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Pharmacological involvement of the calcium channel blocker flunarizine in dopamine transmission at the striatum
Parkinsonism & Related Disorders Parkinsonism and Related Disorders 8 (2001) 33±40
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Pharmacological involvement of the calcium channel blocker ¯unarizine in dopamine transmission at the striatum J.E. Belforte a, C. MagarinÄos-Azcone c, I. Armando b, W. BunÄo c, J.H. Pazo a,* a
Laboratorio de Neuro®siologõÂa, Departamento de FisiologõÂa, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina b Centro de Investigaciones EndocrinoloÂgicas, Hospital de NinÄos Ricardo Gutierrez, Buenos Aires, Argentina c Instituto Cajal (CSIC), Hospital RamoÂn y Cajal, Madrid, Spain
Abstract Single intrastriatal microinjections of 25, 50 and 100 nmol/ml of ¯unarizine in normal rats produced a dose-dependent turning behavior toward the injected side when they were challenged with apomorphine (1 mg/kg, s.c). This effect was seen at 1, 3 and 7 days following administration of the high dose of ¯unarizine, but had subsided by 24 h after administration of the intermediate dose; the low dose was ineffective. However, intrastriatal injection of the high dose of ¯unarizine resulted in a local lesion and thereafter this dose was not used. A similar dose-response relationship was determined for nifedipine, an L-type calcium channel antagonist. Injection of this antagonist did not result in apomorphine-elicited rotational behavior, re¯ecting its lack of antidopaminergic action. Intrastriatal injections of haloperidol (5 mg/ ml), an antagonist of dopamine D2 receptors, or the sodium channel blocker lidocaine (40 mg/ml), were given in order to compare their effects to those observed with ¯unarizine. Intracerebral injection of haloperidol produced ipsilateral turning in response to systemic administration of apomorphine given 60 min after. The same response was obtained with the injection of apomorphine 10 min after the injection of intracerebral lidocaine. This effect was no longer apparent 24 h after the microinjection of haloperidol and 60 min after the injection of lidocaine. In rats rendered hemiparkinsionian by lesioning the nigrostriatal pathway with 6OHDA, intrastriatal microinjection of ¯unarizine (50 nmol/ml) signi®cantly reduced apomorphine (0.2 mg/kg, s.c.)-elicited turning behavior towards the non-lesioned side. These results suggest an antidopaminergic effect of ¯unarizine mediated by antagonistic action of post-synaptic striatal dopamine receptors. However, an action of the drug on sodium channels may not be ruled out. These studies offer additional supporting evidence for the induction or aggravation of extrapyramidal side-effects in patients receiving ¯unarizine. q 2001 Elsevier Science Ltd. All rights reserved. Keywords: Flunarizine; Nifedipine; Striatum; Parkinsonism; Calcium channel blockers; Circling behavior
1. Introduction Calcium channel blockers are widely used in the medical practice for the treatment of cardiovascular, pulmonary, neurological and genitourinary diseases [3]. One of them, ¯unarizine, a piperazine derivative is a mixed T and L type calcium channel blocker and sodium channel antagonist [5,6,34]. It has been found to be effective for cardiovascular and neurological diseases, as well as in the prophylaxis and treatment of migraine headache [13]. Futhermore, ¯unarizine posseses anticonvulsivant properties in both humans and animals [4], and reduces the duration of recurrent hemiplegia of childhood [2]. It is also used as a neuroprotective agent in the treatment of post-cerebrovascular disorders, cerebral infarction and cerebral hemorrhage [17,19]. However, these bene®cial therapeutic effects of ¯unarizine are frequently associated with aggravation or even induction * Corresponding author. Tel.: 154-1-4508-3740. E-mail address: [email protected] (J.H. Pazo).
of extrapyramidal motor signs in chronic treatment in elderly patients [10,14,24]. This side-effect of ¯unarizine is shared with cinnarizine, other calcium channel blocker derived from piperazine and less frequently by other calcium antagonists [22,25]. Despite clinical reports of extrapyramidal side-effects associated with ¯unarizine treatment, experimental studies of the actions of this calcium channel antagonist on dopaminergic neurotransmission are con¯icting. Flunarizine has been suggested to inhibit the dopamine (DA) uptake process [12,23]. Furthermore, ¯unarizine has been reported to inhibit [ 3H]-spiperone binding to D2 and [ 3H]-SCH23390 binding to D1 striatal receptors with the highest af®nity for D2 receptors [1]. On the other hand, it has been demonstrated that systemic ¯unarizine increases striatal extracelular dopamine levels [27,30]. However, other reports examining the repeated administration of ¯unarizine have indicated reduced DA release [23,30]. Similarly, con¯icting studies have been reported on the effects of ¯unarizine on tyrosine hydroxylase [23,31].
1353-8020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved. PII: S 1353-802 0(01)00006-2
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Information concerning the anatomical sites where the calcium blockers exert their pharmacological side-effects are still unclear, since most experimental data have examined the effects of systemic administration of the calcium antagonists or used in vitro approaches. In order to help clarify the actions and site of action of ¯unarizine, we assessed the effects of intrastriatal microinjections of the calcium channel antagonist on nigrostriatal dopaminergic transmission in the rat by monitoring apomorphine-elicited circling behavior. We also studied the effects of another sub-classes of calcium channel blocker antagonists by treating rats with the dihydropyridine derivative, nifedipine, a L-type channel antagonist with very few extrapyramidal side-effects [22]. 2. Material and methods The experiments were carried out in several groups of male rats of the Sprague Dawley strain weighing 300± 350 g, and in strict accordance with the Principles of laboratory animal care (NIH publication no. 86±23, revised 1985). Under pentobarbital anesthesia (45 mg/kg, i.p.), the animals were placed in a stereotaxic frame (DK) and a burr hole was made over the striatum according to the atlas of Paxinos and Watson [28]. A cannula (0.3 mm OD), attached to a 5 ml Hamilton syringe, was lowered into the left or right striatum and the calcium antagonist or the vehicle was injected in a volume of 1 ml in about 2 min. The injection needle was left in place two additional minutes. Dose-response curves for ¯unarizine and nifedipine (kindly supplied by Janssen Labs) were obtained by microinjections of 25, 50 and 100 nmol/ml of ¯unarizine and 50, 100, 200 nmol/ml of nifedipine dissolved in 22% of b-ciclodextrin (RBI) in saline. Three groups of rats were treated with ¯unarizine and the other three with nifedipine The vehicle was administrated to a control group. Nifedipine was used in order to search whether L-channels could be involved in the effects of ¯unarizine. Flunarizine and nifedipine treated animals were then tested with apomorphine (1 mg/kg, 1 ml/kg, s.c, Sigma) disolved in 0.2% ascorbic acid, 1, 3 and 7 days following intrastriatal microinjections. Immediately after the injection of apomorphine, the rats were placed in an automatic rotometer. The full rotations for both side were recorded during 60 min, at 2 min intervals. We thought it of interest to compare the effect of intracerebral ¯unarizine to the effect of haloperidol, a dopamine D2 antagonist. For this purpose, two groups of rats were anesthetized, one was injected into the striatum with haloperidol (5 mg/ml of commercial injectable solution, kindly supplied by Janssen Labs) and the other group was injected with the solvent (kindly supplied by Janssen Labs). Both groups were challenged with apomorphine (1 mg/kg, s.c.) 24 h after the intracerebral microinjections. To test the possibility that the lack of sustained effect of intracerebral injections of nifedipine and haloperidol 24 h after their administration could be due to short half-life of
