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Effects of isatin on rotational behavior and DA levels in caudate putamen in Parkinsonian rats
Brain Research 917 (2001) 127–132 www.elsevier.com / locate / bres
Research report
Effects of isatin on rotational behavior and DA levels in caudate putamen in Parkinsonian rats a,b b a, Yu Zhou , Zhi-Qi Zhao , Jun-Xia Xie * a
Department of Physiology, School of Medicine, Qingdao University, 38 Deng-Zhou Road, Qingdao 266021, PR China b Shanghai Institute of Physiology, Chinese Academy of Sciences, 38 Deng-Zhou Road, Shanghai 200031, PR China Accepted 27 July 2001
Abstract Isatin was a potent endogenous monoamine oxidase (MAO) inhibitor that is more active against MAO-B than MAO-A. The acute effects of isatin on apomorphine (APO)-induced rotations were evaluated in Parkinsonian rats induced by 6-hydroxydopamine (6-OHDA) lesion. Furthermore, the effects of isatin on DA release in caudate putamen (CPu) of model and normal rats were monitored using fast cyclic voltammetry (FCV). The contents of monoamine transmitters and their metabolites in CPu of model and normal rats were also analyzed by high performance liquid chromatography with electrochemical detection after administration of isatin. Here we show that isatin (100 mg / kg, i.p.) apparently inhibited APO-induced rotations of Parkinsonian rats to 39.163.7% of the control (n512), while it had no apparent effects on electrical stimuli-induced DA release either in normal rats or in model rats. In addition, the content of 5-hydroxytryptamine but not DA was increased in both normal rats and model rats after isatin (100 mg / kg, i.p.) was administered (P,0.01, n56). The content of 5-hydroxyindole acetic acid was not changed. These results suggest that isatin cannot increase DA levels in rat CPu. Therefore, the effects of isatin on APO-induced rotations of our Parkinsonian rats could not attribute to its inhibition of DA catabolism as a MAO inhibitor. 2001 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Degenerative disease: Parkinson’s Keywords: Isatin; Dopamine; Rotation; Parkinsonian rat; Fast cyclic voltammetry; High-performance liquid chromatography with electrochemical detection
1. Introduction Isatin (indole-2,3-dione) is widely distributed in mammalian tissues and body fluids. It has a distinct and discontinuous distribution in rat brain. The concentrations in hippocampus and cerebellum are approximately 0.1 mg / g, or about 1 mM [17]. Isatin is well known to have a broad range of biological and pharmacological actions [11,17,18]. In addition to a potent antagonist of the atrial natriuretic peptides (ANP) receptor and ANP-stimulated guanylate cyclase (GC) [8,18], isatin is also an effective endogenous MAO inhibitor in vitro, which is more active against MAO-B than MAO-A [8,10,15,17]. Compared with actions in vitro, the in vitro effects of *Corresponding author. Tel.: 186-532-383-8481; fax: 186-532-3801449. E-mail address: [email protected] (J.-X. Xie).
isatin seem to be more attractive and more complex. Early studies have suggested that isatin, administered i.p. to rats in a dose of 10–200 mg / kg, causes an increase of monoamines, such as serotonin (5-hydroxytryptamine, 5HT) and noradrenaline (NA) in the brain [3,10,16,22]. Other results have elucidated that striatal ACh and DA levels in rat significantly increased after isatin administration [11,19]. Furthermore, isatin could significantly increase striatal DA levels in Parkinsonian rats induced by Japanese encephalitis virus [11,19]. So, isatin may involved in the regulation and metabolism of dopamine in rats and have some protective effect against Parkinson’s disease. However, the mechanisms underlying isatin-induced increase of monoamine transmitters in brain are still not clear [17]. In this study, using Pargyling, a widely used MAO inhibitor [5,6,13,14,21], as the positive control, we investigated the acute effects of isatin on DA levels and apomor-
0006-8993 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )02935-3
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Y. Zhou et al. / Brain Research 917 (2001) 127 – 132
phine (APO)-induced rotations of 6-hydroxydopamine (6OHDA)-lesioned Parkinsonian rats in order to further evaluate the effects of isatin on nigrostriatal dopaminergic system under certain pathological condition.
apparently. Moreover, the depth of stimulation electrode was adjusted appropriately for each experiment to ensure that the release was maximal. After the proper position of stimulation electrode was found and kept unchangeable, DA release from CPu was monitored in normal and Parkinsonian rats in this study.
