C57BL/6J laboratory mice and Formosan wood mice.
Formosan wood mice showed higher levels of explorations and central DAergic activities than C57BL/6J mice after one and two exposures to the open field test
Anxiety-like behavioral tests can be generally applied in rodents with the open field, elevated plus maze and light-dark box tests.[40],[41],[42],[43] There are some subtle differences in the degree of thigmotaxis, exploratory drive and locomotion measured in these tests. The open field test is the best behavioral test to measure locomotor activity and anxiety-like behavioral responses at the same time. Formosan wood mice spent more time in the central area and entered the central area of the open field apparatus more often than C57BL/6J mice [Figure 1]. These data suggest that Formosan wood mice exhibit fewer anxiety-like responses in novel conditions. Furthermore, longer distances travelled, either in the central and peripheral areas or in the total distance travelled, were exhibited by Formosan wood mice than by C57BL/6J mice. These data suggest that Formosan wood mice show higher levels of locomotor activity and explorative behaviors. Similar findings were found in our previous study, showing that Formosan wood mice exhibited increased levels of movement in both the open and closed arms of the elevated plus maze.[5] According to these findings[5] and to the open field test findings in this study, we suggest that Formosan wood mice exhibit comparatively high levels of exploratory behavior and locomotor activity and fewer anxiety-like behavioral responses, as found in a previous report.[27]
Central DAergic systems play an important role in the regulation of locomotion and aggressive, social, and reward-directed behaviors.[44],[45],[46] For example, chronic social stress in mice blunts DA turnover in the NA and reduces reward-directed behavior.[45] DAergic activity in the ST plays a regulatory role in locomotor activity.[46] After acute mild stress, DA turnover in the NA and ST is increased, and spontaneous locomotion is also elevated.[47] Mice with lower basal DA levels in the MPFC show deficits in prepulse inhibition, which is observed in schizophrenic patients,[48] as well as impairments in social behaviors.[49] Moreover, central DAergic activities in the MPFC, NA and ST have been reported to be related to exploratory or novelty-seeking behaviors.[21],[22],[23] In this study, the DAergic turnover in the MPFC, NA and ST was higher in Formosan wood mice than in C57BL/6J mice immediately after the open field test.
Higher levels of novelty seeking or locomotor activities are frequently associated with more novel stimulus, leading an animal to approach.[18],[19] Ordinarily, when animals encounter the same circumstances recurrently, their behavioral responses or reactions are attenuated. A decrease in the total distance travelled after re-exposure to the open field test was reported in C57BL/6 mice.[20],[50] A similar finding was found in C57BL/6J mice after two exposures to the open field tests in the present study. In contrast, Formosan wood mice still showed higher levels of locomotor activity after two exposures to the open field test. Possible explanations for this interesting finding could be that Formosan wood mice either show less-adaptive responses or have a deficiency in learning and memory. Our recent study shows that Formosan wood mice display good performance in learning and memory in the novel object recognition test.[51] Therefore, these wood mice having a less-adaptive response was one of the better explanations. Previous study has speculated that in the natural habitat, where natural resources sometimes become transiently smaller and/or predation pressure is increased, the behavioral characteristics of increased voluntary activity associated with curiosity and exploration and may increase the chances of survival.[52] Further evidence or study is needed before we can make this assumption as the previous study.
We then examined the association between the central DAergic activities and the locomotor activity in C57BL/6J mice and Formosan wood mice after one or two exposures to the open field test. For the total distance travelled and the distance travelled in the central and peripheral areas of the open field test, there was not a significant correlation with the DAergic turnover in the NA, ST, and MPFC in C57BL/6J mice nor in Formosan wood mice. A previous study suggested that central DAergic activities are associated with locomotor activity and exploratory behavior in C57BL/6J mice,[53] and even in prairie voles.[54] Unfortunately, we cannot determine whether this relationship is true from the data in the present study. The smaller sample size is one possible reason. The other possibility is that the environmental impact was lower than that of pharmacological interventions and treatments since previous studies used bisphenol A or amphetamine as treatments.[53],[54]
Conclusion | |  |
The present study showed the differences in locomotor activities and exploration behavior and central DAergic activities between Formosan wood mice and C57BL/6J mice in the open field test. Higher levels of locomotor activity and central DAergic activity still existed in Formosan wood mice even after two exposures to the open field test.
Acknowledgments
The authors appreciate the technical assistance received from R. J. Chen and the Laboratory Animal Center at Tzu Chi University. This work was financially supported in part by the Ministry of Science and Technology in Taiwan (105-2320-B-320-011-MY3 and 108-2320-B-320-005 to KRS and 107-2410-H-277-001 to SCY) and the Tzu Chi Foundation (TCRPP107012 and TCRPP108012 to KRS). The funder had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. This manuscript was English-edited by Nature Research Editing.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1]
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