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Evaluation of functional asymmetry in rats with dose-dependent lesions of dopaminergic nigrostriatal system using elevated body swing test
Physiology & Behavior 82 (2004) 369 – 373
Evaluation of functional asymmetry in rats with dose-dependent lesions of dopaminergic nigrostriatal system using elevated body swing test Tourandokht Baluchnejadmojarada,*, Mehrdad Roghanib a
Department of Physiology, School of Medicine, Iran University of Medical Sciences, Shaheed Hemmat Expressway, P.O. Box 14155-6183, Tehran, Iran b Department of Physiology, School of Medicine, Shahed University, Tehran, Iran Received 6 June 2003; received in revised form 4 April 2004; accepted 5 April 2004
Abstract Although drug-induced rotational behavior has conventionally been used for the assessment of functional asymmetry in 6hydroxydopamine (OHDA)-lesioned rats, a pure behavioral test that can evaluate animals with dose-dependent lesions of the dopaminergic nigrostriatal system in a drug-free state may better reflect a more natural response following lesion. In this study, elevated body swing test (EBST) was used for evaluation of rats with varying lesions of the dopaminergic nigrostriatal system. For this purpose, rats received intrastriatal injection of 8 (L1), 12 (L2), 16 (L3), and 20 (L4) Ag of the neurotoxin 6-OHDA (dissolved in 5 Al of saline – ascorbate). Apomorphine-induced rotational and drug-free elevated body swing behaviors were evaluated before and at different time points after the experiment. The results showed that although there is a significant trend for contralateral rotations and drug-free swings in lesioned rats as compared to sham-operated group, however, there were no significant differences between the L1 and L2 and between the L3 and L4 groups regarding EBST. Therefore, EBST may be valuable for behavioral analysis of rats with mild and/or severe damage of the dopaminergic nigrostriatal system. D 2004 Elsevier Inc. All rights reserved. Keywords: Elevated body swing test; Rotational behavior; 6-hydroxydopamine; Parkinson’s disease; Rat
1. Introduction Striatal 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal system in rat may provide a partial lesion model of the Parkinson’s disease (PD), which is useful for the assessment of neuroprotective treatments and behavioral recovery after therapeutic intervention [1]. Dose-dependent decreases in striatal dopamine levels and tyrosine hydroxylase-immunoreactive cell numbers in the ipsilateral substantia nigra have been shown following the neurotoxin injection [2]. Although apomorphine- and amphetamineinduced rotational behavior in rats with unilateral damage of the dopaminergic nigrostriatal system is a well-established and commonly used approach for the assessment of functional asymmetry [3 – 5], this conventional drug-induced rotational behavior test may lead to misleading results [6]. Furthermore, the development of sensitivity due to repeated administration of dopaminergic agonists, like apo* Corresponding author. Tel.: +98-21-8058709; fax: +98-21-8058719. E-mail address: [email protected] (T. Baluchnejadmojarad). 0031-9384/$ – see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2004.04.005
morphine, might complicate the interpretation of the behavior [7– 9]. Therefore, a pure behavioral test that can evaluate unilaterally lesioned animals in a drug-free state may better reflect a more natural response of the animals to neurotoxin lesion. For this purpose, a battery of drug-free tests including paw retraction [10], adjusting steps [1], staircase [11], locomotor activity [12], reaction time task [13], bar-pressing task [14], and forelimb use asymmetry [15] tests has been developed for the evaluation of behavioral deficits in animal models of PD. However, many are of limited applicability, difficult to perform, or insufficiently objective (movement pattern analyses are subjective for staircase and paw-reaching tests) for evaluation of functional asymmetry following intrastriatal unilateral injection of 6-OHDA [16]. Inasmuch as it has previously been claimed that elevated body swing test (EBST) could be used as a reliable behavioral parameter in hemi-Parkinsonian rats [17 – 19], the present study was carried out to evaluate the efficacy of EBST in dosedependent lesions of the dopaminergic nigrostriatal system. For this purpose, apomorphine-induced rotations and drugfree EBST were conducted prior to and following intra-
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striatal unilateral injection of different doses of the neurotoxin 6-OHDA.