2. Materials and methods
2.1. 6 -OHDA lesion and rotational behavior tests Experiments were performed on adult Wistar rats (180–220 g, female). Rats were anaesthetized with chloralhydrate (400 mg / kg, i.p.) and mounted into a stereotaxic frame. One burr hole (2.5 mm diameter) was drilled in the skull above the region of the medial forebrain bundle (MFB), according to the coordinates of Paxinos and Watson [20]. Two stereotaxi microinjections of 6OHDA (Sigma; 3.6 mg / ml dissolved in saline containing 0.2 mg / ml L-ascorbate) were made into the right MFB [7]: (1) TB: 22.3 mm; AP: 24.4 mm; ML: 1.2 mm; V: 7.8 mm and (2) TB: 13.4 mm; AP: 24.0 mm; ML: 0.8 mm; V: 8.0 mm. Volumes of 2.5 ml and 3.0 ml of 6-OHDA were microinjected at a rate of 1.0 ml / min, respectively. After injection, the microinjection needle was left in place for a further 5 min before slowly retracting it. At 10–12 days after 6-OHDA injections, rats were tested for their rotational behavior with apomorphine (0.05 mg / kg, s.c., Sigma) in automated ‘rotometer’ bowls for 60 min. The rotational behavior was tested successfully for 3–5 times with an interval of 2 weeks. Rats that reached a level of at least five rotations / min were regarded as Parkinsonian rats. The study testing the effects of isatin on APO-induced rotations was carried out with self-control. At 30 min after pretreated with DMSO (dimethylsulfoxide, the solvent of isatin), isatin or pargyline i.p., Parkinsonian rats were tested about the contralateral rotations induced by APO, s.c. Drugs were injected at a 3-day intervals in case of toxic accumulation. Rotational behavior induced by APO, s.c. without pretreatment was regarded as the control, APO-induced rotations after drugs pretreatment were expressed as the percentage of the control. DMSO, isatin and pargyline used were all purchased from Sigma.
2.2. Electrical stimulation of MFB A concentric bipolar electrode was used for electrical stimulation of MFB. The coordinates were anterior 24.3 mm, lateral 1.5 mm relative to bregma and 6.5 mm below the dura. The stimulation electrode was lowered 0.5 mm towards MFB every 10 min. Electrical stimulation (200 ms square wave pulses, 1.5 mA, 100 Hz for 2 s) of the MFB was carried out until DA release was detected in the CPu. It was often the case that when the stimulation electrode was 7.5–8.0 mm below the dura, DA release was detected
2.3. Electrochemical technique: fast cyclic voltammetry ( FCV) The working electrode was prepared from 8 mm carbon fibers (Goodfellow, UK) and was positioned into CPu (AP: 11.1 mm; ML: 2.8 mm; V: 5.5 mm) to measure DA release. All electrochemical measurements were performed using a Millar voltammetric analyser (PD Systems, Survey, UK). Peak heights of the faradaic currents for the DA oxidation at 600 mV versus the reference electrode were recorded digitally on a PC through an interface CED 1401 (Cambridge Electronic). Data was stored and analyzed using CED software (Signal Averager and Chart, PD Systems). Following each experiment in vivo, the working electrode was calibrated in vitro in standard DA solutions (0.01–5.00 mmol / l). Electrode sensitivity and voltammetric signals were routinely assessed. Following histological studies were done to ensure that the position of stimulation electrode and working electrode were appropriate.
2.4. High-performance liquid chromatography with electrochemical detection Rats were decapitated under ethylether anaesthesia 1 h after isatin or DMSO was injected i.p. Both sides of CPu were isolated carefully and transferred to liquid nitrogen. Samples were weighed and then homogenized in 0.5 ml 1 M perchloric acid (HClO 4 ). After being centrifuged twice (12 000 rpm for 6 min at 48C), 20 ml of the supernatant were assayed for content of monoamine transmitters and their metabolites in CPu by HPLC–ECD. Separation was achieved on a PE C 18 reversed-phase column. The mobile phase (0.14 M citromalic acid–sodium citromalic acid buffer, 100 mM EDTA?2 Na, 0.78 mM sodium octane sulphonic acid and containing 10% (v / v) methanol, adjusted to pH 4.2 with HCl) was used at a flow-rate of 0.8 ml / min. A SIL-6A electrochemical detector (PE, USA) was employed and was operated in screen mode. Results were expressed as ng / mg wet weight of brain tissue.
2.5. Statistical analysis Data were expressed as means6S.E.M. The statistical analysis was performed by ANOVA followed by the Student–Newman–Keuls test, or by Student’s t test. A significant difference was considered as P,0.05 or ,0.01.
