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The relation between skin conductance level and plus-maze behavior in male mice
Physiology & Behavior, Vol. 64 No. 4, pp. 573–576, 1998 © 1998 Elsevier Science Inc. All rights reserved. Printed in the U.S.A. 0031-9384/98 $19.00 1 .00
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The Relation Between Skin Conductance Level and Plus-Maze Behavior in Male Mice ¨ ER,1* NAZAN DOLU,* C¸IG ˘ DEM O ¨ ZESMI,* O ¨ ZLEM S¸AHIN* AND AHMET U ¨ LGEN† CEM SU *Department of Physiology, Faculty of Medicine and †Science and Art Faculty, Erciyes University, 38039 Kayseri, Turkey Received 10 February 1997; Accepted 28 January 1998 ¨ ER, C., N. DOLU, C¸. O ¨ ZESMI˙, O ¨ . S¸AHI˙N AND A. U ¨ LGEN. The relation between skin conductance level and plus-maze behavior SU in male mice. PHYSIOL BEHAV 64(4) 573–576, 1998.—The present study examined the correlation between anxiety scores and skin conductance level in 29 male Swiss Albino mice. Skin conductance (SC) was recorded with the SC unit and IBM-AT computer. Anxiety scores of mice were obtained from the elevated plus-maze test. The main result of the present study indicates that SC levels (SCLs) are negatively correlated with plus-maze behavior scores (both entries and time spent on the open arms). Our results are consistent with the findings which suggests that the higher the anxiety level the higher the SCL. This study further demonstrates the utility of SCL as a measurement for identifying anxiety in mouse. The interrelation between SCLs and plus-maze scores and possible explanations of the results are discussed. © 1998 Elsevier Science Inc. Skin conductance
Anxiety
Elevated plus-maze
THE relationship between anxiety and electrodermal activity (EDA) has been widely studied and well known. The results of the studies on human subjects indicate that anxious subjects have greater sweat gland activity (shown by by skin conductance level (SCL) and nonspecific fluctuations) and reactivity (shown by skin conductance (SC) response) than nonanxious or low anxious subjects (2,3,6,7,9,16,29). However, data in animals are lacking. We have only known three studies which have been made on this topic in mice since 1980. In these studies, the SC reaction-habituation test was suggested to assess anxiolytic activities of some drugs (25,35,39). Moreover, there is no study addressing the relationship between anxiety and SCL in mice. The elevated plus-maze test, a well-established animal model of anxiety, is based upon the natural aversion of rodents to heights and open space (27) and has been validated for both rats (30) and mice (21). It is not only sensitive to pharmacological manipulations (4,5,22,28) but has also revealed increases in anxiety produced by prior exposure to stressors (1,32,33,36,40). In this paradigm, mice are placed on an elevated maze that has two arms with sides (closed arms), and two other arms without sides (open arms). Entries from a square center zone into open or closed arms are recorded, as is the time spent on arms. The number of entries is also counted. It is generally found that the number of entries and time spent in the open arms are increased by anxiolytic drugs and decreased by anxiogenic drugs (31). The present study was undertaken: a) to determine the relation1
ship between the behavioral scores of mice tested on the elevated plus-maze test and SCLs; and b) to provide external validation of the maze as a test of anxiety. Based on the results of studies mentioned above, it is predicted that mice having higher levels of anxiety would enter to a lesser extent into the open arms than those having lower levels of anxiety. It is also predicted that SCLs would be higher in mice having higher levels of anxiety than their counterparts with lower anxiety levels. MATERIALS AND METHODS
Animals Twenty-nine male Swiss mice (10 –12 weeks) were housed in groups of 3– 6/cage (60 3 60 3 30 cm polypropylene cages) under a reversed 12-h light:dark cycle (lights on at 0700 hours) and in a constant temperature and humidity controlled environment (21 6 1°C and 30%, respectively). The mice had free access to food and tap water. Elevated Plus-Maze The elevated plus-maze was a modification of that validated for Swiss mice by Lister (21). The elevated plus-maze was made of plexiglas. It consisted of two open arms and two enclosed arms (30 cm long and 5 cm wide) that were opposite each other. The arms extended from a central plate, which is also made of plexiglas (5
