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Behavioural Processes 60 (2002) 181 /190 www.elsevier.


Comparative analysis of the anxiety-related behaviors in four inbred mice

Sanghyeon Kim a, Sukchan Lee a, Sungho Ryu b, Jea-gyun Suk b, Chankyu Park a,*

National Creative Research Initiative Center for Behavioral Genetics, Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Yusong-Ku, Taejon 305-701, Republic of Korea b Division of Computer Science, Korea Advanced Institute of Science and Technology, Yusong-Ku, Taejon 305-701, Republic of Korea Received 26 March 2001; received in revised form 1 October 2001; accepted 3 October 2001

Abstract An anxiety-related behavior is an emotional response of an organism, which is quantitatively measured by several behavioral paradigms. We employed two most frequently used behavioral tests, the open field and light /dark exploration, to comparatively analyze the anxiety-like behaviors in four inbred mice. For an accurate recording of movement, motion analysis software was developed that acquires a real-time video input to generate a behavioral path. Effects of the strains on the test results were evaluated by ANOVA with the Newman-Keuls post hoc comparison. Eight different behavioral indices, four from each tests, were grouped into two classes; the results of duration, center crossing, transition, rearing, and ambulation indicate strain differences of FVB/N /C57BL/6J E/BALB/cA E/CBA/N (I), while stretched-attend posture, peeping, and defecation show the tendency of FVB/N 0/C57BL/6J B/CBA/N 0/BALB/cA (II). The peeping is a novel type of behavior observed in this work. Although there is a variation among behavioral indices in their discrimination between inbred lines, the behaviors are highly correlated one another such that each class I or class II behaviors are clustered on two orthogonal factor planes as a result of the principal component analysis. The polarization of each inbred line toward these two behavioral biases may reflect genetic backgrounds of these strains. # 2002 Elsevier Science B.V. All rights reserved.
Keywords: Anxiety-related behavior; Inbred mouse; Open eld test; Light /dark transition test; Motion analysis

1. Introduction Anxiety-related behavior is one of the frequently observed behavioral phenotypes reflecting differences in genetic backgrounds. The light /dark (L / D) exploration has been used to measure anxietylike behaviors of mice, which is based on a conflict between exploratory tendency toward novel environment and avoiding tendency to brightly lit place

* Corresponding author. Tel.: '/82-42-869-2629; fax: '/8242-869-2610 E-mail address: ckpark@mail.kaist.ac.kr (C. Park).

0376-6357/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 6 - 6 3 5 7 ( 0 2 ) 0 0 0 8 5 - 2


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(Crawley et al., 1997). Pharmacological characterization of the L /D model has shown that anxiolytic drugs affect behaviors including the number of transition and stretched attend posture (Grewal et al., 1997), while other classes of drugs do not. The open field test assesses locomotion activity as well as an emotional reactivity in a novel environment. The task with an elevated plus maze has also been used for anxiety model, which provides broadly consistent data with other test results. Recent explosion of transgenic and knockout studies of mice increases the necessity of behavioral test paradigms that would eventually deal with all the phenotypic differences resulted from neuro-cerebral diversity (Gogos et al., 1998; Crawley, 1999; Mohn et al., 1999). Various behavioral studies related to anxiety were conducted for inbred mice that in most cases include C57BL/6. Diverse genetic backgrounds and their F1 hybrids were also compared for 12 inbred strains including C57BL/6, BALB/c, and FVB/N (Logue et al., 1997). To better describe a particular behavioral phenotype, the choice of appropriate test model and genetic background appears crucial. However, a limited availability of behavioral measures and an apparent diversity of inbred animals make it difficult to select suitable subjects. It is because the most commonly used inbred mice often have extreme behavioral biases. Thus, it will be useful to have behavioral data available for as many animals from genetically diverse origins as one could. We performed the open field and L /D transition tests for the 4 inbred strains of mice in order to assess various anxiety-like behaviors in diverse genetic backgrounds. By exploiting a broad genetic spectrum of inbred lines, we were able to categorize behaviors observed, which are highly correlated with anxiety-like behaviors described previously (Belzung and Le Pape, 1994). In addition, we introduced a more quantitative analysis of behavioral path, which can be applicable in various test situations. The whole system consists of digital recording, tracking, and analysis with the motion analysis software. Based on their discriminative ordering among the 4 inbred mice, the behaviors tested were grouped into two classes,

which are consistent with the results obtained by statistical clustering.