the drugs, and in order to compare the action of lidocaine (kindly supplied by Astra Labs), a short half-life sodium channel blocker [8] to the effect of ¯unarizine, experiments were performed in rats implanted with guide cannulae. This procedure was required for testing drug action at short intervals. Rats anesthetized with pentobarbital (45 mg/kg, i.p) were stereotaxically implanted over the striatum (coordinates: AP 10.48 mm from Bregma, L: 3 mm, D: 25 mm from cranium surface) [28] with stainless steel guide cannulae (26 g., Plastics One) and ®xed in place by screws (3/32, Plastics One) and dental acrylic. Seven days were allowed for recovery. The rats were then divided into several groups. Each group was microinjected into the striatum with one of the following drugs, lidocaine (40 mg/ml in saline), nifedipine (50 nmol/ml in b-ciclodextrin), haloperidol (5 mg/ml, commercial injectable solution) and ¯unarizine (50 nmol/ml in b-ciclodextrin). Control animals were injected with the appropiated solvent. The dose of lidocaine was chosen because it is known to block neurotransmission in the central nervous system [15,21]. The dose of haloperidol used has been reported to produce changes in the activity of the central nervous system [16,20]. Microinjections were made in conscious rats by gently restraining the animal with the hand and inserting the injection needle (33 g, Plastics One) that extended 1 mm beyond the guide cannula tip. Injections were made at a rate of 0.5 ml/60 s and were left in place for at least 60 s to allow the drug to diffuse. Half of the rats injected with lidocaine were tested with apomorphine (1 mg/kg, s.c.) 10 min after the brain injection and the other half 60 min after the brain injection. The other groups (haloperidol, ¯unarizine and nifedipine) were tested with apomorphine (1 mg/kg, s.c.) 60 min after the microinyection of the drugs. In order to ascertain whether the action of ¯unarizine in normal rats could be due to a pre- or post-synaptic effect, experiments were made in rats with unilateral lesion of the nigrostriatal dopaminergic system. Rats anesthetized with pentobarbital (45 mg/kg, i.p.) were placed in a stereotaxic frame, and the nigrostriatal pathway was lesioned on one side by a microinjection into the medial forebrain bundle of 8 mg of 6-hydroxydopamine-HCl (6OHDA), dissolved in 4 ml of 0.2% ascorbic acid in saline. The animals were pre-treated 30 min before the intracerebral administration of 6OHDA with disipramine (25 mg/kg, i.p.) to prevent damage to noradrenergic neurons. The effectiveness of the denervation was evaluated by challenging the rats with apomorphine (0.2 mg/kg, s.c.) two weeks after the lesion. Only those animals with more than 150 full contralateral net turns in 60 min, were used for subsequent experiments. In some rats chosen at random, dopamine and dopac content in the intact and lesioned striatum were measured in dissected tissue, using a high pressure liquid cromatography (HPLC) coupled to amperometric electrochemical detector [9]. Four weeks after 6-OHDA lesioning, the rats were again anesthetized and divided into two groups. One was microinjected into the striatum with ¯unarizine (50 nmol/ml) and the other
J.E. Belforte et al. / Parkinsonism and Related Disorders 8 (2001) 33±40
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Fig. 1. Histological reconstruction of the injection sites in normal and 6OHDA treated rats. Outlines and levels were adopted from Paxinos and Watson [28].
with the vehicle of the drug. The rotatory activity induced by s.c. injection of apomorphine (0.2. mg/kg) was evaluated 1, 3, 7 and 14 days following the intrastriatal microinjections. The sites of injection in the striatum were determined in serial histological sections stained with cresyl violet. Their locations were evaluated with reference to the atlas of Paxinos and Watson [28]. Animals that were found not to have been injected in the target areas were discarded. The following turning behavior parameters were analyzed: (1) Total turns, that is, the number of ipsilateral plus contralateral turns, as a measure of overall locomotor activity, (2) Net turns, are ipsilateral minus contralateral turns and (3) Asymmetric index, that is, ipsilateral turns as percentage of total turns. A ratio of 50% would indicate a non-directional bias, and a higher or lower ratio would indicate ipsilateral or contralateral asymmetry, respectively. Statistical evaluation was carried out by means of one or two-way ANOVA for paired and unpaired measures followed by Newman-Keuls post-hoc test. In some experiments Student's t-test was also applied. Values of P , 0.05 were considered statistically signi®cant. 3. Results Microinjections of ¯unarizine into the dorsal striatum in normal animals (Fig. 1), did not induce spontaneous circling behavior in the inmediate post-operative period. Systemic administration of apomorphine (1 mg/kg, s.c.), 24 h after microinjection of ¯unarizine, produced net rotations towards the injected striatum (ipsilateral turning, Figs. 2(B and C) and 3) at doses of 50 and 100 nmol/ml, but not with 25 nmol/ml. However, with the highest dose (100 nmol/ 1 ml) ipsilateral turnig activity was observed at 3 and 7 days later, but not with the dose of 50 nmol/ml (Fig. 2(B)
Fig. 2. Circling behavior induced by systemic administration of apomorphine (1 mg/kg, s.c.) at different intervals following intrastriatal microinjections of ¯unarizine. Data are means ^ SEM of n rats per group. There was a signi®cant difference in net turns and asymmetric index 24 h after injection of ¯unarizine (50 and 100 nmol/ml) as compared to vehicle treated rats. For net turns, p P , 0.05 and p p P , 0.005 Newman-Keuls after two way ANOVA for repeated measures, treatment F3,29 28.17, P , 0.0001; treatment £ time F6,58 2.44, P , 0.05. For asymmetric index, p P , 0.05 and p p P , 0.005 Newman-Keuls, after two way ANOVA for repeated measures, treatment F3,29 14.32, P , 0.0001; treatment £ time F6,58 3.89, P , 0.05. Abbreviations: ipsi ipsilateral turns, contra contralateral turns.
and (C)). Total turns were not modi®ed by any doses of ¯unarizine (Fig. 2(A)). Microinjections of b-ciclodextrin (22% in saline, 1 ml) produced turning activity to both sides without any preference in response to apomorphine (Fig. 2(B and C)). In addition, systemic administration of the solvent of apomorphine (ascorbic acid) had no effect in circling behavior in ¯unarizine treated rats (Table 1). Histological analyses of injection sites made at the end of the test (7 days) show a lesion in the sites injected with 100 nmol/ml (Fig. 4(A)), while in the sites injected with 25 and 50 nmol/ml no histological damage was observed (Fig. 4(B)). On the basis of this ®ndings, thereafter we chose 50 nmol dose for the following experiments. Intrastriatal microinjections of nifedipine were ineffective in changing any rotational parameters induced by apomorphine when compared to solvent treated animals (Fig. 5). To test the possibility that the lack of ef®cacy
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J.E. Belforte et al. / Parkinsonism and Related Disorders 8 (2001) 33±40
Fig. 3. Dose-response curve for net turns from different doses of ¯unarizine microinjected into the striatum and induced by apomorphine (1 mg/kg, s.c) administrated 24 h later. Data are means ^ SEM of n rats per group taken from experiment of Fig. 2. All turns are toward the injected striatum (ipsilateral turns). There was a signi®cat difference between 50 and 100 nmol/ml when compared to solvent microinjected rats. p P , 0.05 and p p P , 0.005 Newman-Keuls, after two way ANOVA for repeated measures, treatment F3,29 28.17, P , 0.0001. Abbreviations: ipsi ipsilateral turns, contra contralateral turns.