To whom requests for reprints should be addressed. E-mail: [email protected]
573
¨ ER ET AL. SU
574 cm long and 5 cm wide). The walls of the enclosed arms was 15 cm in height and 1 cm in thickness. The plus-maze stood on a stand, so that it could be elevated approximately 40 cm above the ground. The central platform and maze floor were made from opaque plexiglas while the walls of the closed arms were transparent plexiglas. Behavioral Measures Each mouse was individually placed on the center of the platform of the maze facing an open arm. The mouse was allowed a 5-min pretest habituation period to explore the maze (3,13). Following this period, three measures were taken during test period: 1) total arm entries (by all four paws); 2) entries into the open arms; 3) total time spent in open arms. The mouse was removed following a 5-min test session. SCL was recorded, without losing any time. The apparatus was wiped clean with a daub sponge and dry cloths between tests. Behavioral testing was always carried out between 0900 and 1500 hours. The time spent in open arms and the number of entries were calculated by a PC computer. As soon as the mouse entered one of the open arms, experimenter pressed a key. When the front paws of the mouse have crossed into central zone the experimenter pressed another key. The time (in seconds) between pressing the keys and the number of entries were calculated. The same procedure for closed arms was repeated with the other two keys. Because open arms scores are the best measure of general anxiety in the maze, open arm entries and time spent on the open arms were expressed as percentage of total entries and test duration, respectively. Skin Conductance SC was recorded between the paw pads of both hindlimbs using Ag/AgCl electrodes after the maze test. NaCl electrode (0.05 M) jelly was placed between the skin and the electrodes. Electrodes were connected to a SC unit that was built in our laboratory. The technical specifications of this unit have been published elsewhere (38). Briefly, it conforms basically to the Lykken-Venables method (24). SC unit output was digitized and stored on-line by an IBM-AT computer. The mean of SC recorded for 2 min was expressed as SCL [ln (mmho)/cm2 per electrode area]. Statistics For statistical analysis, the data were initially analyzed by one-way ANOVA. When significant intergroup differences were detected, Newman–Keuls tests were applied to compare each pair of groups. Correlation analysis were done using three variables (SCL, percent open arm entries, and percent open arm time spent). All statistical tests were performed with SPSS software. RESULTS
The SCLs and Behavioral Test Scores in Mice with Different Anxiety Levels In average, all mice tested made 48.46% entries into and spent 13.86% of time on the open arms. These mice were subsequently divided into three subgroups according to the scores of elevated plus-maze. In the low-anxiety group, mice had higher scores than its mean values. In the intermediate-anxiety group, one of the scores (percent entry number or percent time spent) was lower than its mean value; the other was higher. In the high-anxiety group, both scores were lower than their own means. Table 1 shows SCLs and behavioral test scores measured from mice with different anxiety status. As shown in Table 1, one-way ANOVA indicated
TABLE 1 SKIN CONDUCTANCE LEVELS AND ELEVATED PLUS-MAZE TEST SCORES IN MICE WITH DIFFERENT ANXIETY LEVEL Anxiety level
n
SCL (mmho)
All mice tested Group 1 Group 2 Group 3 one-way ANOVA F(2,28) p,
29 5 14 10
3.36 6 0.37 2.94 6 0.35 3.40 6 0.33a 3.51 6 0.28a
Entries (%)
time spent (%)
48.46 6 11.43 13.86 6 10.0 63.00 6 8.91 31.58 6 7.57 49.23 6 9.18a 12.16 6 5.91a 39.12 6 5.64a,b 7.37 6 3.10a,b
5.45 0.01
14.99 0.0001
34.30 0.0001
The data are means 6 standard deviations. SCL: skin conductance level; Group 1: mice with low anxiety; Group 2: mice with moderate anxiety; Group 3: mice with high anxiety; a: significantly different when compared with group 1 (Newman–Keuls test); b: significantly different when compared with Group 2 (Newman–Keuls test).