2. Materials and methods 2.1. Animals Four inbred strains of mice, FVB/N (F), C57BL/6J (B6), CBA/N (C), and BALB/cA (BC), were purchased from Daehan Breeding Center Co. Ltd (Seoul, Korea). All mice were raised as a group of five to six per cage in a temperature controlled facility at 22 8C under 12 h of L /D cycle with lights on at 07:00. Humidity was maintained at 55% with food and water freely available. Behavior tests were performed during the day between 09:00 and 17:00 in the facility where mice were raised. Animals were delivered at the age of 4 /5 weeks. For the L /D transition test, mice with 7/8 weeks of age were used while the open field test used 10 /11 week old mice. The same cohorts of animals were used for both the L / D transition and open-field tests of anxiety. Behaviors were recorded with a digital video camera (Sony, mini DV No. DCR-PC3, Sony electronics), transferred to PC with the DV raptor interface (Canopus), and analyzed with video replay on either PC monitor or the regular TV monitor. The use of remotely controlled video setup allowed us to avoid any disturbance frequently caused by an observer. The recorded motions were stored in Sony digital video cassette or on CD-ROM using the software provided with the video interface. All experiments followed the NIH Guideline for the Care and Use of Laboratory Animals. 2.2. Light /dark transition assay A total of 20 mice from each inbred strain with equal number of genders were used in the L /D transition test. The test kit was made of black perspex (40 )/25 cm2 with 16 cm height). The light chamber (25 )/27 cm2) and dark chamber (25 )/13 cm2) were divided by a wall with a small vestibule (circle with the diameter of 10 cm). The large chamber was open, transparent, and brightly

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illuminated by fluorescent lights (180 lux at cage floor), while the small chamber was painted and dark. Mouse was initially placed in the dark chamber. The recorded video files were analyzed by the categories including the duration in the light chamber (DUR) and the numbers of stretchattend posture (SAP, Grewal et al., 1997), peeping (PEE), and transition (TRANS) scored for 5 min. In between each mouse, faecal pellets were removed and the apparatus was thoroughly cleaned using 10% ethanol and dried prior to the next mouse. The SAP was regarded as a posture of mouse with the front legs in the light/dark chamber and the rear ones in the dark/light chamber. The PEE involves an exploration with a head toward the light or dark chamber without the movement of any leg. In most cases, the movements were toward the light chamber.

cleaned using 10% ethanol and dried prior to the next mouse. In the open field test, we observed a gender effect only for the B6 mice. It was reported that estrus cycle of the female may result in sex difference in the anxiety-related behavior (Frye et al., 2000). Since the purpose of our study is to compare strains in terms of their anxiety-related behaviors, we did not include the data of female B6 mice that exhibited statistically significant behavioral difference from those of B6 males. The gender effect was not found in other strains used here.

2.4. General locomotor activity test As a primary screen for inbred line variability, spontaneous locomotor activity was assessed in a stimulus-free test situation. The assay was performed with 5 female and 6 male mice in a standard cage (24 )/18 )/13 cm3) after adaptation for 1 h. The 5-min recording was taken for each mouse, during which mice were constantly mobile without taking a rest. The movements of mice were analyzed with the Mouse tracker software as described.

2.3. Open eld test Ten male and 8 female mice from each inbred strains were used to perform open field test. The test was mostly carried out a week after the L/D test. The open field consisted of a circular arena (60 cm diameter) made of transparent perspex with walls of 20 cm high. Mice were acclimated to a normally illuminated test room for 5 min before testing. The open field was brightly lit with 180 lux at floor. The mice were placed in the center of arena at the start of 5-min test period, and their movement around the arena was recorded using the same videotracking system describe above. The following categories of behavior were recorded: defecation score (boli/session, OFD); vertical counts (rearing activity, OFR); distance traveled (ambulation, OFA); and the number of center crossing (OFC). The center was defined as a circular area (10 cm diameter) in the central part of arena. Entrance of all legs in this area was counted as a crossing. The ambulation was obtained as the sum of traveled distances measured in every 1-s intervals, which was calculated by the 2 motion analysis program (Mouse tracker ver. 1.0) developed for this work. As for the L /D transition, in between each mouse, faecal pellets were removed and the open field was thoroughly

2.5. Video-tracking with motion analysis software For an analysis of mouse behavior, software tracking the movement of mouse was developed. Since recording of behavior is made with the digital video camera, datafile for motion was obtained as a digital format that can easily be handled with the PC program. The Windowsbased program can analyze an AVI file containing mouse behavior taken for 5 min to generate a path data. The area of mouse movement is defined by setting a boundary with a shape of circle or square. After adjusting the contrast to detect an object from the background, initial position of mouse is selected manually by an operator. Tracking the mouse movement is made with a series of algorithms including the process of background smoothing, delation operation to increase signal