Y. Zhou et al. / Brain Research 917 (2001) 127 – 132
3. Results
3.1. Effects of isatin on APO-induced rotations of Parkinsonian rats At 3–5 min after APO was injected s.c., the Parkinsonian rats began to rotate towards the non-lesioned sides. Normally, the rotations were predominant within the initial 30 min after APO administration and disappeared not more than 60 min after injection. After DMSO was pretreated i.p., no abnormal actions were observed in all tested rats within the following 30 min and APO-induced rotations had no significant changes (103.967.7% of the control, n512). Noticeably, after pretreatment with isatin i.p., rats seemed to be silent and unwilling to move. Contralateral rotations did not happen 5–10 min or even 30 min after APO administration. Isatin inhibited APO-induced rotations in a concentration-dependent manner. When isatin was applied at the dose of 40 mg / kg, APO-induced rotations were reduced to 82.564.1% (n512) of the control. Predominantly, the rotations were diminished to 39.163.7% of the control (n512) following the administration of isatin at the higher concentration of 100 mg / kg (Fig. 1). After pretreatment with pargyline (75 mg / kg, i.p.), APO-induced rotations of Parkinsonian rats were similar with the control (99.465.5%, n512). From this study, we saw that APO-induced rotations were largely decreased when pretreated with 100 mg / kg isatin (ANOVA, F529.108, P,0.001); see Fig. 1.
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3.2. Effects of isatin on DA release from CPu in normal and Parkinsonian rats In this study, DA release before drug administration was considered as the control, DA release after drug administration was expressed with the percentage of the control. Ten minutes after injection of pargyline (75 mg / kg, i.p.), DA release began to increase (Fig. 2). In the following 140 min, the release increased constantly and reached the maximum (423.4610.8% of the control, n58). The facilitation of pargyline on DA release was still apparent 4 h after injection. However, DA release in CPu was not potentiated after isatin was injected (n58, Fig. 2). On the contrary, in our experiments, the release seemed to decrease 150 min after injection of isatin (77.267.8% of the control). After DMSO was injected i.p., the case (n56) was similar to that investigated following administration of isatin (Fig. 2). Furthermore, we monitored DA release from CPu in both lesioned and non-lesioned sides of Parkinsonian rats before and after administration of isatin (100 mg / kg, i.p.). In the lesioned sides of Parkinsonian rats, DA release was usually difficult to be detected using FCV. DA release was still not detectable (n56) 2–3 h after injection of isatin. Although DA release detected in non-lesioned sides was much higher than that recorded in the lesioned sides, the release from non-lesioned CPu after isatin administration was not significantly difference from that measured before administration of isatin (n56).
Fig. 1. Effects of drugs administration on APO-induced rotations of 6-OHDA-lesioned parkinsonian rats. APO-induced rotations after drugs pretreatment were expressed as the percentage of the control (rotational behavior induced by APO s.c. without pretreatment). *, P,0.05 and **, P,0.01 significantly different from that pretreatment with DMSO (by one-way ANOVA).
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Fig. 2. Effects of drugs administration on DA release of CPu in normal rats. DA release was monitored with FCV. Arrow in figure indicates drugs administration. DA release after drugs administration was expressed with the percentage of the control (DA release before drugs administration).
3.3. Effects of isatin on the contents of monoamine transmitters and their metabolites in CPu of normal and Parkinsonian rats Contents of DA and its metabolites in CPu were changed neither in normal rats nor in Parkinsonian rats after isatin was injected by 100 mg / kg, compared with the control that measured after DMSO injection; whereas the contents of 5-HT were higher than control in both normal rats and parkinsonian rats after isatin (100 mg / kg, i.p.) administration (P,0.01, n56). The content of 5-hydroxyindole acetic acid (5-HIAA) was unchanged (Fig. 3).
4. Discussion This experiment showed that isatin (100 mg / kg, i.p.) apparently inhibited APO-induced rotations of 6-OHDA lesioned Parkinsonian rats, while pargyline, an effective MAO-A and -B inhibitor with apparent effect on DA level in vivo, had no such effect. This result suggests that such an inhibitory action of isatin may have no relationship with its MAO inhibition. In addition, isatin had no apparent effects on electrical stimuli-induced DA release in vivo. Consistently, the content of DA detected with HPLC–ECD
was not increased after isatin administration. These results further indicate that isatin inhibiting APO-induced rotations of Parkinsonian rats cannot be attributed to its inhibition of DA catabolism. Previous studies have elucidated that small amounts of isatin (15–20 mg / kg, i.p.) are anxiogenic and proconvulsant in rodent models, but higher doses (60–80 mg / kg, i.p.) cause sedation and possess anticonvulsant action [4,9]. Another behavioral study also indicated that male rats injected with isatin (0–160 mg / kg, i.p.) ambulated less in the open field and were more immobile in the forced swim test than controls [1]. Neither the axiongenic action nor anticonvulsant action of isatin is likely to be related to its MAO inhibiting activity [4]. Therefore, the effect of isatin on APO-induced rotations of the Parkinsonian rats may be caused by its anticonvulsant action and could not be attributed to inhibition of endogenous MAO activity and of DA catabolism. Our results seem to contradict previous findings indicating that isatin administered in vivo significantly elevated the striatal DA level in Parkinsonian rats [11,19]. The different models of Parkinson’s disease and different strains of rats may contribute to this contradiction. Alternatively, a previous study has indicated that DA metabolism under basal conditions is primarily mediated by MAO-A, but both MAO-A and MAO-B mediate DA
Y. Zhou et al. / Brain Research 917 (2001) 127 – 132
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Fig. 3. Effects of isatin on monoamine transmitters and their metabolites in CPu of normal rats (A) and lesioned sides (B) and non-lesioned sides (C) of Parkinsonian rats. **, P,0.01 significantly different from pretreatment with DMSO (by Student’s t test).