2. Methods 2.1. Animals Adult male Wistar rats (Pasteur’s Institute, Tehran), weighing 215– 255 g at the start of the experiment, were housed three to four per cage in a temperature-controlled colony room under a light– dark cycle with free access to tap water and standard pelleted food. The experimental protocol for animal care and handling was according to the guidelines of the National Institutes of Health for the use of live animals and those of the research council of Iran University of Medical Sciences (Tehran, Iran). 2.2. Experimental procedure Only rats not showing biased swing behavior (defined as showing net swings not greater than 3/45 s) and biased rotational behavior (net rotations less than 30/h) following injection of apomorphine hydrochloride (0.5 mg/kg ip) were selected for the present study. The animals (n=77) were randomly divided into five experimental groups: sham-operated group (SH, n=16) and lesion groups including L 1 (n=15), L 2 (n=15), L 3 (n=17), and L4 (n=14). The rats were anesthetized with a combination of ketamin (100 mg/kg ip) and xylazine (5 mg/kg ip), had their heads shaved, and were placed in a Stoelting stereotaxic apparatus (incisor bar 3.3 mm, ear bars positioned symmetrically). The scalp was cleaned with iodine solution and incised on the midline, and a burr hole was drilled through the skull at coordinates L 3 mm, AP +9.2 mm, V+4.5 mm from the center of interaural line, according to the atlas of Paxinos and Watson [20]. The lesion groups including L1, L2, L3, and L4 received a single injection of 5 Al of 0.9% saline containing 1.5, 2.5, 3, and 4 Ag/Al of 6-OHDA-HCL (Sigma) and 0.2% ascorbic acid (W/V), respectively. The SH group received an identical volume of ascorbate – saline solution. 2.3. Behavioral testing The animals were tested for rotational behavior by apomorphine hydrochloride (0.5 mg/kg ip) 1 week before (baseline) and on the 4th and 8th weeks after the surgery. The animals were tested for drug-free elevated-body swing behavior 1 week before (baseline) and on the 1st, 2nd, 3rd, 4th, and 8th weeks postsurgery. The rotations were measured according to the method as described previously [5,21]. The swing test was performed at least 1 day before rotational test according to a slightly modified method as described before [17].
Briefly, the animal was placed in a Plexiglas box (404035 cm), allowed to habituate for 10 min, and attain a neutral position, defined as having all four paws on the floor. Then, the animal was held approximately 2 cm from the base of its tail. It was then elevated to 2 cm above the surface on which it was resting. The animal was held in the vertical axis, defined as no deviation of more than 10j to either side. A swing was recorded whenever the animal moved its head out of the vertical axis to either side. Before attempting another swing, the animal must return to the vertical position for the next swing to be counted. When the animal did not commence swing behavior after being elevated for more than 5 s, a gentle pinch to the tail induced the behavior. Swings were counted for a period of 45 s. One observer was responsible for timing the test session, determining and recording the direction and the frequency of swings, while another observer held the rat. All tests were conducted blind to the groups. The total number of swings made to each side was measured, and the net number of swings was calculated. A positive value was used for right-handed rotations and swings. 2.4. Statistical analysis All data were expressed as meanFS.E.M. For withingroup and intergroup comparisons, two-tailed paired and unpaired Student’s t tests were used respectively. Oneway ANOVA, followed by Tukey post hoc test, was used for each group at different time points. In all analyses, the null hypothesis was rejected at the level of .05.
3. Results The effect of 6-OHDA-induced damage of dopaminergic nigrostriatal system was evaluated on apomorphineinduced rotations (Table 1). At baseline testing (before surgery), all rats exhibited less than 30 rotations in 60 min following intraperitoneal administration of apomorphine. Statistical analysis of the net total number of rotations over a 60-min period on the 4th and 8th weeks postsurgery
Table 1 Net total number of rotations in the different groups
SH L1 L2 L3 L4
4th week
8th week
4.67F2.29 77.2F8.02* 138.47F17.7** 266.27F27.65** 261.27F29.17**
3.6F1.62 60.6F6.38* 115F17.11** 231.13F26.9** 235.8F27.6**
Net total number of rotations (meanFS.E.M.) induced by apomorphine (0.5 mg/kg ip) over a period of 60 min. Note that positive values indicate turns contralateral to the side of lesion. * P<.05 (in comparison with SH). ** P<.001 (in comparison with SH).