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to noise ratio, and calculating the center of mass. The path of mouse can be obtained as a sum of average distances for the unit time intervals of 1/ 30, 0.1, 0.5, 1, or 2 s. These data are stored in a format to be used for any kind of spreadsheet program. The current version, the Mouse tracker 1.0, runs in the Windows95 and 98 environments. Although the program was successfully exploited in the present work to analyze an open-field activity, it can also be used for other test situations including the L /D transition test, the Morris learning paradigm, etc. The capability of adding a new window for analysis provides versatility for more sophisticated behavioral analysis. The current version of our program for PCWindows uses any AVI file as an input to generate data compatible with the spreadsheet program. After processing a digitally recorded behavioral data, the obtained information can be interpreted either quantitatively as a form of distance traveled

(ambulation) or qualitatively as a behavioral track shown in Fig. 1.

2.6. Statistical analysis Data were initially assessed by the Shapiro-Wilk test in the SAS statistical program (SAS Institute Inc.) to determine the homogeneity of variances and its suitability for parametric analysis. Given the results of this preliminary test regarding the number of independent factors involved, sample size, and the quantitative nature of the data, all data were subsequently analyzed using one and two-way analysis of variance (ANOVA). Therefore, a one-way ANOVA was performed with strain as the grouping variable. For comparing differences in parameters between inbred strains, the Newman-Keuls test was conducted for post hoc comparison (SAS Institute Inc.). The Pearson correlation coefficients for actual data were esti-

Fig. 1. Behavioral tracks observed in the open eld arena. These are the graphical outputs of the motion analysis program (Mouse tracker v. 1.0) developed here. The behaviors were initially recorded with digital camcorder, which were analyzed and plotted with Excel and Sigmaplot, respectively. The movements were segmented into 1-min intervals. Strains with high anxiety level, CBA and BALB/c, display distinct patterns of thigmotaxis with reduced movements. The testing conditions are as described in Section 2.

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mated using the SAS program. In addition, principal component analysis was performed with the SAS program, in which the rotation was accomplished by the Varimax procedure.

3. Result 3.1. Comparative analysis of behavioral data obtained by the L /D transition and open-eld tests We performed the L /D transition and openfield tests for the 4 inbred strains including FVB/ N, C57BL/6J, BALB/cA, and CBA/N, and obtained results on eight different behaviors (Table 1). Four behaviors, DUR, TRANS, PEE, and SAP, are from the L /D test, while defecation (OFD), center crossing (OFC), rearing (OFR), and ambulation (OFA) are from the open-field test. ANOVA indicates that the effects of strains are all significant (TRANS, F (3, 76) 0/83.87; OFR, F (3, 68) 0/122.31; OFA, F (3, 68)0/119.92; DUR, F (3, 76) 0/16.05; OFC, F (3, 68) 0/42.50; OFD, F (3, 68) 0/32.32; PEE, F (3, 76) 0/52.22; SAP, F (3, 76) 0/30.83, all P B/0.001). A post hoc comparison with Newman-Keuls test classify the behaviors into two different groups in terms of their biases of either increasing or decreasing tendencies in the four inbred lines. The FVB/N /C57BL/6JE/ BALB/cA E/CBA/N (class I) pattern of inbred line differences were obtained in four behaviors including DUR, OFC, TRANS, OFR, and OFA. When we analyzed the behavioral paths of each inbred mice more carefully with the Mouse tracker
Table 1 Results of various behavioral tests in the four inbred mice Strain L /D transition DUR FVB/N C57BL/6J BALB/cA CBA/N 183.49/3.4 110.89/6.2# 111.59/20.7# 55.59/14.4 TRANS 19.49/1.1 10.69/0.8 2.99/0.7# 2.89/0.8# PEE 4.09/0.7 6.69/0.7# 27.29/2.4 12.39/1.2

(Fig. 1), the pattern, consistent with the data of OFC, was observed. The SAP, PEE, and OFD behaviors shows a slightly shuffled order of FVB/ N 0/C57BL/6JB/CBA/N 0/BALB/cA (class II), in which CBA and BALB/c are reversed from the class I. In each class, discriminative abilities vary among different behaviors with OFA the highest (P B/0.01). The PEE behavior, newly described here, shows a very similar pattern to SAP. In the class I behaviors, similar patterns were also observed between DUR and OFC, and between TRANS and OFR.