significant differences between groups for all variables (F 5 5.45, p , 0.01, for SCL; F 5 14.99, p , 0.0001, for open arm entries; and F 5 34.29, p , 0.0001, for time spent in open arms). SCL values were found to be significantly lower in low-anxiety group when compared with both moderate- and high-anxiety groups (p , 0.05). No significant difference was observed between moderate and high anxiety groups, when SCL values were compared. Both plus-maze scores were also found to be significantly higher in the low-anxiety group when compared with both the moderate- and high-anxiety groups (p , 0.05). There were significant differences between the moderate- and high-anxiety groups (p , 0.05). The Correlation Between SCLs and Behavioral Test Scores The correlation matrix reveals powerful correlation between SCL, entries into the open arms, and time spent on them. A significant negative correlation in the SCL was found regarding the percent entries into open arm (r 5 20.707, p , 0.001) and the percent time into open arms (r 5 [minusa]0.559, p , 0.002). These correlations are shown in Fig. 1. There was also a significant correlation between the percentage of entries into the open arms and the percentage of time spent on the open arms (r 5 0.527, p , 0.003). We also found a weak correlation between closed arm entries and SCL (r 5 0.39, p , 0.04) when the number of entries into the closed arms was mentioned as frequency. DISCUSSION
The elevated plus-maze introduced by Pellow et al. (31) is often used in studies to identify anxiolytic and anxiogenic drugs (4,5,21,22,28). Because the open arms evoke a greater strength of fear drive than the closed arms, mice are willing to enter into enclosed arms and stay a longer time on enclosed arms during test period (26). Anxiolytic drugs, such as benzodiazepines, barbiturates, and ethanol, increase the number of entries into open arms and time spent on open arms; in contrast, anxiogenic drugs, such as yohimbine, decrease both (31). The elevated plus-maze is also sensitive on the anxiety level of rodents (26,32). In the present study, the mice who exhibit increased anxiety-like behavior, that is, they made more frequent entries into closed arms and stayed a longer time in them, showed higher SCLs than those who exhibit decreased anxiety-like behavior. Moreover, there was a correlation between SCLs and anxiety behavior measures with elevated plusmaze. Because elevated SCL is already established as a correlate
EDA AND PLUS-MAZE BEHAVIOR IN MICE
575
FIG. 1. The relationship between skin conductance level (SCL, mmho) and percent time spent (dotted line, triangle dot) and percent entries (solid line, circle dot) into open arms.
of anxiety (20,23), the observed SCL increase is perhaps explained with anxiety associated with anticipation of an aversive event. Our findings indicate the utility of SC as a measurement for identifying anxiety in mice. Several studies (25,35,39) suggested the utility of SC habituation, another electrodermal activity parameter, as a tool to assess anxiolytic and anxiogenic activities of some drugs. However, habituation can be measured by several different methods, such as three nonresponding criteria, two nonresponding criteria, and inversion criteria. There are two problems with these criteria: 1) they do not make use of all data, and 2) they cannot discriminate between subjects who failed to meet in the total number of trials administered but who may nonetheless vary greatly in the amount of habituation shown. Therefore, we concluded that the SCL would appear to be more suitable in identifying anxiety than SC habituation tests in mice. Involvement of neurotransmitter systems in behavioral changes in elevated plus-maze (10 –12,15,17–19,21) and in electrodermal activity (37) has been studied in rodents. The results obtained from elevated plus-maze experiments indicated that activation of the noradrenergic system may induce anxiogenic-like behavior (10,19, 21). Furthermore, activated Nagic system may also induce electrodermal changes (37). File et al. (11,12) indicated that exposure to the plus-maze decreased cortical GABA function and increased
hippocampal 5-HT function in rats. Therefore, we concluded that central catecholaminergic hyperactivity may be the underlying cause of correlation between increased SCL and increased anxietylike behavior, but this cannot be the sole explanation. Observed relation in our study between plus-maze scores and SCL may involves general locomotor activity, as well as anxiety. File (14), Cruz (8), and Rodgers and Johnson (34) reported that the frequency of closed arm entries was the most widely accepted measure of locomotor activity in the maze. Therefore, we computed the correlation between closed arm entry (in frequency) and SCL. As the correlation coefficient decreased from 0.707 to 0.39, we concluded that observed correlation between maze scores and SCL may have had more to do with anxiety differences than locomotor activity. Consequently, together with data obtained from human anxiety studies, our data indicate an universal relationship between SCL and anxiety level. However, such a relationship must be supported by findings from studies with other animal species. We also stress that our data along with the work on plasma corticosterone response (13) provide an important source of external validation for the plus-maze as a test of anxiety. We also suggest the utility of SCL as a measurement for identifying anxiety in mice.