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showed that apomorphine caused a marked significant contralateral turning in the rats of lesioned groups as compared to the SH group. In addition, there was a 6OHDA dose-dependent increase in the number of apomorphine-induced rotations, especially for lower doses of the neurotoxin (i.e., for the L1, L2, and L3 groups). In addition, there was a nonsignificant reduction in rotations in lesioned groups on the 8th week after the experiment as compared to data on the 4th week postlesion. This observation was consistent with our previous data in this laboratory. Meanwhile, on the 4th week postlesion, groups L2 ( P<.05), L3 ( P<.01), and L4 ( P<.01) showed significant higher rotations in comparison with the L1 group, and it was interesting that there was no significant difference between the L3 and L4 groups. A similar pattern was found for the data for the 8th week postsurgery. The effect of dose-dependent 6-OHDA-induced dopaminergic nigrostriatal damage was also investigated on drugfree EBST made over a 45-s period (Fig. 1). There was no significant difference between the SH and lesioned groups at baseline (presurgery). Statistical analysis of the net total number of swings showed that there existed a dose-dependent increase in contralateral swings in the lesioned groups as compared to the SH group in all of the postsurgery weeks. In this respect, elevated body swings were maximal for 15 Ag of 6-OHDA. Excluding the 1st week postsurgery, the maximum dose of the neurotoxin (20 Ag) was associated with a relative decrease or no marked change in the number of swings and with no observable change in the magnitude of stereotypic movements. There was also a nonsignificant reduction in swings in the L1, L2, and L4 groups on the 8th week, as compared to the 4th week postsurgery. In addition, the observed differences between L1 and L2 (except for the 2nd and 3rd weeks postlesion) and between L3 and L4 were nonsignificant. Meanwhile, the data for the L1 and L2 groups were nearer to each other, and a similar condition existed for the L3 and L4 groups. Finally, the existing
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difference between the L2 and L3 groups was only significant ( P<.05) on the 8th week postsurgery.
4. Discussion The objective of the present study was to evaluate the applicability of EBST for the analysis of motor asymmetry in dose-dependent lesions of the dopaminergic nigrostriatal system in rat. For this purpose, drug-free elevated body swing and apomorphine-induced rotational behaviors were quantitatively analyzed. There are three major conclusions to be drawn from the results. First, similar to previous reports [17 – 19], 6-OHDAlesioned rats with damage of the dopaminergic nigrostriatal system showed a net biased and significant trend for elevated body swings and drug-induced rotational behavior. In this respect, both rotational and swing tests were able to detect precisely motor asymmetry at different postlesion time points with a high sensitivity, especially at lower doses of the neurotoxin. The results also showed that an invertedU relationship exists between the swings and the neurotoxin dosage. In agreement with this finding, it has been demonstrated that lesioning with 6-OHDA elicits a specific profile for apomorphine-induced rotations (i.e., the rotations show an inverted-U dose –response curve), with maximal rotations occurring at lower doses of the neurotoxin. In addition, higher doses of the neurotoxin (17 Ag) may induce some postsynaptic changes in the dopaminergic system of the lesioned striatum that may interfere with apomorphine stimulation of the dopaminergic receptors. Alternatively, other structures involved in rotation may be affected by high doses of the neurotoxin, counteracting apomorphine’s effect [4]. Furthermore, higher doses of the neurotoxin 6OHDA may induce nonspecific damage and may lead to the loss of intrinsic striatal neurons [22]. In this respect, a similar condition may have occurred for a relative reduction
Fig. 1. Net total number of swings (meanFS.E.M.) recorded over a period of 45 s. Note that the positive values indicate swings contralateral to the side of lesion. * P<.01, ** P<.005 (in comparison with SH).