Fig. 4. Photomicrographs of coronal sections through the striatum one week following microinjections of ¯unarizine (arrowheads). (A) a microinjection of 100 nmol/ml of ¯unarizine. Note the lesion around the site of injection. (B) a microinjection of 50 nmol/ml of ¯unarizine. Note the track left by the injection cannula Magni®cation £ 5.
seen in nifedipine could be due to its short half-life, experiments were performed in animals with implanted cannulae in the striatum. The intrastriatal administration of nifedipine did not produce spontaneous motor activity in the immediate post-injection period. The motor responses of these animals to apomorphine, injected i.p 60 min after the nifedipine, were similarly to those of control rats injected with the solvent (Table 2). Histological analyses did not show any lesions in the striatum at the doses of nifedipine used. Taken together, these data indicate that nifedipine did not affect dopaminergic transmission and for this reason was not used in the following experiments. Table 1 Effect of the solvent of apomorphine (ascorbic acid 0.2%) injected s.c 1 ml/ kg in rats microinjected 24 h before with ¯unarizine (50 nmol/ml) or its solvent into the striatum. Data are the means ^ SEM of n rats per group. There were no signi®cant (ns) differences between the groups, Student's t-test. Negative sign contralateral turns Treatment Solvent (n 3) Flunarizine (n 5)
Total turns/h
Net turns/h
Asymmetric index %
19.6 ^ 9.5 25.4 ^ 9.2
2 3 ^ 4.5 2 0.2 ^ 4.1
51.6 ^ 9.7 49.3 ^ 8.5
Table 2 Turning behavior in rats injected with ¯unarizine (50 nmol/ml) and nifedipine (50 nmol/ml) into the striatum and tested 60 min later by systemic administration of apomorphine (1 mg/kg, s.c), compared to rats injected with the solvent. Data are the means ^ SEM of n rats per group. There were no signi®cant differences after one way ANOVA, for total F2,23 1.47, P ns, for nets F2,23 2.26, P ns, for index F2,23 2.65, P ns. Negative sign contralateral turns Treatment
Total turns/h
Solvent (n 12) 152.3 ^ 19.1 Nifedipine (n 8) 183.6 ^ 17.5 Flunarizine (n 6) 141.5 ^ 18.2
Net turns/h Asymmetric index % 7.3 ^ 9.2 2 8.0 ^ 12.9 43.5 ^ 30.2
52.6 ^ 3.4 48.7 ^ 4.2 65.9 ^ 8.6
Fig. 5. Turning activity induced by systemic administration of apomorphine (1 mg/kg, s.c.) at different intervals following intrastriatal microinjection of nifedipine. Data are means ^ SEM of n rats per group. There is not significant difference in any of the motor parameters studied when compared to control rats injected with solvent. For total turns, treatment two way ANOVA for repeated measures, F3,14 1.79, P ns; for net F3,14 0.14, P ns; for index F3,14 0.11, P ns. Abbreviations: ipsi ipsilateral turns, contra contralateral turns.
153.2 ^ 16.2 (n 5) 2 11.2 ^ 28.0 45.5 ^ 8.8 a
109.4 ^ 18.4 (n 5) 2 26.2 ^ 21.8 44.9 ^ 14.1
P , 0.05. P , 0.001 Newman-Keuls, after one-way ANOVA, for nets F5,28 6.85; P , 0.0005, for index F5,28 4.71, P , 0.005.
152.8 ^ 31.8 (n 5) 61.6 ^ 17.9 a 73.6 ^ 6.3 a b
1h
156.0 ^ 15.9 (n 4) 112.0 ^ 27.4 b 84.4 ^ 5.5 a 149.0 ^ 22.0 (n 8) 2 3.3 ^ 10.6 46.8 ^ 4.0 Total turns/h Net turns/h Asymmetric index %
24 h
Solvent
159.7 ^ 22.2 (n 7) 2 2.0 ^ 12.2 47.4 ^ 4.5
10 min
Lidocaine
60 min
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Haloperidol Solvent
In order to determine whether the effects of ¯unarizine could be similar to the action of haloperidol, a dopamine receptor antagonist, and lidocaine, a sodium channel blocker, we examine their effects in rats implanted with guide cannulae. A microinjection of haloperidol (5 mg/ml) was given into the striatum 60 min before an i.p injection of apomorphine (1 mg/kg, s.c). Apomorphine induced a significant turning activity ipsilateral to the injected striatum and increases of the asymmetric index (Table 3), as compared to animals microinjected with haloperidol vehicle. There was no change in total turning. Nevertheless, in the other group of rats, administration s.c of apomorphine 24 h after intrastriatal injection of haloperidol failed to modify circling activity (Table 3). Two groups of rats were intrastriatally injected with lidocaine (40 mg/ml) through implanted cannulaes and tested with apomorphine (1 mg/kg, s.c). One group was tested 10 min after intracranial microinjections and the other 60 min later. Signi®cant motor changes were only produced in the 10 min group. The rats rotated to the side ipsilateral to the injection site and the asymmetric index was increased as compared to animals microinjected with solvent (Table 3). Total turns remained unchanged. To compare the effects at short intervals of nifedipine, haloperidol and lidocaine to the action of ¯unarizine at short interval after its intracerebral microinjection. Flunarizine (50 nmol/ml) was injected into the striatum through implanted guide cannulae in one group of rats and tested 60 min later with apomorphine (Table 2). Flunarizine had no effect on any of the parameters studied, although a tendency towards an increase in the number of net turns was observed. However, it did not reach signi®cance, perhaps due to the wide-ranging variability. The range of net turns was from 237 (contralateral turns) to 83 (ipsilateral turns). No lesions were observed at the striatal sites microinjected with haloperidol or lidocaine. In hemiparkinsonian rats made by unilateral lesioning of the nigrostriatal pathway with 6OHDA, systemic administration of apomorphine (0.2 mg/kg, s.c) produced turning behavior contralateral to the lesioned nigrostriatal pathway. These rats were divided into two groups with similar rotational behavior (no signi®cant differences between groups in net turns induced by apomorphine) and microinjected into the lesioned striatum. One group was injected with 50 nmol/ml of ¯unarizine and the other with solvent. Intrastriatal administration of ¯unarizine produced a signi®cant reduction in the net contralateral turns induced by systemic administration of apomorphine as compared to animals injected with the solvent (Fig. 6(B)). This was re¯ected by a decrease in the total turns (Fig. 6(A)). The effect was observed 1, 3, 7 and 14 days after intrastriatal microinjection of the calcium antagonist. The asymmetric index was only signi®cant 24 h after treatment with ¯unarizine, which indicates a less asymmetric activity in the ¯unarizine treated rats as compared to the solvent-injected animals (Fig. 6(C)). Histological analyses of hemiparkinsonian
Table 3 Turns induced by apomorphine (1 mg/kg, s.c) at different intervals after intrastriatal microinjections of haloperidol (5 mg/ml) and lidocaine (40 mg/ml) Data are the means ^ SEM of n rats per group. The data for the solvent group of haloperidol at 60 min and 24 h were quantitatively similar and have therefore been combined, as is the case for the control group for lidocaine. There were signi®cant differences for net turns and asymmetric index at 60 min for haloperidol and at 10 min for lidocaine when compared to solvent groups. Negative sign contralateral turns
J.E. Belforte et al. / Parkinsonism and Related Disorders 8 (2001) 33±40
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J.E. Belforte et al. / Parkinsonism and Related Disorders 8 (2001) 33±40
Fig. 1 shows the histological reconstruction of the injection sites in the different groups of animals.
4. Discussion
Fig. 6. Effect of intrastriatal microinjection of 50 nmol/ml of ¯unarizine in rats with unilateral lesion of nigrostriatal pathway by 6OHDA, on the turning behavior induced by apomorphine (0.2 mg/kg, s.c.) injected at different times after intracerebral microinjection. The animals turn to the side opposite to the lesioned striatum (contralateral turns). Data are means ^ SEM of n rats per group. The global activity (A) was signi®cantly reduced by ¯unarizine as compared to control group at all studied intervals. Two way ANOVA for repeated measures, treatment £ time F4,76 4.51, P , 0.005. The net turns (B) were also reduced by ¯unarizine. Two way ANOVA for repeated measures, treatment £ time F4,76 5.12 P , 0.001. The asymmetric index (C) was greater than control animals 24 h after intrastriatal injection, that is less asymmmetry. Two way ANOVA, treatment £ time F4,76 3, P , 0.05. Newman-Keuls for comparisons between means p P , 0.05, p p P , 0.01 and p p p P , 0.001. Pre before ¯unarizine, i.e. rats tested with apomorphine alone. Flunarizine was microinjected at time zero.
animals did not show lesions in the sites injected with ¯unarizine. The levels of dopamine and dopac in eight 6OHDA lesioned rats, taken at random from each group, showed a decrease of 96 and 92.7%, respectively, in the lesioned striatum as compared to the intact side (Table 4).