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formation when L-DOPA is administered exogenously [5]. If this is so, as a potent MAO-B inhibitor, isatin ought to have little effect on DA and its metabolites. Besides, an early study has demonstrated that isatin, administered i.p. with an anticonvulsant dose of 50 or 100 mg / kg, resulted in a concentration of about 9 mg / kg or about 60 mM in rat brain [2]. However, this concentration of isatin is not likely to result in non-selective inhibition of MAO [4]. Therefore, the effects of isatin on DA and its metabolites observed previously may not relate to a MAO-B or-A blocking action. Consistent with previous studies [11,12,15,22], our study using HPLC also showed that the level of 5-HT in CPu of rats became higher after isatin was injected. Since the level of 5-HIAA did not change, and isatin did not influence the 5-HT and 5-HIAA content of the pineal gland [22], alternative non-MAO-inhibitor mechanisms are necessary to account for isatin-induced increase in monoamine levels in brain. Isatin in vivo might have a direct effect on the firing of monoamine neurons, which could also explain the anxiongenic effect of isatin [4,17]. In addition, it is also possible that its antagonist action at the ANP receptor results in an increase in the 5-HT level. ANP has been shown to inhibit arginine vasopressin (AVP) release in the brain, and AVP is known to potentiate the release of both catecholamines and cortisol via its action at the ANP receptor [17]. Unlike the effects of isatin on 5-HT level, the effects of isatin on DA level in vivo seemed to be complex and contradictory. The mechanism for this is uncertain and it is not yet clear what role the inhibition of MAO plays in the action. Whether isatin inhibition of MAO, particularly MAO-B, is physiologically relevant, remains to be determined.
[5]
[6]
[7]
[8]
[9]
[10]
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[13]
[14]
[15]
[16]
Acknowledgements
[17]
Supported by grants from National Plan of Tackling Key Problems in Science and Technology (969060508) of China and from National Foundation of Shandong Province (Y98D01049, L2000C01).
[18]
[19]
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anticonvulsant activity of isatin, a putative biological factor, in rats, Ind. J. Exp. Biol. 36 (1998) 118–121. T. Brannan, A. Prikhojan, J. Martinez-Tica, M.D. Yahr, In vivo comparison of the effects of inhibition of MAO-A versus MAO-B on striatal L-DOPA and dopamine metabolism, J. Neural. Transm. Park. Dis. Dement. Sect. 10 (1995) 79–89. C. Desvignes, L. Bert, L. Vinet, L. Denoroy, B. Renaud, L. LambasSenas, Evidence that the neuronal nitric oxide synthase inhibitor 7-nitroindazole inhibits monoamine oxidase in the rat: in vivo effects on extracellular striatal dopamine and 3,4-dihydroxyphenylacetic acid, Neurosci. Lett. 264 (1999) 5–8. C.D. Earl, T. Reum, J.X. Xie, Foetal nigral cell suspension grafts influence dopamine release in the non-grafted side in the 6-OHDA rat model of Parkinson’s disease: in vivo voltammetric data, Exp. Brain Res. 109 (1996) 179–184. V. Glover, S.K. Bhattacharya, A. Charkrabarti, M. Sandler, The psychopharmacology of isatin: a brief review, Stress Med. 14 (1998) 225–229. V. Glover, A.E. Medvedev, M. Sandler, Isatin: possible role in functional interaction of natriuretic peptides and monoamines, Vopr. Med. KhimNov. 43 (1997) 515–521. N. Hamaue, M. Minami, Y. Kanamaru, M. Ishikura, N. Yamazaki, H. Saito, S.H. Parvez, Identification of isatin, an endogenous MAO inhibitor, in the brain of stroke prone SHR, Biog. Amines 10 (1994) 99–110. N. Hamaue, Pharmacological role of isatin, an endogenous MAO inhibitor, Yakugaku Zasshi 120 (2000) 352–362. N. Hamaue, T. Endo, M. Hirafuji, N. Yamazaki, H. Togashi, H. Saito, M. Minami, Role of an endogenous monoamine oxidase inhibitor, isatin, in SHRSP brain, Clin. Exp. Pharmacol. Physiol. 22 (Suppl. 