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or no increase in swings and rotations in the group receiving 20 Ag of the neurotoxin. Second, in agreement with previous reports [18,19], the direction of biased swing behavior taken by animals with striatal lesions corresponded with the direction of asymmetrical apomorphine-induced rotational behavior. The results of previous studies have shown that the direction of apomorphine-induced rotational behavior is rather dependent on the location of (anterior/posterior coordinates) neurotoxin injection into the striatum. In this respect, rats receiving the more posterior lesion rotated ipsilateral to lesioned side of the brain, whereas the majority of rats receiving the more anterior lesion rotated contralateral to lesioned side [23]. Our results are consistent with the fact that injection of the neurotoxin into the anterior region of dorsal striatum leads to contralateral swings and drug-induced rotations, as has previously been demonstrated using immunohistochemical and tract-tracing methods [5]. Third, EBST, according to our results, does not seem to be an appropriate test for evaluation and differentiation of animals with dose-dependent (varying) lesions of the dopaminergic nigrostriatal system. Despite this limitation, it can be used as a reliable and reproducible test for evaluation of functional asymmetry in 6-OHDA-induced models of PD as long as single doses of the neurotoxin are concerned. Other valuable attributes of EBST are its positive correlation with apomorphine-induced rotational behavior and its nature as a motor response, not a sensorimotor one [18]. In addition, this test can be quickly and easily performed. Thus, it could be included in a larger battery of tests for evaluation of the 6-OHDA-treated animals or as a quick screening tool to verify the presence of partial and/or severe dopamine depletion. Although similar behavioral tests for 6-OHDAlesioned animals in a drug-free state have been reported, the development of sensitivity due to electrical stimulation (treadmill apparatus to make the lesioned animals run uphill by electrostimulation), being expensive (automated recording of open-field locomotor activities), complexity and inherent subjective scaling (movement pattern analyses of paw-reaching test), and negative correlation with apomorphine-induced rotations (paw-reaching test and recording of asymmetrical orientation to edges of an open field) have limited their application [18]. Taken together, although EBST is a reliable and reproducible experimental tool for evaluation of motor asymmetry in animals with lesions of the dopaminergic nigrostriatal system, it could not be considered as an appropriate test for evaluation and differentiation of animals with dose-dependent lesions of the system.
Acknowledgements This study was supported by a grant-in-aid from the IUMS University (Tehran, Iran). We also gratefully appre-
ciate the personnel of the Department of Physiology of IUMS University for their excellent technical assistance.
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T. Baluchnejadmojarad, M. Roghani / Physiology & Behavior 82 (2004) 369–373 body swing test in the early model of Parkinson’s disease in rat. Physiol Behav 2002;76:507 – 10. [20] Paxinos G, Watson C. The rat brain in stereotaxic coordinates. Academic Press. San Diego; 1986. [21] Fujita M, Nishino H, Kumazaki M, Shimada S, Tohyama M, Nishimura T. Expression of dopamine transporter mRNA and its binding site in fetal nigral cells transplanted into the striatum of 6-OHDA lesioned rat. Mol Brain Res 1996;39:127 – 36. [22] Javoy F, Agid Y, Sotelo C. Specific and non-specific catecholamin-
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Evaluation of functional asymmetry in rats with dose-dependent lesions of dopaminergic nigrostriatal system using elevated body swing test Tourandokht Baluchnejadmojarada,*, Mehrdad Roghanib a
Department of Physiology, School of Medicine, Iran University of Medical Sciences, Shaheed Hemmat Expressway, P.O. Box 14155-6183, Tehran, Iran b Department of Physiology, School of Medicine, Shahed University, Tehran, Iran Received 6 June 2003; received in revised form 4 April 2004; accepted 5 April 2004
Abstract Although drug-induced rotational behavior has conventionally been used for the assessment of functional asymmetry in 6hydroxydopamine (OHDA)-lesioned rats, a pure behavioral test that can evaluate animals with dose-dependent lesions of the dopaminergic nigrostriatal system in a drug-free state may better reflect a more natural response following lesion. In this study, elevated body swing test (EBST) was used for evaluation of rats with varying lesions of the dopaminergic nigrostriatal system. For this purpose, rats received intrastriatal injection of 8 (L1), 12 (L2), 16 (L3), and 20 (L4) Ag of the neurotoxin 6-OHDA (dissolved in 5 Al of saline – ascorbate). Apomorphine-induced rotational and drug-free elevated body swing behaviors were evaluated before and at different time points after the experiment. The results showed that although there is a significant trend for contralateral rotations and drug-free swings in lesioned rats as compared to sham-operated group, however, there were no significant differences between the L1 and L2 and between the L3 and L4 groups regarding EBST. Therefore, EBST may be valuable for behavioral analysis of rats with mild and/or severe damage of the dopaminergic nigrostriatal system. D 2004 Elsevier Inc. All rights reserved. Keywords: Elevated body swing test; Rotational behavior; 6-hydroxydopamine; Parkinson’s disease; Rat