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in a defined situational arousal. Eur. Arch. Psychiatry Clin. Neurosci. 244:86 –92; 1994. Bruce, M.; Scott, N.; Shine, P.; Lader, M.. Anxiogenic effects of caffeine in patients with anxiety disorders. Arch. Gen. Psychiatry 49:867– 869; 1992. Cruz, A. P.; Frei, F.; Graeff, F. G. Ethopharmacological analysis of rat behavior on the elevated plus-maze. Pharmacol. Biochem. Behav. 49:171–176; 1994. Emanuele, S.; Dale, J. A.; Klions, H. L. Psychology of computer use: XLII. Problem solving and humor as a function of computer anxiety. Percept. Mot. Skills. 84:147–156; 1997. File, S. E. The contribution of behavioral studies to the neuropharmacology of anxiety. Neuropharmacology 26:877– 888; 1997. File, S. E.; Andrews, N.; Wu, P. Y.; Zharkovsky, A.; Zangrossi, H., Jr. Modification of clordiazepoxides behavioral and neurochemical effects by handling and plus-maze experience. Eur. J. Pharmacol. 218: 9 –14; 1992.
576 12. File, S. E.; Zangrossi, H. Jr.; Andrews, N. Social interaction and elevated plus maze tests: Changes in release and uptake of 5-HT and GABA. Neuropharmacology 32:217–221; 1993. 13. File, S. E.; Zangrossi, H. Jr.; Sanders, F. L.; Mabbutt, P. S. Raised corticosterone in the rat after exposure to the elevated plus-maze. Psychopharmacology (Berl.) 113:543–546; 1994. 14. File, S. E; Zangrossi, H. Jr; Viana, M. Trial 2 in the elevated plusmaze: A different form of fear? Psychopharmacology (Berl.) 111:491– 494; 1993. 15. File, S. E.; Zharkovsky, A.; Gulati, K. Effects of baclofen and nitrendipine on ethanol withdrawal responses in the rat. Neuropharmacology 30:183–190; 1991. 16. Go´mez-Amor, J.; Martı´nez-Selva, J. M.; Roma´n, F.; Zamora, S.; Sastre, J. F. Electrodermal activity, hormonal levels and subjective experience during the menstrual cycle. Biol. Psychol. 30:125–139; 1990. 17. Gonzalez, L. E.; Andrews, N.; File, S. E. 5-HT1A and benzodiazepine receptors in the basolateral amygdala modulate anxiety in the social interaction test, but not in the elevated plus-maze. Brain Res. 732:145– 153; 1996. 18. Gonzalez, L. E.; File, S. E. A five-minute experience in the elevated plus-maze alters the state of the benzodiazepine receptor in the dorsal raphe nucleus. J. Neurosci. 17:1505–1511; 1997. 19. Ida, Y.; Tanaka, M.; Tsuda, A.; Tsujimasu, S.; Nagasaki, N. Attenuating effect of diazepam on stress-induced increases in noradrenaline turnover in specific brain regions of rats. Antagonism by Ro15–1788. Life Sci. 37:2491–2498; 1985. 20. Lader, M. H. Palmar skin conductance measures in anxiety and phobic states. J. Abnorm. Psych. 70:442– 445; 1967. 21. Lister, R. G. The use of a plus-maze to measure anxiety in the mouse. Psychopharmacology (Berl.) 92:180 –185; 1987. 22. Lister, R. G. Etiologically based animal models of anxiety disorders. Pharmacol. Ther. 46:321–340; 1990. 23. Lovibond, P. F. Tonic and phasic electrodermal measures of human aversive conditioning with long duration stimuli. Psychophysiology 29:621– 632; 1992. 24. Lykken, D. T.; Venables, P. H. Direct measurement of skin conductance: A proposal for standardization. Psychophysiology 8:656 – 671; 1971. 25. Marcy, R.; Quermonne, M. A.; Raoul, J.; Nammathao, B. and Smida, A. Skin Conductance Reaction (SCR)-Habituation Test, a tool to detect anxiolytic activity. Its justification by the correlation between SCR-Habituation Test activities and specific binding potencies in benzodiazepines. Prog. Neuropsychopharmacol. Biol. Psychiat. 9:387– 391; 1985.