1) (1995) S86–87. C.C. Huang, J.J. Tsai, K.S. Hsu, L-Deprenyl (selegiline) limits the repetitive firing of action potentials in rat hippocampal CA1 neurons via a dopaminergic mechanism, Brain. Res. 753 (1997) 27–35. Y. Kishimoto, M. Geffard, R. Arai, Catecholamine degradation by monoamine oxidase in locus coeruleus neurons of the rat. An immunohistochemical study, Brain Res. 859 (2000) 373–377. R. Kumar, R.C. Bansal, A. Mahmood, In vivo effects of isatin on certain enzymes, lipids and serotonergic system of rat brain, Ind. J. Med. Res. 100 (1994) 246–250. I.M. McIntyre, T.R. Norman, Serotonergic effects of isatin, an endogenous MAO inhibitor related to tribulin, J. Neural. Transm. 79 (1990) 35–40. A.E. Medvedev, A. Clow, M. Sandler, V. Glover, Isatin: a link between natriuretic peptides and monoamines?, Biochem. Pharmacol. 52 (1996) 385–391. A.E. Medvedev, Monoamine oxidase, tribulin, isatin: basic and applied medical aspects, Vestn. Ross. Akad. Med. Nauk. 285 (1999) 45–48. M. Minami, N. Hamaue, T. Endo, M. Hirafuji, M. Terado, H. Ide, N. Yamazaki, M. Yoshioka, A. Ogata, K. Tashiro, Effects of isatin, an endogenous MAO inhibitor, on dopamine (DA) and acetylcholine (ACh) concentrations in rats, Nippon Yakurigaku Zasshi 114 (Suppl 1) (1999) 186–191. G. Paxions, C. Watson (Eds.), The Rat Brain in Stereotaxic Coordinates, Academic Press, London, 1986, pp. 1–104. H.K. Wayment, J.O. Schenk, B.A. Sorg, Characterization of extracellular dopamine clearance in the medial prefrontal cortex: role of monoamine uptake and monoamine oxidase inhibition, J. Neurosci. 21 (2001) 35–44. A. Yuwiler, The effect of isatin (tribulin) on metabolism of indoles in the rat brain and pineal: in vitro and in vivo studies, Neurochem. Res. 15 (1990) 95–100.
Research report
Effects of isatin on rotational behavior and DA levels in caudate putamen in Parkinsonian rats a,b b a, Yu Zhou , Zhi-Qi Zhao , Jun-Xia Xie * a
Department of Physiology, School of Medicine, Qingdao University, 38 Deng-Zhou Road, Qingdao 266021, PR China b Shanghai Institute of Physiology, Chinese Academy of Sciences, 38 Deng-Zhou Road, Shanghai 200031, PR China Accepted 27 July 2001
Abstract Isatin was a potent endogenous monoamine oxidase (MAO) inhibitor that is more active against MAO-B than MAO-A. The acute effects of isatin on apomorphine (APO)-induced rotations were evaluated in Parkinsonian rats induced by 6-hydroxydopamine (6-OHDA) lesion. Furthermore, the effects of isatin on DA release in caudate putamen (CPu) of model and normal rats were monitored using fast cyclic voltammetry (FCV). The contents of monoamine transmitters and their metabolites in CPu of model and normal rats were also analyzed by high performance liquid chromatography with electrochemical detection after administration of isatin. Here we show that isatin (100 mg / kg, i.p.) apparently inhibited APO-induced rotations of Parkinsonian rats to 39.163.7% of the control (n512), while it had no apparent effects on electrical stimuli-induced DA release either in normal rats or in model rats. In addition, the content of 5-hydroxytryptamine but not DA was increased in both normal rats and model rats after isatin (100 mg / kg, i.p.) was administered (P,0.01, n56). The content of 5-hydroxyindole acetic acid was not changed. These results suggest that isatin cannot increase DA levels in rat CPu. Therefore, the effects of isatin on APO-induced rotations of our Parkinsonian rats could not attribute to its inhibition of DA catabolism as a MAO inhibitor. 2001 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Degenerative disease: Parkinson’s Keywords: Isatin; Dopamine; Rotation; Parkinsonian rat; Fast cyclic voltammetry; High-performance liquid chromatography with electrochemical detection
1. Introduction Isatin (indole-2,3-dione) is widely distributed in mammalian tissues and body fluids. It has a distinct and discontinuous distribution in rat brain. The concentrations in hippocampus and cerebellum are approximately 0.1 mg / g, or about 1 mM [17]. Isatin is well known to have a broad range of biological and pharmacological actions [11,17,18]. In addition to a potent antagonist of the atrial natriuretic peptides (ANP) receptor and ANP-stimulated guanylate cyclase (GC) [8,18], isatin is also an effective endogenous MAO inhibitor in vitro, which is more active against MAO-B than MAO-A [8,10,15,17]. Compared with actions in vitro, the in vitro effects of *Corresponding author. Tel.: 186-532-383-8481; fax: 186-532-3801449. E-mail address: [email protected] (J.-X. Xie).