1. Introduction Striatal 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal system in rat may provide a partial lesion model of the Parkinson’s disease (PD), which is useful for the assessment of neuroprotective treatments and behavioral recovery after therapeutic intervention [1]. Dose-dependent decreases in striatal dopamine levels and tyrosine hydroxylase-immunoreactive cell numbers in the ipsilateral substantia nigra have been shown following the neurotoxin injection [2]. Although apomorphine- and amphetamineinduced rotational behavior in rats with unilateral damage of the dopaminergic nigrostriatal system is a well-established and commonly used approach for the assessment of functional asymmetry [3 – 5], this conventional drug-induced rotational behavior test may lead to misleading results [6]. Furthermore, the development of sensitivity due to repeated administration of dopaminergic agonists, like apo* Corresponding author. Tel.: +98-21-8058709; fax: +98-21-8058719. E-mail address: [email protected] (T. Baluchnejadmojarad). 0031-9384/$ – see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2004.04.005
morphine, might complicate the interpretation of the behavior [7– 9]. Therefore, a pure behavioral test that can evaluate unilaterally lesioned animals in a drug-free state may better reflect a more natural response of the animals to neurotoxin lesion. For this purpose, a battery of drug-free tests including paw retraction [10], adjusting steps [1], staircase [11], locomotor activity [12], reaction time task [13], bar-pressing task [14], and forelimb use asymmetry [15] tests has been developed for the evaluation of behavioral deficits in animal models of PD. However, many are of limited applicability, difficult to perform, or insufficiently objective (movement pattern analyses are subjective for staircase and paw-reaching tests) for evaluation of functional asymmetry following intrastriatal unilateral injection of 6-OHDA [16]. Inasmuch as it has previously been claimed that elevated body swing test (EBST) could be used as a reliable behavioral parameter in hemi-Parkinsonian rats [17 – 19], the present study was carried out to evaluate the efficacy of EBST in dosedependent lesions of the dopaminergic nigrostriatal system. For this purpose, apomorphine-induced rotations and drugfree EBST were conducted prior to and following intra-
370
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striatal unilateral injection of different doses of the neurotoxin 6-OHDA.
2. Methods 2.1. Animals Adult male Wistar rats (Pasteur’s Institute, Tehran), weighing 215– 255 g at the start of the experiment, were housed three to four per cage in a temperature-controlled colony room under a light– dark cycle with free access to tap water and standard pelleted food. The experimental protocol for animal care and handling was according to the guidelines of the National Institutes of Health for the use of live animals and those of the research council of Iran University of Medical Sciences (Tehran, Iran). 2.2. Experimental procedure Only rats not showing biased swing behavior (defined as showing net swings not greater than 3/45 s) and biased rotational behavior (net rotations less than 30/h) following injection of apomorphine hydrochloride (0.5 mg/kg ip) were selected for the present study. The animals (n=77) were randomly divided into five experimental groups: sham-operated group (SH, n=16) and lesion groups including L 1 (n=15), L 2 (n=15), L 3 (n=17), and L4 (n=14). The rats were anesthetized with a combination of ketamin (100 mg/kg ip) and xylazine (5 mg/kg ip), had their heads shaved, and were placed in a Stoelting stereotaxic apparatus (incisor bar 3.3 mm, ear bars positioned symmetrically). The scalp was cleaned with iodine solution and incised on the midline, and a burr hole was drilled through the skull at coordinates L 3 mm, AP +9.2 mm, V+4.5 mm from the center of interaural line, according to the atlas of Paxinos and Watson [20]. The lesion groups including L1, L2, L3, and L4 received a single injection of 5 Al of 0.9% saline containing 1.5, 2.5, 3, and 4 Ag/Al of 6-OHDA-HCL (Sigma) and 0.2% ascorbic acid (W/V), respectively. The SH group received an identical volume of ascorbate – saline solution. 2.3. Behavioral testing The animals were tested for rotational behavior by apomorphine hydrochloride (0.5 mg/kg ip) 1 week before (baseline) and on the 4th and 8th weeks after the surgery. The animals were tested for drug-free elevated-body swing behavior 1 week before (baseline) and on the 1st, 2nd, 3rd, 4th, and 8th weeks postsurgery. The rotations were measured according to the method as described previously [5,21]. The swing test was performed at least 1 day before rotational test according to a slightly modified method as described before [17].