¨ ER ET AL. SU 26. Miyamoto, M.; Kiyota, Y.; Nishiyama, M.; Nagaoka, A. Senescenceaccelerated mouse (SAM): Age-related reduced anxiety-like behavior in the SAM-P/8 strain. Physiol. Behav. 51:979 –985; 1992. 27. Montgomery, K. C. The relation between fear induced by novelty stimulation and exploratory behavior. J. Comp. Physiol. Psychol. 48:254 –260; 1958. 28. Moser, P. C. An evaluation of the elevated plus-maze using the novel anxiolytic buspirone. Psychopharmacology (Berl.) 99:48 –53; 1989. 29. Ohman A; Soares JJ. Unconscious anxiety: Phobic responses to masked stimuli. J. Abnorm. Psychol. 103:231–240; 1994. 30. Pellow, S.; Chopin, P.; File, S. E.; Briley, M. Validation of open: closed arm entries in an elevated plus-maze as a measure of anxiety in the rat. J. Neurosci. Methods 14:149 –167; 1985 . 31. Pellow, S.; File S. E. Anxiolytic and anxiogenic drug effects in exploratory activity in an elevated plus-maze: A novel test of anxiety in the rat. Pharmacol. Biochem. Behav. 24:525–529; 1986. 32. Rodgers, R. J.; Cole, J. C. Anxiety enhancement in the murine elevated plus-maze by immediate prior exposure to social stressors. Physiol. Behav. 53:383–388; 1993. 33. Rodgers, R. J.; Cole, J. C.; Harrison Phillips, D. J. ‘Cohort Removal’ induces hyperthermia but fails to influence plus-maze behavior in male mice. Physiol. Behav. 55:189 –192; 1994. 34. Rodgers, R. J.; Johnson, N. J. Factor analysis of spatiotemporal and ethnological measures in the murine elevated plus-maze test of anxiety. Pharmacol. Biochem. Behav. 52:297–303; 1995. 35. Quermonne, M. A.; Nammathao, B.; Louchah[undot]i-Raou, I. J.; Marcy, R. Skin conductance reaction– habituation test, an elementary model of anxiety as a tool to assess anxiogenic activities of some drugs. Prog. Neuropsychopharmacol. Biol. Psychiatry 17:311–318; 1993. 36. Steenbergen, H. L.; Farabollini, F.; Heinsbroek, R. P. W.; Van de Pol, N. E. Sex-dependent effects of aversive stimulation on haelboard and elevated plus-maze behavior. Behav. Brain Res. 43:159 –165; 1991. ¨ zesmi C¸., Temoc¸in S., Dog˘an P., Ciliv G. The effects of 37. Su¨er C.; O immobilization stress on electrodermal activity and brain catecholamine levels in rats. Int. J. Neurosci. 65:91–101; 1992. ¨ lgen, A.; O ¨ zesmi C¸.; Y[undot]ilmaz A. A technique for 38. Su¨er, C.; U measuring computerized skin conductance. Tr. J. Med. Sci. 24:191– 194; 1995. 39. Valdman A. V.; Zaikonnikova I. V.; Kozlovskaia M. M.; Zimakova I. E. Characteristics of the psychotropic spectrum of action of mebicar. Biull. Eksp. Biol. Med. 89:568 –570; 1980. 40. Zangrossi, H; File, S. E. Behavioral consequence in animal tests of anxiety and exploration of exposure to cat odor. Brain Res. Bull. 29:381–388; 1972.