The calcium channel blockers, particularly those derived from piperazine, ¯unarizine, are able to produce abnormal motor signs similar to those seen in parkinsonism [22,25]. In some cases this effect is transient and disappears following the withdrawal of the drug. However, in other cases, the effect remains despite withdrawal of the drug [23]. The mechanism of this action is not totally understood. On the basis of experimental results, many hypotheses have been proposed concerning the mechanisms and sites of action of the calcium blockers in the central nervous system [14,25]. In order to rule out whether a calcium blocker, such as nifedipine, with different chemical structure and antagonistic action, injected intrastrially would have a comparable effect to that of ¯unarizine, it was microinjected into the striatum and tested to different intervals with apomorphine given s.c. However, the turning response elicited by administration of apomorphine remained unaffected. This could be interpreted as this L channel blocker having no effect on dopaminergic neurotransmission, which is in agreement with previous reports of its weak antidopaminergic action and reduced likelihood in producing drug-induced parkinsonism [23,30]. On the other hand, acute unilateral intrastriatal injection of 50 nmol/ml of ¯unarizine, a T and L calcium channel blocker induces a transient turning activity 24 h following its administration, in response to systemic injection of apomorphine. This was expressed as signi®cant increases in net turns towards the injected side as compared to solvent-injected rats as well as in the asymmetric index. However, the highest dose used of ¯unarizine (100 nmol/ml) produced an unexpected lesion at the level of the site of injection into the striatum. This was responsible for the persistency of the apomorphine-induced turning activity during all test sessions (Fig. 2). This is in agreement with previous studies in neuroblatoma cells in which the observed cell death was attributed to the effect of ¯unarizine [23]. Intrastriatal administration of haloperidol and lidocaine produced an ipsiversive circling behavior in response to apomorphine. However, the pharmacodynamic of both drugs were different because we were still able to elicit the effect of haloperidol 60 min after its intracerebral injection while the
Table 4 Concentration of dopamine and dopac in the striatum of the lesioned and contralateral intac side
Dopamine (n 8) (pg/mg tissue) Dopac (n 8) (pg/mg tissue) a b
Lesioned side
Intact side
% decreased
976.7 ^ 268 502.6 ^ 153
24711.6 ^ 1039 b 6944.3 ^ 1221 a
96.0 92.7
P , 0.001. P , 0.00001, Student's t-test, when was compared intact vs lesioned side.
J.E. Belforte et al. / Parkinsonism and Related Disorders 8 (2001) 33±40
effect of lidocaine only lasted 10 min after microinjection. In addition, the effect of haloperidol disapeared 24 h after its intracerebral injection and the lidocaine after 60 min. The unilateral imbalance between basal ganglia on both sides produced by these drugs and brought on by stimulation of striatal dopamine receptors [29] could be attributed to their activity as receptors and channel blockers. Haloperidol is an antagonist of the D2 receptors and lidocaine of sodium channels [8,20,30]. The effects of haloperidol and lidocaine were similar to those observed when ¯unarizine was administrated into the striatum. However, the time course of action was different. The haloperidol and lidocaine have a rapid onset of effect and are short acting, while ¯unarizine has a slow onset of effect and long action. However, there is good evidence that ¯unarizine blocks dopamine receptors mainly of D2 type [1,23,30] and its effect is similar to the action of haloperidol. These facts strongly suggest that the effect of the drug could mainly be attributed to interference with dopaminergic transmission in the striatum. Likewise, an effect of ¯unarizine on voltage-dependent sodium channels may be not excluded from our experiments. In order to elucidate whether the effects observed by intrastriatal microinjection of ¯unarizine would be consequence of a pre- or post-synaptic action of the drug, experiments were performed in rats with unilateral dopaminergic denervation of the striatum by intracerebral administration of 6OHDA into the nigrostriatal pathway. These hemiparkinsonian rats develop an up-regulation of striatal dopaminergic receptors, mainly of type D2 [33], which was accompanied by spontaneous circling behavior, contralateral to the denervated striatum. This activity could also be evoked by systemic administration of apomorphine. In these animals, unilateral injection of ¯unarizine into the denervated striatum signi®cantly decreased the total turns in response to apomorphine as the result of a decrease in contralateral rotations. These effects were observed during all testing sessions (Fig. 6). The asymmetric index was only modi®ed at 24 h after intrastriatal injection of ¯unarizine.This means there was less asymmetric motor activity in rats treated with ¯unarizine than in animals injected with solvent. Intrastriatal administration of ¯unarizine did not modify the time-course of the development of supersensitivity in the striatum of 6OHDA treated rats. The curves followed an almost parallel course, with the only difference being that the rats treated with ¯unarizine began from a lower activity at 24 h. (Fig. 6(A)). The fact that the effect of ¯unarizine was long-lasting in 6OHDA lesioned rats as compared to animals with normosensitive striatum may be attributed to biochemical changes that occurs in dopamine receptors after treatment with 6OHDA [7,18]. These results in 6OHDA treated rats demonstrated a post-synaptic effect of ¯unarizine, since they were observed in a denervated striatum. This neuroleptic-like action of ¯unarizine on post-synaptic dopamine receptors in the striatum may explain the rapid
39
onset of motor side-effects in patients [26]. Likewise, the long duration effect of ¯unarizine on altered dopamine receptors like those in 6OHDA rats could give an explanation of the drug effect in elderly patients, where an agerelated decline of dopamine cells occurs in the substantia nigra pars compacta [11], or in Parkinson's disease. However, cellular lesions may be also considered in light of neurotoxic action of ¯unarizine at high doses. Taken together, these factors could be related to the permanent effect of the calcium blocker in some patients in addition to the other mechanisms described for these side-effects [27,30,32]. In conclusion, our observations con®rm and extend previous reports on the neuroleptic-like action of ¯unarizine, and they are the ®rst description of the effects of the drug directly microinjected into the striatum. This action is probably exerted by blocking the post-synaptic striatal dopamine receptors mainly of D2 type and it has long duration in modifying dopamine receptors. However, an action on sodium channels or other receptors could not be ruled out.
Acknowledgements This work was supported by grants from CONICET, UBA, and the National Agency for Scienti®c and Technological Promotion (FONCYT). References [1] Ambrosio S, Stefanini E. Interaction of ¯unarizine with dopamine D2 and D1 receptors. Eur J Pharmacol 1991;197:221±3. [2] Andermann E, Andermann F, Silver K, Arnold D. Benign familial nocturnal alternating hemiplegia of childhood. Neurology 1994;44:1812±4. [3] Barnes PJ. Clinical studies with calcium antagonists in asthma. Br J Clin Pharmacol 1985;20:9±13. [4] Barnes PJ, Bleck TP. New anticonvulsant drugs. Focus on ¯unarizine, fosphenytoin, midazolam and stiripentol. Drug 1994;48:153±71. [5] Ragsdale DS, Scheuer T, Catterall WA. Frequency and voltagedependent inhibition of type IIA Na 1 channels, expressed in a mammalian cell line, by local anesthetic, antiarrhythmic, and anticonvulsant drugs. Mol Pharmacol 1991;40:756±65. [6] Blackburn-Munro G, Fleetwood-Walker SM. The effects of Na 1 channels blokers on somatosensory processing by rat dorsal neurons. NeuroReport 1997;8:1549±54. [7] Bezard E, Gross CE. Compensatory mechanisms in experimental and human parkinsonism: towards a dynamic approach. Prog Neurobiol 1998;55:1±24. [8] Bingmann D, Lipinski HG, Hagemann G, Speckmann EJ, Tetsch P. Time courses of lidocaine effects on sodium membrane currents in small and large neurons. Gen Physiol Biophys 1990;9:331±42. [9] Buchholz J, Tsai H, Foucart S, Duckles SP. Advancing age alters intracellular calcium buffering in rat adrenergic nerves. Neurobiol Aging 1996;16:885±92. [10] Chousa C, Scaramelli A, CaamanÄo JL, DeMedina O, Aljanati R, Romero S. Parkinson, tardive dyskinesia, akathisia, and depression induced by ¯unarizine. Lancet 1986;1:1303±4. [11] Clough CG. Parkinson's disease: management. Lancet 1991;337:1324±7.