3.2. Correlation analysis and statistical clustering of the behaviors tested Based on high scores of inter-correlation (Henderson, 1967), behaviors observed in the open field and L /D tests were generally assumed to be associated with anxiety. However, the more strict definition of anxiety behavior was obtained for ones altered by an anxiolytic drug, diazepam, e.g. the TRANS behavior measured in L /D test (Crawley et al., 1997). All the behavioral indices analysed in this work exhibit statistically significant correlation with the transition score (Table 2, Pearson correlation coefficients r /0.42, P B/ 0.0001). Moreover, SAP, suggested as an anxiety behavior in mouse and rat (Grewal et al., 1997), shows correlation with all behaviors tested except for duration (r /0.31, P B/0.01). Therefore, the behavioral indices obtained here are, to some extent, likely to have anxiety component.

Open field SAP 0.89/0.2 0.29/0.1# 6.69/0.8 3.09/0.6


OFD 0.19/0.1 0.69/0.3# 4.99/0.6* 3.49/0.4*


OFC 11.69/1.0 4.99/0.5# 4.49/0.6# 1.99/0.4

OFR 68.69/3.0 30.39/1.6 8.89/1.2# 14.99/3.3#

OFA 4061.79/130.4 2686.89/113.5 1837.99/75.4 1422.69/93.3

Behavioral data were presented with the same units (number), except for DUR (second), OFD (no. of boli), and OFA (cm). Standard errors are also shown.#*The marks represent a pair of data, not significantly different from each other as a result of the post hoc analysis.


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Table 2 Correlation between various behavioral indices Behavior L /D transition TRANS TRANS DUR PEE SAP OFD OFC OFR OFA DUR PEE SAP Open field OFD OFC OFR OFA

0.59 (/0.54 (/0.53 (/0.61 0.64 0.80 0.80

(/0.16 (/0.18 (/0.25 0.43 0.42 0.50

0.59 0.73 (/0.30 (/0.57 (/0.52

0.50 (/0.32 (/0.46 (/0.45

(/0.41 (/0.61 (/0.60

0.70 0.78


Actual data obtained for 8 females and 10 males of each inbred strains were used.The coefficients more than 0.31 are marked with italicised characters.Critical values for r can be assessed; P 0/0.01 if r E/0.302, P 0/0.001 if r E/0.380, and P0/0.0001 if r E/0.442.

Since it is believed that many behavioral indices share underlying locomotor activity, we estimated the locomotor effect by conducting a simple experiment in a conflict-free test situation. Locomotor activities tested for each inbred lines were F (722.99/84.4 cm), B6(476.99/61.4), C(136.99/ 32.5), and BC(393.69/31.7), which are comparable with the data obtained by others (Rogers et al., 1999). Although they used different method, an interruption of infrared beams, the numbers obtained for B6, C, and BC were quantitatively similar. When correlation of the locomotor activity was analyzed with other behaviors tested, we obtained two different classes (Table 2); TRANS (r0/0.69, P B/0.00001), OFA (0.65, P B/0.00001), OFR (0.63, P B/0.00001), OFC (0.56, P B/0.0001), and DUR (0.51, P B/0.0005) as highly correlated, whereas OFD ((/0.46, P /0.001), SAP ((/0.22, P /0.1), and PEE ((/0.21, P /0.1) as less or little correlated. While most behaviors are, to some degree, associated with locomotor activity, the PEE and SAP seem to have no correlation with this activity, although they seem to share other anxiety-related component. The principal component analysis of the above behavioral indices allows a clustering of behaviors into two different groups (Fig. 2). The first group with five behaviors has high degree of correlation with the factor 1 explaining about 60% of the variance, while the second cluster is more associated with the factor 2 that corresponds to 15% of the variance. It is noted that the previous classi-

fication of behaviors based on their discrimination pattern among the four inbred lines generates essentially the same conclusion as that of the principal component analysis.

4. Discussion Behavioral paradigms measuring fear-like reactions of animal has been used as a model to study human emotion. In mouse, several tests were developed to detect anxiety level in various fearinducing conditions. Here, we chose the two most commonly employed tests, the L /D transition and open field, to assess mouse behaviors with a particular emphasis on their ability to discriminate diverse genetic spectrum of inbred lines. Behaviors observed in the tests are highly correlated each other, perhaps reflecting their sharing of the anxiety-associated component. The genetic diversity represented by the four inbred lines provides a wide spectrum of test subjects that enabled us to easily categorize various behaviors measured in our test. Indeed, the pattern of strain discrimination allows a classification of these behaviors into two groups, which are coincided with the results of principal component analysis. The pattern of behavioral differences among the inbred lines is similar to that of previous analysis (Crawley et al., 1997; Rogers et al., 1999) primarily carried out for C57BL/6J and BALB/cA. In the class I behaviors, the open-field ambulation is the