PII S0031-9384(98)00075-4
BRIEF COMMUNICATION
The Relation Between Skin Conductance Level and Plus-Maze Behavior in Male Mice ¨ ER,1* NAZAN DOLU,* C¸IG ˘ DEM O ¨ ZESMI,* O ¨ ZLEM S¸AHIN* AND AHMET U ¨ LGEN† CEM SU *Department of Physiology, Faculty of Medicine and †Science and Art Faculty, Erciyes University, 38039 Kayseri, Turkey Received 10 February 1997; Accepted 28 January 1998 ¨ ER, C., N. DOLU, C¸. O ¨ ZESMI˙, O ¨ . S¸AHI˙N AND A. U ¨ LGEN. The relation between skin conductance level and plus-maze behavior SU in male mice. PHYSIOL BEHAV 64(4) 573–576, 1998.—The present study examined the correlation between anxiety scores and skin conductance level in 29 male Swiss Albino mice. Skin conductance (SC) was recorded with the SC unit and IBM-AT computer. Anxiety scores of mice were obtained from the elevated plus-maze test. The main result of the present study indicates that SC levels (SCLs) are negatively correlated with plus-maze behavior scores (both entries and time spent on the open arms). Our results are consistent with the findings which suggests that the higher the anxiety level the higher the SCL. This study further demonstrates the utility of SCL as a measurement for identifying anxiety in mouse. The interrelation between SCLs and plus-maze scores and possible explanations of the results are discussed. © 1998 Elsevier Science Inc. Skin conductance
Anxiety
Elevated plus-maze
THE relationship between anxiety and electrodermal activity (EDA) has been widely studied and well known. The results of the studies on human subjects indicate that anxious subjects have greater sweat gland activity (shown by by skin conductance level (SCL) and nonspecific fluctuations) and reactivity (shown by skin conductance (SC) response) than nonanxious or low anxious subjects (2,3,6,7,9,16,29). However, data in animals are lacking. We have only known three studies which have been made on this topic in mice since 1980. In these studies, the SC reaction-habituation test was suggested to assess anxiolytic activities of some drugs (25,35,39). Moreover, there is no study addressing the relationship between anxiety and SCL in mice. The elevated plus-maze test, a well-established animal model of anxiety, is based upon the natural aversion of rodents to heights and open space (27) and has been validated for both rats (30) and mice (21). It is not only sensitive to pharmacological manipulations (4,5,22,28) but has also revealed increases in anxiety produced by prior exposure to stressors (1,32,33,36,40). In this paradigm, mice are placed on an elevated maze that has two arms with sides (closed arms), and two other arms without sides (open arms). Entries from a square center zone into open or closed arms are recorded, as is the time spent on arms. The number of entries is also counted. It is generally found that the number of entries and time spent in the open arms are increased by anxiolytic drugs and decreased by anxiogenic drugs (31). The present study was undertaken: a) to determine the relation1
ship between the behavioral scores of mice tested on the elevated plus-maze test and SCLs; and b) to provide external validation of the maze as a test of anxiety. Based on the results of studies mentioned above, it is predicted that mice having higher levels of anxiety would enter to a lesser extent into the open arms than those having lower levels of anxiety. It is also predicted that SCLs would be higher in mice having higher levels of anxiety than their counterparts with lower anxiety levels. MATERIALS AND METHODS
Animals Twenty-nine male Swiss mice (10 –12 weeks) were housed in groups of 3– 6/cage (60 3 60 3 30 cm polypropylene cages) under a reversed 12-h light:dark cycle (lights on at 0700 hours) and in a constant temperature and humidity controlled environment (21 6 1°C and 30%, respectively). The mice had free access to food and tap water. Elevated Plus-Maze The elevated plus-maze was a modification of that validated for Swiss mice by Lister (21). The elevated plus-maze was made of plexiglas. It consisted of two open arms and two enclosed arms (30 cm long and 5 cm wide) that were opposite each other. The arms extended from a central plate, which is also made of plexiglas (5