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[12] Devoto P, Pani L, Kuzmin A, De Montis G. Inhibition of [3H] dopamine uptake by ¯unarizine. Eur J Pharmacol 1991;203:67±69. [13] Diener HC. A review of current treatment of migraine. Eur Neurol 1994;34(2):18±25. [14] GarcõÂa Ruiz PJ, GarcõÂa de YeÂbenes J, JimeÂnez JimeÂnez FJ, VaÂzquez A, GarcõÂa Urra D, Morales B. Parkinsonism associate with calcium channel blockers: a prospective follow-up study. Clin Neuropharmacol 1992;15:19±26. [15] Janss AJ, Gebhart GF. Brainstem and spinal pathways mediating descending inhibition from the medullary lateral reticular nucleus in the rat. Brain Res 1988;440:109±22. [16] Jurna I, Heinz G. Anti-nociceptive effect of morphine, opioid analgesics and haloperidol injected into the caudate nucleus of the rat. Arch Phamacol 1979;309:145±51. [17] Kobayashi T, Mori Y. Ca 11 channel antagonists and neuroprotection from cerebral ischeemia. Eur J Pharmacol 1998;363:1±15. [18] Kostrezewa RM. Dopamine receptor supersensitivity. Neurosc Biobehav Rev 1995;19:1±17. [19] Kuroki M, Nagamachi S, Hoshi H, Flores LG, Ohnishi T, Jinnouchi L, Futami S, Watanaba K. Cerebral perfusion imagin evalutes pharmacologic treatment of unilateral moyamoya disease. J Nucl Med 1996;37:84±86. [20] Magnusson EJ, Fisher K. The involvement of dopamine in nociception: the role of D1 and D2 in the dorsolateral striatum. Brain Res 2000;855:260±6. [21] Martin JH. Autoradiographic estimation of the extent of reversible inactivation produced by microinjection of lidocaine and muscimol in the rat. Neurosc Lett 1991;127:160±4. [22] Marti-Masso JF, Poza JJ, Lopez de Munain A. Drugs inducing aggravating parkinsonisms: a review. Therapie 1996;51:568±77. [23] Mena MA, GarcõÂa de YeÂbenes MJ, Tabernero C, Casarejos MJ, Pardo B, GarcõÂa de YeÂbenes J. Effects of calcium antagonists on the dopamine system. Clin Neuropharmacol 1995;18:410±26. [24] Micheli FE, Fernandez Pardal MN, Gianaula R, Gatto M, Casas
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www.elsevier.com/locate/parkreldis
Pharmacological involvement of the calcium channel blocker ¯unarizine in dopamine transmission at the striatum J.E. Belforte a, C. MagarinÄos-Azcone c, I. Armando b, W. BunÄo c, J.H. Pazo a,* a
Laboratorio de Neuro®siologõÂa, Departamento de FisiologõÂa, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina b Centro de Investigaciones EndocrinoloÂgicas, Hospital de NinÄos Ricardo Gutierrez, Buenos Aires, Argentina c Instituto Cajal (CSIC), Hospital RamoÂn y Cajal, Madrid, Spain
Abstract Single intrastriatal microinjections of 25, 50 and 100 nmol/ml of ¯unarizine in normal rats produced a dose-dependent turning behavior toward the injected side when they were challenged with apomorphine (1 mg/kg, s.c). This effect was seen at 1, 3 and 7 days following administration of the high dose of ¯unarizine, but had subsided by 24 h after administration of the intermediate dose; the low dose was ineffective. However, intrastriatal injection of the high dose of ¯unarizine resulted in a local lesion and thereafter this dose was not used. A similar dose-response relationship was determined for nifedipine, an L-type calcium channel antagonist. Injection of this antagonist did not result in apomorphine-elicited rotational behavior, re¯ecting its lack of antidopaminergic action. Intrastriatal injections of haloperidol (5 mg/ ml), an antagonist of dopamine D2 receptors, or the sodium channel blocker lidocaine (40 mg/ml), were given in order to compare their effects to those observed with ¯unarizine. Intracerebral injection of haloperidol produced ipsilateral turning in response to systemic administration of apomorphine given 60 min after. The same response was obtained with the injection of apomorphine 10 min after the injection of intracerebral lidocaine. This effect was no longer apparent 24 h after the microinjection of haloperidol and 60 min after the injection of lidocaine. In rats rendered hemiparkinsionian by lesioning the nigrostriatal pathway with 6OHDA, intrastriatal microinjection of ¯unarizine (50 nmol/ml) signi®cantly reduced apomorphine (0.2 mg/kg, s.c.)-elicited turning behavior towards the non-lesioned side. These results suggest an antidopaminergic effect of ¯unarizine mediated by antagonistic action of post-synaptic striatal dopamine receptors. However, an action of the drug on sodium channels may not be ruled out. These studies offer additional supporting evidence for the induction or aggravation of extrapyramidal side-effects in patients receiving ¯unarizine. q 2001 Elsevier Science Ltd. All rights reserved. Keywords: Flunarizine; Nifedipine; Striatum; Parkinsonism; Calcium channel blockers; Circling behavior
1. Introduction Calcium channel blockers are widely used in the medical practice for the treatment of cardiovascular, pulmonary, neurological and genitourinary diseases [3]. One of them, ¯unarizine, a piperazine derivative is a mixed T and L type calcium channel blocker and sodium channel antagonist [5,6,34]. It has been found to be effective for cardiovascular and neurological diseases, as well as in the prophylaxis and treatment of migraine headache [13]. Futhermore, ¯unarizine posseses anticonvulsivant properties in both humans and animals [4], and reduces the duration of recurrent hemiplegia of childhood [2]. It is also used as a neuroprotective agent in the treatment of post-cerebrovascular disorders, cerebral infarction and cerebral hemorrhage [17,19]. However, these bene®cial therapeutic effects of ¯unarizine are frequently associated with aggravation or even induction * Corresponding author. Tel.: 154-1-4508-3740. E-mail address: [email protected] (J.H. Pazo).
of extrapyramidal motor signs in chronic treatment in elderly patients [10,14,24]. This side-effect of ¯unarizine is shared with cinnarizine, other calcium channel blocker derived from piperazine and less frequently by other calcium antagonists [22,25]. Despite clinical reports of extrapyramidal side-effects associated with ¯unarizine treatment, experimental studies of the actions of this calcium channel antagonist on dopaminergic neurotransmission are con¯icting. Flunarizine has been suggested to inhibit the dopamine (DA) uptake process [12,23]. Furthermore, ¯unarizine has been reported to inhibit [ 3H]-spiperone binding to D2 and [ 3H]-SCH23390 binding to D1 striatal receptors with the highest af®nity for D2 receptors [1]. On the other hand, it has been demonstrated that systemic ¯unarizine increases striatal extracelular dopamine levels [27,30]. However, other reports examining the repeated administration of ¯unarizine have indicated reduced DA release [23,30]. Similarly, con¯icting studies have been reported on the effects of ¯unarizine on tyrosine hydroxylase [23,31].