isatin seem to be more attractive and more complex. Early studies have suggested that isatin, administered i.p. to rats in a dose of 10–200 mg / kg, causes an increase of monoamines, such as serotonin (5-hydroxytryptamine, 5HT) and noradrenaline (NA) in the brain [3,10,16,22]. Other results have elucidated that striatal ACh and DA levels in rat significantly increased after isatin administration [11,19]. Furthermore, isatin could significantly increase striatal DA levels in Parkinsonian rats induced by Japanese encephalitis virus [11,19]. So, isatin may involved in the regulation and metabolism of dopamine in rats and have some protective effect against Parkinson’s disease. However, the mechanisms underlying isatin-induced increase of monoamine transmitters in brain are still not clear [17]. In this study, using Pargyling, a widely used MAO inhibitor [5,6,13,14,21], as the positive control, we investigated the acute effects of isatin on DA levels and apomor-
0006-8993 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )02935-3
128
Y. Zhou et al. / Brain Research 917 (2001) 127 – 132
phine (APO)-induced rotations of 6-hydroxydopamine (6OHDA)-lesioned Parkinsonian rats in order to further evaluate the effects of isatin on nigrostriatal dopaminergic system under certain pathological condition.
apparently. Moreover, the depth of stimulation electrode was adjusted appropriately for each experiment to ensure that the release was maximal. After the proper position of stimulation electrode was found and kept unchangeable, DA release from CPu was monitored in normal and Parkinsonian rats in this study.
2. Materials and methods
2.1. 6 -OHDA lesion and rotational behavior tests Experiments were performed on adult Wistar rats (180–220 g, female). Rats were anaesthetized with chloralhydrate (400 mg / kg, i.p.) and mounted into a stereotaxic frame. One burr hole (2.5 mm diameter) was drilled in the skull above the region of the medial forebrain bundle (MFB), according to the coordinates of Paxinos and Watson [20]. Two stereotaxi microinjections of 6OHDA (Sigma; 3.6 mg / ml dissolved in saline containing 0.2 mg / ml L-ascorbate) were made into the right MFB [7]: (1) TB: 22.3 mm; AP: 24.4 mm; ML: 1.2 mm; V: 7.8 mm and (2) TB: 13.4 mm; AP: 24.0 mm; ML: 0.8 mm; V: 8.0 mm. Volumes of 2.5 ml and 3.0 ml of 6-OHDA were microinjected at a rate of 1.0 ml / min, respectively. After injection, the microinjection needle was left in place for a further 5 min before slowly retracting it. At 10–12 days after 6-OHDA injections, rats were tested for their rotational behavior with apomorphine (0.05 mg / kg, s.c., Sigma) in automated ‘rotometer’ bowls for 60 min. The rotational behavior was tested successfully for 3–5 times with an interval of 2 weeks. Rats that reached a level of at least five rotations / min were regarded as Parkinsonian rats. The study testing the effects of isatin on APO-induced rotations was carried out with self-control. At 30 min after pretreated with DMSO (dimethylsulfoxide, the solvent of isatin), isatin or pargyline i.p., Parkinsonian rats were tested about the contralateral rotations induced by APO, s.c. Drugs were injected at a 3-day intervals in case of toxic accumulation. Rotational behavior induced by APO, s.c. without pretreatment was regarded as the control, APO-induced rotations after drugs pretreatment were expressed as the percentage of the control. DMSO, isatin and pargyline used were all purchased from Sigma.
2.2. Electrical stimulation of MFB A concentric bipolar electrode was used for electrical stimulation of MFB. The coordinates were anterior 24.3 mm, lateral 1.5 mm relative to bregma and 6.5 mm below the dura. The stimulation electrode was lowered 0.5 mm towards MFB every 10 min. Electrical stimulation (200 ms square wave pulses, 1.5 mA, 100 Hz for 2 s) of the MFB was carried out until DA release was detected in the CPu. It was often the case that when the stimulation electrode was 7.5–8.0 mm below the dura, DA release was detected
2.3. Electrochemical technique: fast cyclic voltammetry ( FCV) The working electrode was prepared from 8 mm carbon fibers (Goodfellow, UK) and was positioned into CPu (AP: 11.1 mm; ML: 2.8 mm; V: 5.5 mm) to measure DA release. All electrochemical measurements were performed using a Millar voltammetric analyser (PD Systems, Survey, UK). Peak heights of the faradaic currents for the DA oxidation at 600 mV versus the reference electrode were recorded digitally on a PC through an interface CED 1401 (Cambridge Electronic). Data was stored and analyzed using CED software (Signal Averager and Chart, PD Systems). Following each experiment in vivo, the working electrode was calibrated in vitro in standard DA solutions (0.01–5.00 mmol / l). Electrode sensitivity and voltammetric signals were routinely assessed. Following histological studies were done to ensure that the position of stimulation electrode and working electrode were appropriate.