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Fig. 2. Results of principal component analysis. The two factors are suggested to explain most of the variances. The rotation was accomplished by the Varimax method in the SAS package. The variations covered by the factor 1 and factors 2 are 60 and 15%, respectively.

most discriminative while others divide inbred strains into three groups. Since it is possible that OFA may, to some extent, depend upon the general locomotor activity, i.e. high correlation of 0.65 (P B/0.00001), we examine whether such discriminative capability comes from the locomotor differences. Even with a simple locomotor experiment, the degrees of locomotion were indistinguishable between C57 and BALB/c (after post hoc analysis) unlike OFA. Indeed, a previous QTL analysis indicated that the major loci linked to

OFA were not linked to the ones for general locomotion (Flint et al., 1995), implying that OFA has an anxiety component that is not fully associated with general locomotion. In contrast to the class I behaviors that are considerably associated with the general locomotion (more than 0.5 of correlation), the class II behaviors have much less degree of correlation with the general locomotion. In fact, PEE and SAP were not associated with it, thereby providing a unique category of behavior observed with L /D


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Fig. 3. Polarization of behavioral scores in the four inbred backgrounds. Radial plot was generated with the data in Table 1 after the maximum scores were set to 1. The behaviors are arranged according to their clustering and the degree of correlation with one another. For example, OFA and PEE, the most highly correlated with the factor 1 (0.81) and 2 (0.89), respectively, are horizontally located.

test. PEE, a novel type of behavior observed in this work, appears to be very similar to SAP, although they are phenotypically distinguishable. Perhaps in some testing apparatus with a photocell attached to the vestibule of L/D boundary (Gershenfeld and Paul, 1997), the PEE, besides SAP, might have been scored together with the number of transition that is automatically detected as an interruption of the light beam. The motion analysis system introduced here can be used as an alternative to obtain more accurate recording with better resolution. An open field activity has been recorded with the photocell beam equipment. In such system, ambulations of highly

emotional individuals are generally over-estimated due to a frequent body movement without a significant displacement, which is still detected by the equipment. In our analysis with the Mouse tracker v. 1.0, this artifact can be abolished by increasing the time interval of sampling data. As a matter of fact, increasing such interval from 1/30 to 1/2 s adjusts the mean values of total travel distances for 5 min in the open field from 2491.09/ 90.1 cm for CBA and 2643.99/91.8 cm for BALB/c to 1422.69/93.3 and 1656.59/70.5, respectively. In addition to its advantage of both quantitative and qualitative evaluations, the power of the program has been further expanded to a more sophisticated

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behavioral analysis by creating a separate window. This is particularly useful in measuring the center crossing, duration, etc. An acquisition of highresolution behavioral track may allow an analysis of various behavioral patterns in the future after appropriate mathematical transformation. The degree of correlation between behaviors may reflect the degree of sharing between genetic traits underlying each behavior. Indeed, various QTL analysis revealed different sets of associated loci with some overlap with one another. The chromosomal loci primarily screened for ambulation were mapped to six different locations (Chromosome # 1, 4, 12, 15, 17, 18), which are not equally associated with other behavioral measures including defecation (Flint et al., 1995). A similar study with transition and center crossing revealed QTLs mapped to chromosome 10/6/15/19/X and 1/ 6/14, respectively (Gershenfeld and Paul, 1997). Thus, an exact behavioral phenotyping is likely to provide a solid ground for identifying sets of gene as well as their localizations. The inbred lines selected in our test seem to represent at least some genetic spectrum in terms of anxiety levels (Fig. 3). In particular, the FVB/N strain exhibits an extremely lower level of anxiety, which is comparable with the previous data (Voikar et al., 2001). Since the FVB/N stain contain the retinal degeneration mutation (Taketo et al., 1991), there is a possibility that this mutation may affect the ability of sensory perception required for behavioral tests. However, it does not seem to be the case because the C3H strain carrying the same mutation does not exhibit an extremely lower level of anxiety (Kopp et al., 1999). The FVB/N inbred line was fairly recently established and reported to have an advantage in generating a large and prominent pronuclei in zygote for microinjection (Taketo et al., 1991). The lower level of anxiety makes it useful for this mouse to be used as a transgenic or knock-out host for genes expected to increase anxiety. Occasionally, effects of genetically manipulated organisms were masked by an extreme behavioral bias of a test organism (Kustova et al., 1998). The fact that FVB/N, an albino strain, lies in other extreme of BALB/c in terms of anxiety level indicates that albinism is not significantly asso-

ciated with anxiety-related behavior. This is consistent with the results obtained for the congenic pigmented BALB/c strain (Lassalle et al., 1994).

Acknowledgements This work is supported by a grant from the Creative Research Initiative Program.

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