1353-8020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved. PII: S 1353-802 0(01)00006-2
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Information concerning the anatomical sites where the calcium blockers exert their pharmacological side-effects are still unclear, since most experimental data have examined the effects of systemic administration of the calcium antagonists or used in vitro approaches. In order to help clarify the actions and site of action of ¯unarizine, we assessed the effects of intrastriatal microinjections of the calcium channel antagonist on nigrostriatal dopaminergic transmission in the rat by monitoring apomorphine-elicited circling behavior. We also studied the effects of another sub-classes of calcium channel blocker antagonists by treating rats with the dihydropyridine derivative, nifedipine, a L-type channel antagonist with very few extrapyramidal side-effects [22]. 2. Material and methods The experiments were carried out in several groups of male rats of the Sprague Dawley strain weighing 300± 350 g, and in strict accordance with the Principles of laboratory animal care (NIH publication no. 86±23, revised 1985). Under pentobarbital anesthesia (45 mg/kg, i.p.), the animals were placed in a stereotaxic frame (DK) and a burr hole was made over the striatum according to the atlas of Paxinos and Watson [28]. A cannula (0.3 mm OD), attached to a 5 ml Hamilton syringe, was lowered into the left or right striatum and the calcium antagonist or the vehicle was injected in a volume of 1 ml in about 2 min. The injection needle was left in place two additional minutes. Dose-response curves for ¯unarizine and nifedipine (kindly supplied by Janssen Labs) were obtained by microinjections of 25, 50 and 100 nmol/ml of ¯unarizine and 50, 100, 200 nmol/ml of nifedipine dissolved in 22% of b-ciclodextrin (RBI) in saline. Three groups of rats were treated with ¯unarizine and the other three with nifedipine The vehicle was administrated to a control group. Nifedipine was used in order to search whether L-channels could be involved in the effects of ¯unarizine. Flunarizine and nifedipine treated animals were then tested with apomorphine (1 mg/kg, 1 ml/kg, s.c, Sigma) disolved in 0.2% ascorbic acid, 1, 3 and 7 days following intrastriatal microinjections. Immediately after the injection of apomorphine, the rats were placed in an automatic rotometer. The full rotations for both side were recorded during 60 min, at 2 min intervals. We thought it of interest to compare the effect of intracerebral ¯unarizine to the effect of haloperidol, a dopamine D2 antagonist. For this purpose, two groups of rats were anesthetized, one was injected into the striatum with haloperidol (5 mg/ml of commercial injectable solution, kindly supplied by Janssen Labs) and the other group was injected with the solvent (kindly supplied by Janssen Labs). Both groups were challenged with apomorphine (1 mg/kg, s.c.) 24 h after the intracerebral microinjections. To test the possibility that the lack of sustained effect of intracerebral injections of nifedipine and haloperidol 24 h after their administration could be due to short half-life of
the drugs, and in order to compare the action of lidocaine (kindly supplied by Astra Labs), a short half-life sodium channel blocker [8] to the effect of ¯unarizine, experiments were performed in rats implanted with guide cannulae. This procedure was required for testing drug action at short intervals. Rats anesthetized with pentobarbital (45 mg/kg, i.p) were stereotaxically implanted over the striatum (coordinates: AP 10.48 mm from Bregma, L: 3 mm, D: 25 mm from cranium surface) [28] with stainless steel guide cannulae (26 g., Plastics One) and ®xed in place by screws (3/32, Plastics One) and dental acrylic. Seven days were allowed for recovery. The rats were then divided into several groups. Each group was microinjected into the striatum with one of the following drugs, lidocaine (40 mg/ml in saline), nifedipine (50 nmol/ml in b-ciclodextrin), haloperidol (5 mg/ml, commercial injectable solution) and ¯unarizine (50 nmol/ml in b-ciclodextrin). Control animals were injected with the appropiated solvent. The dose of lidocaine was chosen because it is known to block neurotransmission in the central nervous system [15,21]. The dose of haloperidol used has been reported to produce changes in the activity of the central nervous system [16,20]. Microinjections were made in conscious rats by gently restraining the animal with the hand and inserting the injection needle (33 g, Plastics One) that extended 1 mm beyond the guide cannula tip. Injections were made at a rate of 0.5 ml/60 s and were left in place for at least 60 s to allow the drug to diffuse. Half of the rats injected with lidocaine were tested with apomorphine (1 mg/kg, s.c.) 10 min after the brain injection and the other half 60 min after the brain injection. The other groups (haloperidol, ¯unarizine and nifedipine) were tested with apomorphine (1 mg/kg, s.c.) 60 min after the microinyection of the drugs. In order to ascertain whether the action of ¯unarizine in normal rats could be due to a pre- or post-synaptic effect, experiments were made in rats with unilateral lesion of the nigrostriatal dopaminergic system. Rats anesthetized with pentobarbital (45 mg/kg, i.p.) were placed in a stereotaxic frame, and the nigrostriatal pathway was lesioned on one side by a microinjection into the medial forebrain bundle of 8 mg of 6-hydroxydopamine-HCl (6OHDA), dissolved in 4 ml of 0.2% ascorbic acid in saline. The animals were pre-treated 30 min before the intracerebral administration of 6OHDA with disipramine (25 mg/kg, i.p.) to prevent damage to noradrenergic neurons. The effectiveness of the denervation was evaluated by challenging the rats with apomorphine (0.2 mg/kg, s.c.) two weeks after the lesion. Only those animals with more than 150 full contralateral net turns in 60 min, were used for subsequent experiments. In some rats chosen at random, dopamine and dopac content in the intact and lesioned striatum were measured in dissected tissue, using a high pressure liquid cromatography (HPLC) coupled to amperometric electrochemical detector [9]. Four weeks after 6-OHDA lesioning, the rats were again anesthetized and divided into two groups. One was microinjected into the striatum with ¯unarizine (50 nmol/ml) and the other
J.E. Belforte et al. / Parkinsonism and Related Disorders 8 (2001) 33±40
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Fig. 1. Histological reconstruction of the injection sites in normal and 6OHDA treated rats. Outlines and levels were adopted from Paxinos and Watson [28].
with the vehicle of the drug. The rotatory activity induced by s.c. injection of apomorphine (0.2. mg/kg) was evaluated 1, 3, 7 and 14 days following the intrastriatal microinjections. The sites of injection in the striatum were determined in serial histological sections stained with cresyl violet. Their locations were evaluated with reference to the atlas of Paxinos and Watson [28]. Animals that were found not to have been injected in the target areas were discarded. The following turning behavior parameters were analyzed: (1) Total turns, that is, the number of ipsilateral plus contralateral turns, as a measure of overall locomotor activity, (2) Net turns, are ipsilateral minus contralateral turns and (3) Asymmetric index, that is, ipsilateral turns as percentage of total turns. A ratio of 50% would indicate a non-directional bias, and a higher or lower ratio would indicate ipsilateral or contralateral asymmetry, respectively. Statistical evaluation was carried out by means of one or two-way ANOVA for paired and unpaired measures followed by Newman-Keuls post-hoc test. In some experiments Student's t-test was also applied. Values of P , 0.05 were considered statistically signi®cant. 3. Results Microinjections of ¯unarizine into the dorsal striatum in normal animals (Fig. 1), did not induce spontaneous circling behavior in the inmediate post-operative period. Systemic administration of apomorphine (1 mg/kg, s.c.), 24 h after microinjection of ¯unarizine, produced net rotations towards the injected striatum (ipsilateral turning, Figs. 2(B and C) and 3) at doses of 50 and 100 nmol/ml, but not with 25 nmol/ml. However, with the highest dose (100 nmol/ 1 ml) ipsilateral turnig activity was observed at 3 and 7 days later, but not with the dose of 50 nmol/ml (Fig. 2(B)
Fig. 2. Circling behavior induced by systemic administration of apomorphine (1 mg/kg, s.c.) at different intervals following intrastriatal microinjections of ¯unarizine. Data are means ^ SEM of n rats per group. There was a signi®cant difference in net turns and asymmetric index 24 h after injection of ¯unarizine (50 and 100 nmol/ml) as compared to vehicle treated rats. For net turns, p P , 0.05 and p p P , 0.005 Newman-Keuls after two way ANOVA for repeated measures, treatment F3,29 28.17, P , 0.0001; treatment £ time F6,58 2.44, P , 0.05. For asymmetric index, p P , 0.05 and p p P , 0.005 Newman-Keuls, after two way ANOVA for repeated measures, treatment F3,29 14.32, P , 0.0001; treatment £ time F6,58 3.89, P , 0.05. Abbreviations: ipsi ipsilateral turns, contra contralateral turns.
and (C)). Total turns were not modi®ed by any doses of ¯unarizine (Fig. 2(A)). Microinjections of b-ciclodextrin (22% in saline, 1 ml) produced turning activity to both sides without any preference in response to apomorphine (Fig. 2(B and C)). In addition, systemic administration of the solvent of apomorphine (ascorbic acid) had no effect in circling behavior in ¯unarizine treated rats (Table 1). Histological analyses of injection sites made at the end of the test (7 days) show a lesion in the sites injected with 100 nmol/ml (Fig. 4(A)), while in the sites injected with 25 and 50 nmol/ml no histological damage was observed (Fig. 4(B)). On the basis of this ®ndings, thereafter we chose 50 nmol dose for the following experiments. Intrastriatal microinjections of nifedipine were ineffective in changing any rotational parameters induced by apomorphine when compared to solvent treated animals (Fig. 5). To test the possibility that the lack of ef®cacy
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J.E. Belforte et al. / Parkinsonism and Related Disorders 8 (2001) 33±40
Fig. 3. Dose-response curve for net turns from different doses of ¯unarizine microinjected into the striatum and induced by apomorphine (1 mg/kg, s.c) administrated 24 h later. Data are means ^ SEM of n rats per group taken from experiment of Fig. 2. All turns are toward the injected striatum (ipsilateral turns). There was a signi®cat difference between 50 and 100 nmol/ml when compared to solvent microinjected rats. p P , 0.05 and p p P , 0.005 Newman-Keuls, after two way ANOVA for repeated measures, treatment F3,29 28.17, P , 0.0001. Abbreviations: ipsi ipsilateral turns, contra contralateral turns.