2.4. High-performance liquid chromatography with electrochemical detection Rats were decapitated under ethylether anaesthesia 1 h after isatin or DMSO was injected i.p. Both sides of CPu were isolated carefully and transferred to liquid nitrogen. Samples were weighed and then homogenized in 0.5 ml 1 M perchloric acid (HClO 4 ). After being centrifuged twice (12 000 rpm for 6 min at 48C), 20 ml of the supernatant were assayed for content of monoamine transmitters and their metabolites in CPu by HPLC–ECD. Separation was achieved on a PE C 18 reversed-phase column. The mobile phase (0.14 M citromalic acid–sodium citromalic acid buffer, 100 mM EDTA?2 Na, 0.78 mM sodium octane sulphonic acid and containing 10% (v / v) methanol, adjusted to pH 4.2 with HCl) was used at a flow-rate of 0.8 ml / min. A SIL-6A electrochemical detector (PE, USA) was employed and was operated in screen mode. Results were expressed as ng / mg wet weight of brain tissue.
2.5. Statistical analysis Data were expressed as means6S.E.M. The statistical analysis was performed by ANOVA followed by the Student–Newman–Keuls test, or by Student’s t test. A significant difference was considered as P,0.05 or ,0.01.
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3. Results
3.1. Effects of isatin on APO-induced rotations of Parkinsonian rats At 3–5 min after APO was injected s.c., the Parkinsonian rats began to rotate towards the non-lesioned sides. Normally, the rotations were predominant within the initial 30 min after APO administration and disappeared not more than 60 min after injection. After DMSO was pretreated i.p., no abnormal actions were observed in all tested rats within the following 30 min and APO-induced rotations had no significant changes (103.967.7% of the control, n512). Noticeably, after pretreatment with isatin i.p., rats seemed to be silent and unwilling to move. Contralateral rotations did not happen 5–10 min or even 30 min after APO administration. Isatin inhibited APO-induced rotations in a concentration-dependent manner. When isatin was applied at the dose of 40 mg / kg, APO-induced rotations were reduced to 82.564.1% (n512) of the control. Predominantly, the rotations were diminished to 39.163.7% of the control (n512) following the administration of isatin at the higher concentration of 100 mg / kg (Fig. 1). After pretreatment with pargyline (75 mg / kg, i.p.), APO-induced rotations of Parkinsonian rats were similar with the control (99.465.5%, n512). From this study, we saw that APO-induced rotations were largely decreased when pretreated with 100 mg / kg isatin (ANOVA, F529.108, P,0.001); see Fig. 1.
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3.2. Effects of isatin on DA release from CPu in normal and Parkinsonian rats In this study, DA release before drug administration was considered as the control, DA release after drug administration was expressed with the percentage of the control. Ten minutes after injection of pargyline (75 mg / kg, i.p.), DA release began to increase (Fig. 2). In the following 140 min, the release increased constantly and reached the maximum (423.4610.8% of the control, n58). The facilitation of pargyline on DA release was still apparent 4 h after injection. However, DA release in CPu was not potentiated after isatin was injected (n58, Fig. 2). On the contrary, in our experiments, the release seemed to decrease 150 min after injection of isatin (77.267.8% of the control). After DMSO was injected i.p., the case (n56) was similar to that investigated following administration of isatin (Fig. 2). Furthermore, we monitored DA release from CPu in both lesioned and non-lesioned sides of Parkinsonian rats before and after administration of isatin (100 mg / kg, i.p.). In the lesioned sides of Parkinsonian rats, DA release was usually difficult to be detected using FCV. DA release was still not detectable (n56) 2–3 h after injection of isatin. Although DA release detected in non-lesioned sides was much higher than that recorded in the lesioned sides, the release from non-lesioned CPu after isatin administration was not significantly difference from that measured before administration of isatin (n56).
Fig. 1. Effects of drugs administration on APO-induced rotations of 6-OHDA-lesioned parkinsonian rats. APO-induced rotations after drugs pretreatment were expressed as the percentage of the control (rotational behavior induced by APO s.c. without pretreatment). *, P,0.05 and **, P,0.01 significantly different from that pretreatment with DMSO (by one-way ANOVA).