Briefly, the animal was placed in a Plexiglas box (404035 cm), allowed to habituate for 10 min, and attain a neutral position, defined as having all four paws on the floor. Then, the animal was held approximately 2 cm from the base of its tail. It was then elevated to 2 cm above the surface on which it was resting. The animal was held in the vertical axis, defined as no deviation of more than 10j to either side. A swing was recorded whenever the animal moved its head out of the vertical axis to either side. Before attempting another swing, the animal must return to the vertical position for the next swing to be counted. When the animal did not commence swing behavior after being elevated for more than 5 s, a gentle pinch to the tail induced the behavior. Swings were counted for a period of 45 s. One observer was responsible for timing the test session, determining and recording the direction and the frequency of swings, while another observer held the rat. All tests were conducted blind to the groups. The total number of swings made to each side was measured, and the net number of swings was calculated. A positive value was used for right-handed rotations and swings. 2.4. Statistical analysis All data were expressed as meanFS.E.M. For withingroup and intergroup comparisons, two-tailed paired and unpaired Student’s t tests were used respectively. Oneway ANOVA, followed by Tukey post hoc test, was used for each group at different time points. In all analyses, the null hypothesis was rejected at the level of .05.
3. Results The effect of 6-OHDA-induced damage of dopaminergic nigrostriatal system was evaluated on apomorphineinduced rotations (Table 1). At baseline testing (before surgery), all rats exhibited less than 30 rotations in 60 min following intraperitoneal administration of apomorphine. Statistical analysis of the net total number of rotations over a 60-min period on the 4th and 8th weeks postsurgery
Table 1 Net total number of rotations in the different groups
SH L1 L2 L3 L4
4th week
8th week
4.67F2.29 77.2F8.02* 138.47F17.7** 266.27F27.65** 261.27F29.17**
3.6F1.62 60.6F6.38* 115F17.11** 231.13F26.9** 235.8F27.6**
Net total number of rotations (meanFS.E.M.) induced by apomorphine (0.5 mg/kg ip) over a period of 60 min. Note that positive values indicate turns contralateral to the side of lesion. * P<.05 (in comparison with SH). ** P<.001 (in comparison with SH).
T. Baluchnejadmojarad, M. Roghani / Physiology & Behavior 82 (2004) 369–373
showed that apomorphine caused a marked significant contralateral turning in the rats of lesioned groups as compared to the SH group. In addition, there was a 6OHDA dose-dependent increase in the number of apomorphine-induced rotations, especially for lower doses of the neurotoxin (i.e., for the L1, L2, and L3 groups). In addition, there was a nonsignificant reduction in rotations in lesioned groups on the 8th week after the experiment as compared to data on the 4th week postlesion. This observation was consistent with our previous data in this laboratory. Meanwhile, on the 4th week postlesion, groups L2 ( P<.05), L3 ( P<.01), and L4 ( P<.01) showed significant higher rotations in comparison with the L1 group, and it was interesting that there was no significant difference between the L3 and L4 groups. A similar pattern was found for the data for the 8th week postsurgery. The effect of dose-dependent 6-OHDA-induced dopaminergic nigrostriatal damage was also investigated on drugfree EBST made over a 45-s period (Fig. 1). There was no significant difference between the SH and lesioned groups at baseline (presurgery). Statistical analysis of the net total number of swings showed that there existed a dose-dependent increase in contralateral swings in the lesioned groups as compared to the SH group in all of the postsurgery weeks. In this respect, elevated body swings were maximal for 15 Ag of 6-OHDA. Excluding the 1st week postsurgery, the maximum dose of the neurotoxin (20 Ag) was associated with a relative decrease or no marked change in the number of swings and with no observable change in the magnitude of stereotypic movements. There was also a nonsignificant reduction in swings in the L1, L2, and L4 groups on the 8th week, as compared to the 4th week postsurgery. In addition, the observed differences between L1 and L2 (except for the 2nd and 3rd weeks postlesion) and between L3 and L4 were nonsignificant. Meanwhile, the data for the L1 and L2 groups were nearer to each other, and a similar condition existed for the L3 and L4 groups. Finally, the existing
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difference between the L2 and L3 groups was only significant ( P<.05) on the 8th week postsurgery.