Fig. 4. Photomicrographs of coronal sections through the striatum one week following microinjections of ¯unarizine (arrowheads). (A) a microinjection of 100 nmol/ml of ¯unarizine. Note the lesion around the site of injection. (B) a microinjection of 50 nmol/ml of ¯unarizine. Note the track left by the injection cannula Magni®cation £ 5.
seen in nifedipine could be due to its short half-life, experiments were performed in animals with implanted cannulae in the striatum. The intrastriatal administration of nifedipine did not produce spontaneous motor activity in the immediate post-injection period. The motor responses of these animals to apomorphine, injected i.p 60 min after the nifedipine, were similarly to those of control rats injected with the solvent (Table 2). Histological analyses did not show any lesions in the striatum at the doses of nifedipine used. Taken together, these data indicate that nifedipine did not affect dopaminergic transmission and for this reason was not used in the following experiments. Table 1 Effect of the solvent of apomorphine (ascorbic acid 0.2%) injected s.c 1 ml/ kg in rats microinjected 24 h before with ¯unarizine (50 nmol/ml) or its solvent into the striatum. Data are the means ^ SEM of n rats per group. There were no signi®cant (ns) differences between the groups, Student's t-test. Negative sign contralateral turns Treatment Solvent (n 3) Flunarizine (n 5)
Total turns/h
Net turns/h
Asymmetric index %
19.6 ^ 9.5 25.4 ^ 9.2
2 3 ^ 4.5 2 0.2 ^ 4.1
51.6 ^ 9.7 49.3 ^ 8.5
Table 2 Turning behavior in rats injected with ¯unarizine (50 nmol/ml) and nifedipine (50 nmol/ml) into the striatum and tested 60 min later by systemic administration of apomorphine (1 mg/kg, s.c), compared to rats injected with the solvent. Data are the means ^ SEM of n rats per group. There were no signi®cant differences after one way ANOVA, for total F2,23 1.47, P ns, for nets F2,23 2.26, P ns, for index F2,23 2.65, P ns. Negative sign contralateral turns Treatment
Total turns/h
Solvent (n 12) 152.3 ^ 19.1 Nifedipine (n 8) 183.6 ^ 17.5 Flunarizine (n 6) 141.5 ^ 18.2
Net turns/h Asymmetric index % 7.3 ^ 9.2 2 8.0 ^ 12.9 43.5 ^ 30.2
52.6 ^ 3.4 48.7 ^ 4.2 65.9 ^ 8.6
Fig. 5. Turning activity induced by systemic administration of apomorphine (1 mg/kg, s.c.) at different intervals following intrastriatal microinjection of nifedipine. Data are means ^ SEM of n rats per group. There is not significant difference in any of the motor parameters studied when compared to control rats injected with solvent. For total turns, treatment two way ANOVA for repeated measures, F3,14 1.79, P ns; for net F3,14 0.14, P ns; for index F3,14 0.11, P ns. Abbreviations: ipsi ipsilateral turns, contra contralateral turns.
153.2 ^ 16.2 (n 5) 2 11.2 ^ 28.0 45.5 ^ 8.8 a
109.4 ^ 18.4 (n 5) 2 26.2 ^ 21.8 44.9 ^ 14.1
P , 0.05. P , 0.001 Newman-Keuls, after one-way ANOVA, for nets F5,28 6.85; P , 0.0005, for index F5,28 4.71, P , 0.005.
152.8 ^ 31.8 (n 5) 61.6 ^ 17.9 a 73.6 ^ 6.3 a b
1h
156.0 ^ 15.9 (n 4) 112.0 ^ 27.4 b 84.4 ^ 5.5 a 149.0 ^ 22.0 (n 8) 2 3.3 ^ 10.6 46.8 ^ 4.0 Total turns/h Net turns/h Asymmetric index %
24 h
Solvent
159.7 ^ 22.2 (n 7) 2 2.0 ^ 12.2 47.4 ^ 4.5
10 min
Lidocaine
60 min
37
Haloperidol Solvent
In order to determine whether the effects of ¯unarizine could be similar to the action of haloperidol, a dopamine receptor antagonist, and lidocaine, a sodium channel blocker, we examine their effects in rats implanted with guide cannulae. A microinjection of haloperidol (5 mg/ml) was given into the striatum 60 min before an i.p injection of apomorphine (1 mg/kg, s.c). Apomorphine induced a significant turning activity ipsilateral to the injected striatum and increases of the asymmetric index (Table 3), as compared to animals microinjected with haloperidol vehicle. There was no change in total turning. Nevertheless, in the other group of rats, administration s.c of apomorphine 24 h after intrastriatal injection of haloperidol failed to modify circling activity (Table 3). Two groups of rats were intrastriatally injected with lidocaine (40 mg/ml) through implanted cannulaes and tested with apomorphine (1 mg/kg, s.c). One group was tested 10 min after intracranial microinjections and the other 60 min later. Signi®cant motor changes were only produced in the 10 min group. The rats rotated to the side ipsilateral to the injection site and the asymmetric index was increased as compared to animals microinjected with solvent (Table 3). Total turns remained unchanged. To compare the effects at short intervals of nifedipine, haloperidol and lidocaine to the action of ¯unarizine at short interval after its intracerebral microinjection. Flunarizine (50 nmol/ml) was injected into the striatum through implanted guide cannulae in one group of rats and tested 60 min later with apomorphine (Table 2). Flunarizine had no effect on any of the parameters studied, although a tendency towards an increase in the number of net turns was observed. However, it did not reach signi®cance, perhaps due to the wide-ranging variability. The range of net turns was from 237 (contralateral turns) to 83 (ipsilateral turns). No lesions were observed at the striatal sites microinjected with haloperidol or lidocaine. In hemiparkinsonian rats made by unilateral lesioning of the nigrostriatal pathway with 6OHDA, systemic administration of apomorphine (0.2 mg/kg, s.c) produced turning behavior contralateral to the lesioned nigrostriatal pathway. These rats were divided into two groups with similar rotational behavior (no signi®cant differences between groups in net turns induced by apomorphine) and microinjected into the lesioned striatum. One group was injected with 50 nmol/ml of ¯unarizine and the other with solvent. Intrastriatal administration of ¯unarizine produced a signi®cant reduction in the net contralateral turns induced by systemic administration of apomorphine as compared to animals injected with the solvent (Fig. 6(B)). This was re¯ected by a decrease in the total turns (Fig. 6(A)). The effect was observed 1, 3, 7 and 14 days after intrastriatal microinjection of the calcium antagonist. The asymmetric index was only signi®cant 24 h after treatment with ¯unarizine, which indicates a less asymmetric activity in the ¯unarizine treated rats as compared to the solvent-injected animals (Fig. 6(C)). Histological analyses of hemiparkinsonian
Table 3 Turns induced by apomorphine (1 mg/kg, s.c) at different intervals after intrastriatal microinjections of haloperidol (5 mg/ml) and lidocaine (40 mg/ml) Data are the means ^ SEM of n rats per group. The data for the solvent group of haloperidol at 60 min and 24 h were quantitatively similar and have therefore been combined, as is the case for the control group for lidocaine. There were signi®cant differences for net turns and asymmetric index at 60 min for haloperidol and at 10 min for lidocaine when compared to solvent groups. Negative sign contralateral turns
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J.E. Belforte et al. / Parkinsonism and Related Disorders 8 (2001) 33±40
Fig. 1 shows the histological reconstruction of the injection sites in the different groups of animals.