To whom requests for reprints should be addressed. E-mail: [email protected]
573
¨ ER ET AL. SU
574 cm long and 5 cm wide). The walls of the enclosed arms was 15 cm in height and 1 cm in thickness. The plus-maze stood on a stand, so that it could be elevated approximately 40 cm above the ground. The central platform and maze floor were made from opaque plexiglas while the walls of the closed arms were transparent plexiglas. Behavioral Measures Each mouse was individually placed on the center of the platform of the maze facing an open arm. The mouse was allowed a 5-min pretest habituation period to explore the maze (3,13). Following this period, three measures were taken during test period: 1) total arm entries (by all four paws); 2) entries into the open arms; 3) total time spent in open arms. The mouse was removed following a 5-min test session. SCL was recorded, without losing any time. The apparatus was wiped clean with a daub sponge and dry cloths between tests. Behavioral testing was always carried out between 0900 and 1500 hours. The time spent in open arms and the number of entries were calculated by a PC computer. As soon as the mouse entered one of the open arms, experimenter pressed a key. When the front paws of the mouse have crossed into central zone the experimenter pressed another key. The time (in seconds) between pressing the keys and the number of entries were calculated. The same procedure for closed arms was repeated with the other two keys. Because open arms scores are the best measure of general anxiety in the maze, open arm entries and time spent on the open arms were expressed as percentage of total entries and test duration, respectively. Skin Conductance SC was recorded between the paw pads of both hindlimbs using Ag/AgCl electrodes after the maze test. NaCl electrode (0.05 M) jelly was placed between the skin and the electrodes. Electrodes were connected to a SC unit that was built in our laboratory. The technical specifications of this unit have been published elsewhere (38). Briefly, it conforms basically to the Lykken-Venables method (24). SC unit output was digitized and stored on-line by an IBM-AT computer. The mean of SC recorded for 2 min was expressed as SCL [ln (mmho)/cm2 per electrode area]. Statistics For statistical analysis, the data were initially analyzed by one-way ANOVA. When significant intergroup differences were detected, Newman–Keuls tests were applied to compare each pair of groups. Correlation analysis were done using three variables (SCL, percent open arm entries, and percent open arm time spent). All statistical tests were performed with SPSS software. RESULTS
The SCLs and Behavioral Test Scores in Mice with Different Anxiety Levels In average, all mice tested made 48.46% entries into and spent 13.86% of time on the open arms. These mice were subsequently divided into three subgroups according to the scores of elevated plus-maze. In the low-anxiety group, mice had higher scores than its mean values. In the intermediate-anxiety group, one of the scores (percent entry number or percent time spent) was lower than its mean value; the other was higher. In the high-anxiety group, both scores were lower than their own means. Table 1 shows SCLs and behavioral test scores measured from mice with different anxiety status. As shown in Table 1, one-way ANOVA indicated
TABLE 1 SKIN CONDUCTANCE LEVELS AND ELEVATED PLUS-MAZE TEST SCORES IN MICE WITH DIFFERENT ANXIETY LEVEL Anxiety level
n
SCL (mmho)
All mice tested Group 1 Group 2 Group 3 one-way ANOVA F(2,28) p,
29 5 14 10
3.36 6 0.37 2.94 6 0.35 3.40 6 0.33a 3.51 6 0.28a
Entries (%)
time spent (%)
48.46 6 11.43 13.86 6 10.0 63.00 6 8.91 31.58 6 7.57 49.23 6 9.18a 12.16 6 5.91a 39.12 6 5.64a,b 7.37 6 3.10a,b
5.45 0.01
14.99 0.0001
34.30 0.0001
The data are means 6 standard deviations. SCL: skin conductance level; Group 1: mice with low anxiety; Group 2: mice with moderate anxiety; Group 3: mice with high anxiety; a: significantly different when compared with group 1 (Newman–Keuls test); b: significantly different when compared with Group 2 (Newman–Keuls test).