4. Discussion The objective of the present study was to evaluate the applicability of EBST for the analysis of motor asymmetry in dose-dependent lesions of the dopaminergic nigrostriatal system in rat. For this purpose, drug-free elevated body swing and apomorphine-induced rotational behaviors were quantitatively analyzed. There are three major conclusions to be drawn from the results. First, similar to previous reports [17 – 19], 6-OHDAlesioned rats with damage of the dopaminergic nigrostriatal system showed a net biased and significant trend for elevated body swings and drug-induced rotational behavior. In this respect, both rotational and swing tests were able to detect precisely motor asymmetry at different postlesion time points with a high sensitivity, especially at lower doses of the neurotoxin. The results also showed that an invertedU relationship exists between the swings and the neurotoxin dosage. In agreement with this finding, it has been demonstrated that lesioning with 6-OHDA elicits a specific profile for apomorphine-induced rotations (i.e., the rotations show an inverted-U dose –response curve), with maximal rotations occurring at lower doses of the neurotoxin. In addition, higher doses of the neurotoxin (17 Ag) may induce some postsynaptic changes in the dopaminergic system of the lesioned striatum that may interfere with apomorphine stimulation of the dopaminergic receptors. Alternatively, other structures involved in rotation may be affected by high doses of the neurotoxin, counteracting apomorphine’s effect [4]. Furthermore, higher doses of the neurotoxin 6OHDA may induce nonspecific damage and may lead to the loss of intrinsic striatal neurons [22]. In this respect, a similar condition may have occurred for a relative reduction
Fig. 1. Net total number of swings (meanFS.E.M.) recorded over a period of 45 s. Note that the positive values indicate swings contralateral to the side of lesion. * P<.01, ** P<.005 (in comparison with SH).
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or no increase in swings and rotations in the group receiving 20 Ag of the neurotoxin. Second, in agreement with previous reports [18,19], the direction of biased swing behavior taken by animals with striatal lesions corresponded with the direction of asymmetrical apomorphine-induced rotational behavior. The results of previous studies have shown that the direction of apomorphine-induced rotational behavior is rather dependent on the location of (anterior/posterior coordinates) neurotoxin injection into the striatum. In this respect, rats receiving the more posterior lesion rotated ipsilateral to lesioned side of the brain, whereas the majority of rats receiving the more anterior lesion rotated contralateral to lesioned side [23]. Our results are consistent with the fact that injection of the neurotoxin into the anterior region of dorsal striatum leads to contralateral swings and drug-induced rotations, as has previously been demonstrated using immunohistochemical and tract-tracing methods [5]. Third, EBST, according to our results, does not seem to be an appropriate test for evaluation and differentiation of animals with dose-dependent (varying) lesions of the dopaminergic nigrostriatal system. Despite this limitation, it can be used as a reliable and reproducible test for evaluation of functional asymmetry in 6-OHDA-induced models of PD as long as single doses of the neurotoxin are concerned. Other valuable attributes of EBST are its positive correlation with apomorphine-induced rotational behavior and its nature as a motor response, not a sensorimotor one [18]. In addition, this test can be quickly and easily performed. Thus, it could be included in a larger battery of tests for evaluation of the 6-OHDA-treated animals or as a quick screening tool to verify the presence of partial and/or severe dopamine depletion. Although similar behavioral tests for 6-OHDAlesioned animals in a drug-free state have been reported, the development of sensitivity due to electrical stimulation (treadmill apparatus to make the lesioned animals run uphill by electrostimulation), being expensive (automated recording of open-field locomotor activities), complexity and inherent subjective scaling (movement pattern analyses of paw-reaching test), and negative correlation with apomorphine-induced rotations (paw-reaching test and recording of asymmetrical orientation to edges of an open field) have limited their application [18]. Taken together, although EBST is a reliable and reproducible experimental tool for evaluation of motor asymmetry in animals with lesions of the dopaminergic nigrostriatal system, it could not be considered as an appropriate test for evaluation and differentiation of animals with dose-dependent lesions of the system.
Acknowledgements This study was supported by a grant-in-aid from the IUMS University (Tehran, Iran). We also gratefully appre-
ciate the personnel of the Department of Physiology of IUMS University for their excellent technical assistance.
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