4. Discussion
Fig. 6. Effect of intrastriatal microinjection of 50 nmol/ml of ¯unarizine in rats with unilateral lesion of nigrostriatal pathway by 6OHDA, on the turning behavior induced by apomorphine (0.2 mg/kg, s.c.) injected at different times after intracerebral microinjection. The animals turn to the side opposite to the lesioned striatum (contralateral turns). Data are means ^ SEM of n rats per group. The global activity (A) was signi®cantly reduced by ¯unarizine as compared to control group at all studied intervals. Two way ANOVA for repeated measures, treatment £ time F4,76 4.51, P , 0.005. The net turns (B) were also reduced by ¯unarizine. Two way ANOVA for repeated measures, treatment £ time F4,76 5.12 P , 0.001. The asymmetric index (C) was greater than control animals 24 h after intrastriatal injection, that is less asymmmetry. Two way ANOVA, treatment £ time F4,76 3, P , 0.05. Newman-Keuls for comparisons between means p P , 0.05, p p P , 0.01 and p p p P , 0.001. Pre before ¯unarizine, i.e. rats tested with apomorphine alone. Flunarizine was microinjected at time zero.
animals did not show lesions in the sites injected with ¯unarizine. The levels of dopamine and dopac in eight 6OHDA lesioned rats, taken at random from each group, showed a decrease of 96 and 92.7%, respectively, in the lesioned striatum as compared to the intact side (Table 4).
The calcium channel blockers, particularly those derived from piperazine, ¯unarizine, are able to produce abnormal motor signs similar to those seen in parkinsonism [22,25]. In some cases this effect is transient and disappears following the withdrawal of the drug. However, in other cases, the effect remains despite withdrawal of the drug [23]. The mechanism of this action is not totally understood. On the basis of experimental results, many hypotheses have been proposed concerning the mechanisms and sites of action of the calcium blockers in the central nervous system [14,25]. In order to rule out whether a calcium blocker, such as nifedipine, with different chemical structure and antagonistic action, injected intrastrially would have a comparable effect to that of ¯unarizine, it was microinjected into the striatum and tested to different intervals with apomorphine given s.c. However, the turning response elicited by administration of apomorphine remained unaffected. This could be interpreted as this L channel blocker having no effect on dopaminergic neurotransmission, which is in agreement with previous reports of its weak antidopaminergic action and reduced likelihood in producing drug-induced parkinsonism [23,30]. On the other hand, acute unilateral intrastriatal injection of 50 nmol/ml of ¯unarizine, a T and L calcium channel blocker induces a transient turning activity 24 h following its administration, in response to systemic injection of apomorphine. This was expressed as signi®cant increases in net turns towards the injected side as compared to solvent-injected rats as well as in the asymmetric index. However, the highest dose used of ¯unarizine (100 nmol/ml) produced an unexpected lesion at the level of the site of injection into the striatum. This was responsible for the persistency of the apomorphine-induced turning activity during all test sessions (Fig. 2). This is in agreement with previous studies in neuroblatoma cells in which the observed cell death was attributed to the effect of ¯unarizine [23]. Intrastriatal administration of haloperidol and lidocaine produced an ipsiversive circling behavior in response to apomorphine. However, the pharmacodynamic of both drugs were different because we were still able to elicit the effect of haloperidol 60 min after its intracerebral injection while the
Table 4 Concentration of dopamine and dopac in the striatum of the lesioned and contralateral intac side
Dopamine (n 8) (pg/mg tissue) Dopac (n 8) (pg/mg tissue) a b
Lesioned side
Intact side
% decreased
976.7 ^ 268 502.6 ^ 153
24711.6 ^ 1039 b 6944.3 ^ 1221 a
96.0 92.7
P , 0.001. P , 0.00001, Student's t-test, when was compared intact vs lesioned side.
J.E. Belforte et al. / Parkinsonism and Related Disorders 8 (2001) 33±40
effect of lidocaine only lasted 10 min after microinjection. In addition, the effect of haloperidol disapeared 24 h after its intracerebral injection and the lidocaine after 60 min. The unilateral imbalance between basal ganglia on both sides produced by these drugs and brought on by stimulation of striatal dopamine receptors [29] could be attributed to their activity as receptors and channel blockers. Haloperidol is an antagonist of the D2 receptors and lidocaine of sodium channels [8,20,30]. The effects of haloperidol and lidocaine were similar to those observed when ¯unarizine was administrated into the striatum. However, the time course of action was different. The haloperidol and lidocaine have a rapid onset of effect and are short acting, while ¯unarizine has a slow onset of effect and long action. However, there is good evidence that ¯unarizine blocks dopamine receptors mainly of D2 type [1,23,30] and its effect is similar to the action of haloperidol. These facts strongly suggest that the effect of the drug could mainly be attributed to interference with dopaminergic transmission in the striatum. Likewise, an effect of ¯unarizine on voltage-dependent sodium channels may be not excluded from our experiments. In order to elucidate whether the effects observed by intrastriatal microinjection of ¯unarizine would be consequence of a pre- or post-synaptic action of the drug, experiments were performed in rats with unilateral dopaminergic denervation of the striatum by intracerebral administration of 6OHDA into the nigrostriatal pathway. These hemiparkinsonian rats develop an up-regulation of striatal dopaminergic receptors, mainly of type D2 [33], which was accompanied by spontaneous circling behavior, contralateral to the denervated striatum. This activity could also be evoked by systemic administration of apomorphine. In these animals, unilateral injection of ¯unarizine into the denervated striatum signi®cantly decreased the total turns in response to apomorphine as the result of a decrease in contralateral rotations. These effects were observed during all testing sessions (Fig. 6). The asymmetric index was only modi®ed at 24 h after intrastriatal injection of ¯unarizine.This means there was less asymmetric motor activity in rats treated with ¯unarizine than in animals injected with solvent. Intrastriatal administration of ¯unarizine did not modify the time-course of the development of supersensitivity in the striatum of 6OHDA treated rats. The curves followed an almost parallel course, with the only difference being that the rats treated with ¯unarizine began from a lower activity at 24 h. (Fig. 6(A)). The fact that the effect of ¯unarizine was long-lasting in 6OHDA lesioned rats as compared to animals with normosensitive striatum may be attributed to biochemical changes that occurs in dopamine receptors after treatment with 6OHDA [7,18]. These results in 6OHDA treated rats demonstrated a post-synaptic effect of ¯unarizine, since they were observed in a denervated striatum. This neuroleptic-like action of ¯unarizine on post-synaptic dopamine receptors in the striatum may explain the rapid
39
onset of motor side-effects in patients [26]. Likewise, the long duration effect of ¯unarizine on altered dopamine receptors like those in 6OHDA rats could give an explanation of the drug effect in elderly patients, where an agerelated decline of dopamine cells occurs in the substantia nigra pars compacta [11], or in Parkinson's disease. However, cellular lesions may be also considered in light of neurotoxic action of ¯unarizine at high doses. Taken together, these factors could be related to the permanent effect of the calcium blocker in some patients in addition to the other mechanisms described for these side-effects [27,30,32]. In conclusion, our observations con®rm and extend previous reports on the neuroleptic-like action of ¯unarizine, and they are the ®rst description of the effects of the drug directly microinjected into the striatum. This action is probably exerted by blocking the post-synaptic striatal dopamine receptors mainly of D2 type and it has long duration in modifying dopamine receptors. However, an action on sodium channels or other receptors could not be ruled out.
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