significant differences between groups for all variables (F 5 5.45, p , 0.01, for SCL; F 5 14.99, p , 0.0001, for open arm entries; and F 5 34.29, p , 0.0001, for time spent in open arms). SCL values were found to be significantly lower in low-anxiety group when compared with both moderate- and high-anxiety groups (p , 0.05). No significant difference was observed between moderate and high anxiety groups, when SCL values were compared. Both plus-maze scores were also found to be significantly higher in the low-anxiety group when compared with both the moderate- and high-anxiety groups (p , 0.05). There were significant differences between the moderate- and high-anxiety groups (p , 0.05). The Correlation Between SCLs and Behavioral Test Scores The correlation matrix reveals powerful correlation between SCL, entries into the open arms, and time spent on them. A significant negative correlation in the SCL was found regarding the percent entries into open arm (r 5 20.707, p , 0.001) and the percent time into open arms (r 5 [minusa]0.559, p , 0.002). These correlations are shown in Fig. 1. There was also a significant correlation between the percentage of entries into the open arms and the percentage of time spent on the open arms (r 5 0.527, p , 0.003). We also found a weak correlation between closed arm entries and SCL (r 5 0.39, p , 0.04) when the number of entries into the closed arms was mentioned as frequency. DISCUSSION
The elevated plus-maze introduced by Pellow et al. (31) is often used in studies to identify anxiolytic and anxiogenic drugs (4,5,21,22,28). Because the open arms evoke a greater strength of fear drive than the closed arms, mice are willing to enter into enclosed arms and stay a longer time on enclosed arms during test period (26). Anxiolytic drugs, such as benzodiazepines, barbiturates, and ethanol, increase the number of entries into open arms and time spent on open arms; in contrast, anxiogenic drugs, such as yohimbine, decrease both (31). The elevated plus-maze is also sensitive on the anxiety level of rodents (26,32). In the present study, the mice who exhibit increased anxiety-like behavior, that is, they made more frequent entries into closed arms and stayed a longer time in them, showed higher SCLs than those who exhibit decreased anxiety-like behavior. Moreover, there was a correlation between SCLs and anxiety behavior measures with elevated plusmaze. Because elevated SCL is already established as a correlate
EDA AND PLUS-MAZE BEHAVIOR IN MICE
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FIG. 1. The relationship between skin conductance level (SCL, mmho) and percent time spent (dotted line, triangle dot) and percent entries (solid line, circle dot) into open arms.
of anxiety (20,23), the observed SCL increase is perhaps explained with anxiety associated with anticipation of an aversive event. Our findings indicate the utility of SC as a measurement for identifying anxiety in mice. Several studies (25,35,39) suggested the utility of SC habituation, another electrodermal activity parameter, as a tool to assess anxiolytic and anxiogenic activities of some drugs. However, habituation can be measured by several different methods, such as three nonresponding criteria, two nonresponding criteria, and inversion criteria. There are two problems with these criteria: 1) they do not make use of all data, and 2) they cannot discriminate between subjects who failed to meet in the total number of trials administered but who may nonetheless vary greatly in the amount of habituation shown. Therefore, we concluded that the SCL would appear to be more suitable in identifying anxiety than SC habituation tests in mice. Involvement of neurotransmitter systems in behavioral changes in elevated plus-maze (10 –12,15,17–19,21) and in electrodermal activity (37) has been studied in rodents. The results obtained from elevated plus-maze experiments indicated that activation of the noradrenergic system may induce anxiogenic-like behavior (10,19, 21). Furthermore, activated Nagic system may also induce electrodermal changes (37). File et al. (11,12) indicated that exposure to the plus-maze decreased cortical GABA function and increased
hippocampal 5-HT function in rats. Therefore, we concluded that central catecholaminergic hyperactivity may be the underlying cause of correlation between increased SCL and increased anxietylike behavior, but this cannot be the sole explanation. Observed relation in our study between plus-maze scores and SCL may involves general locomotor activity, as well as anxiety. File (14), Cruz (8), and Rodgers and Johnson (34) reported that the frequency of closed arm entries was the most widely accepted measure of locomotor activity in the maze. Therefore, we computed the correlation between closed arm entry (in frequency) and SCL. As the correlation coefficient decreased from 0.707 to 0.39, we concluded that observed correlation between maze scores and SCL may have had more to do with anxiety differences than locomotor activity. Consequently, together with data obtained from human anxiety studies, our data indicate an universal relationship between SCL and anxiety level. However, such a relationship must be supported by findings from studies with other animal species. We also stress that our data along with the work on plasma corticosterone response (13) provide an important source of external validation for the plus-maze as a test of anxiety. We also suggest the utility of SCL as a measurement for identifying anxiety in mice.
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