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Fig. 2. Effects of drugs administration on DA release of CPu in normal rats. DA release was monitored with FCV. Arrow in figure indicates drugs administration. DA release after drugs administration was expressed with the percentage of the control (DA release before drugs administration).
3.3. Effects of isatin on the contents of monoamine transmitters and their metabolites in CPu of normal and Parkinsonian rats Contents of DA and its metabolites in CPu were changed neither in normal rats nor in Parkinsonian rats after isatin was injected by 100 mg / kg, compared with the control that measured after DMSO injection; whereas the contents of 5-HT were higher than control in both normal rats and parkinsonian rats after isatin (100 mg / kg, i.p.) administration (P,0.01, n56). The content of 5-hydroxyindole acetic acid (5-HIAA) was unchanged (Fig. 3).
4. Discussion This experiment showed that isatin (100 mg / kg, i.p.) apparently inhibited APO-induced rotations of 6-OHDA lesioned Parkinsonian rats, while pargyline, an effective MAO-A and -B inhibitor with apparent effect on DA level in vivo, had no such effect. This result suggests that such an inhibitory action of isatin may have no relationship with its MAO inhibition. In addition, isatin had no apparent effects on electrical stimuli-induced DA release in vivo. Consistently, the content of DA detected with HPLC–ECD
was not increased after isatin administration. These results further indicate that isatin inhibiting APO-induced rotations of Parkinsonian rats cannot be attributed to its inhibition of DA catabolism. Previous studies have elucidated that small amounts of isatin (15–20 mg / kg, i.p.) are anxiogenic and proconvulsant in rodent models, but higher doses (60–80 mg / kg, i.p.) cause sedation and possess anticonvulsant action [4,9]. Another behavioral study also indicated that male rats injected with isatin (0–160 mg / kg, i.p.) ambulated less in the open field and were more immobile in the forced swim test than controls [1]. Neither the axiongenic action nor anticonvulsant action of isatin is likely to be related to its MAO inhibiting activity [4]. Therefore, the effect of isatin on APO-induced rotations of the Parkinsonian rats may be caused by its anticonvulsant action and could not be attributed to inhibition of endogenous MAO activity and of DA catabolism. Our results seem to contradict previous findings indicating that isatin administered in vivo significantly elevated the striatal DA level in Parkinsonian rats [11,19]. The different models of Parkinson’s disease and different strains of rats may contribute to this contradiction. Alternatively, a previous study has indicated that DA metabolism under basal conditions is primarily mediated by MAO-A, but both MAO-A and MAO-B mediate DA
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Fig. 3. Effects of isatin on monoamine transmitters and their metabolites in CPu of normal rats (A) and lesioned sides (B) and non-lesioned sides (C) of Parkinsonian rats. **, P,0.01 significantly different from pretreatment with DMSO (by Student’s t test).
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formation when L-DOPA is administered exogenously [5]. If this is so, as a potent MAO-B inhibitor, isatin ought to have little effect on DA and its metabolites. Besides, an early study has demonstrated that isatin, administered i.p. with an anticonvulsant dose of 50 or 100 mg / kg, resulted in a concentration of about 9 mg / kg or about 60 mM in rat brain [2]. However, this concentration of isatin is not likely to result in non-selective inhibition of MAO [4]. Therefore, the effects of isatin on DA and its metabolites observed previously may not relate to a MAO-B or-A blocking action. Consistent with previous studies [11,12,15,22], our study using HPLC also showed that the level of 5-HT in CPu of rats became higher after isatin was injected. Since the level of 5-HIAA did not change, and isatin did not influence the 5-HT and 5-HIAA content of the pineal gland [22], alternative non-MAO-inhibitor mechanisms are necessary to account for isatin-induced increase in monoamine levels in brain. Isatin in vivo might have a direct effect on the firing of monoamine neurons, which could also explain the anxiongenic effect of isatin [4,17]. In addition, it is also possible that its antagonist action at the ANP receptor results in an increase in the 5-HT level. ANP has been shown to inhibit arginine vasopressin (AVP) release in the brain, and AVP is known to potentiate the release of both catecholamines and cortisol via its action at the ANP receptor [17]. Unlike the effects of isatin on 5-HT level, the effects of isatin on DA level in vivo seemed to be complex and contradictory. The mechanism for this is uncertain and it is not yet clear what role the inhibition of MAO plays in the action. Whether isatin inhibition of MAO, particularly MAO-B, is physiologically relevant, remains to be determined.
[5]
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Acknowledgements
[17]
Supported by grants from National Plan of Tackling Key Problems in Science and Technology (969060508) of China and from National Foundation of Shandong Province (Y98D01049, L2000C01).
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