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Effects of histamine H3-ligands on the levodopa-induced turning behavior of hemiparkinsonian rats
Parkinsonism & Related Disorders Parkinsonism and Related Disorders 6 (2000) 159–164 www.elsevier.com/locate/parkreldis
Effects of histamine H3-ligands on the levodopa-induced turning behavior of hemiparkinsonian rats M. Huotari, K. Kukkonen, N. Liikka, T. Potasev, A. Raasmaja, P.T. Ma¨nnisto¨* University of Kuopio, Department of Pharmacology and Toxicology, P.O. Box 1627, FIN-70211 Kuopio, Finland
Abstract Histamine H3-receptors act as heteroreceptors on many neurons. The effects of H3-ligands (an agonist, R-a-methylhistamine and an antagonist, thioperamide) on levodopa-induced turning behavior in a rat model of Parkinson’s disease were quite similar to those seen with a2-adrenoceptor ligands (dexmedetomidine and atipamezole). R-a-methylhistamine clearly reduced contralateral turning behavior but the increase of turning behavior after thioperamide was less clear. The lack of effect of H3-ligands, in contrast to a2-ligands, on the amphetamineinduced ipsilateral turning behavior points to different roles or neuronal distribution of these two presynaptic receptors. We propose that in this lesion model, H3-receptors modify those pathways participating striatal outflow. 䉷 2000 Elsevier Science Ltd. All rights reserved. Keywords: Histamine H3-receptors; Thioperamide; R-a-methylhistamine; Adrenergic a2-receptors; Dexmedetomidine; Atipamezole; Turning behavior; Rat Parkinson model
1. Introduction Since the early 1980s, histamine has been reported to have important actions on central neurotransmission [1,2]. At about the same time a new receptor subtype, the histamine H3-receptor, was characterized [3] and novel ligands for this receptor were synthesized [4]. Recently the histamine H3-receptor has been cloned and functionally expressed [5]. In addition to functioning as an autoreceptor in histaminergic neurons, histamine H3-receptors can also act as inhibitory presynaptic heteroreceptors on a number of other neurons [2]. However, very little is known about the functional role of this histamine receptor subtype [2]. Histamine H3-receptors are found in several brain regions, notably in the striatum and substantia nigra [5–9]. It is also known that activation of histamine H3-receptors inhibits dopamine release in the mouse striatum [10]. Asymmetric lesioning of the dopaminergic nigrostriatal pathways by 6-OH-dopamine (6-OHDA) is an established rat model of unilateral Parkinson’s disease [11]. After a unilateral infusion of 6-OHDA into the substantia nigra, nigral dopamine cell bodies are destroyed, and supersensitivity of the postsynaptic, striatal dopamine receptors on the lesioned side develops. The rats rotate in a direction away from the lesion (contralaterally) when direct dopamine * Corresponding author. Tel.: ⫹ 358-17-162-401; fax: ⫹ 358-17-162424. E-mail address: [email protected] (P.T. Ma¨nnisto¨).
agonists (e.g. apomorphine or bromocriptine) or the dopamine precursor, levodopa, are given. Indirectly acting dopamine-releasing compounds (e.g. amphetamine) cause rotation in a direction towards the lesioned side (ipsilateral turnings) [11,12]. This model is a very sensitive way of testing changes in the dopaminergic tone in the brain. Recently it was demonstrated that after 6-OHDA lesions, the numbers of H3-receptors were greatly increased in the ipsilateral striatum and substantia nigra [13,14]. These findings indicate that histamine H3-receptors may be targets for the drug treatment of Parkinson’s disease. However, clinical findings have shown that central histaminergic neurons are preserved in Parkinson’s disease [15]. In this study we wanted to examine whether a histamine H3receptor agonist, R-a-methylhistamine, or an antagonist, thioperamide, would modify the turning behavior induced by either levodopa or amphetamine in the unilaterally lesioned rats. An established a2-adrenergic agonist, dexmedetomidine, and an a2-adrenergic antagonist, atipamezole, were used as control treatments reflecting the modification of presynaptic receptors. The effects of some other a2-adrenergic ligands in the rat turning model were recently reported [16].
2. Material and methods 2.1. Animals
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Male albino rats (Han/Kuo), weighing 230–300 g, were
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obtained from the National Laboratory Animal Center, Kuopio, Finland. The rats were kept under standard laboratory conditions (temperature 20 ^ 3⬚C in groups (3–5 rats/ group) in a 12 h dark/light cycle (lights on 7.00 a.m.). The rats had free and continuous access to fresh tap water and mice/rat food pellets. All animal procedures were reviewed by the Animal Ethics Committee of the University of Kuopio and approved by the local Provincial Government. 2.1.1. Lesion on the medial forebrain bundle The procedure was performed according to Ungerstedt [11] with some modifications [17,18]. The details are given below briefly. 6-OHDA was dissolved in isotonic NaCl solution containing 0.2% of ascorbic acid (2.5 mg of 6-OHDA in 1 ml). For administration of 6-OHDA into the right substantia nigra, the rats were anesthetized with chloral hydrate anesthesia (350 mg/kg, i.p.). A stainless steel guide cannula (made from a 23 G needle) was lowered 2.0 mm above the target. An infusion needle (made from a 30 G needle) was lowered 2.0 mm below the lower tip of the guide cannula. The coordinates for the final infusion site near substantia nigra were AP 4.4, L 1.2, DV ⫺8.3 [19]. The volume of the infusion was 4 ml, and it was given during 8 min. We used Hamilton syringes (1 ml) connected to PE 10 plastic tubing for infusion. The syringes were run by a motor-driven slow-motion syringe pump (CMA/102, Carnegie Medicin, Sweden). The needle was retained in position for an extra 4 min after the infusion of 6-OHDA. During the 1 week recovery period from anesthesia, the animals were housed singly and after that in groups of 3–5 rats. 2.1.2. Testing of turning behavior The rats were placed in individual hemispherical plastic bowls (diameter 35 cm). They were clothed with vests having two holes for the front legs. The vest was connected to an automatic eight-channel rotameter (Coulbourn Instruments, Inc., Allentown, PA). The rotameter registered right and left full turns separately. The rats were allowed to adapt to the bowls for about 30 min before the drug treatments. 2.1.3. Analysis of the striatal dopamine levels To confirm the success of the unilateral lesion, dopamine levels in both striata were analyzed by HPLC using electrochemical detection as earlier described [20]. The rats were decapitated at about 7 days after the last turning experiment. The brains were exposed within 30 s, cooled in liquid nitrogen and both striata were dissected according to Glowinski and Iversen [21]. The dissected tissues were frozen in liquid nitrogen and stored at ⫺80⬚C until analyzed. 2.2. Treatments 2.2.1. Apomorphine test To test the success of the lesioning, apomorphine (0.1 mg/kg) was given subcutaneously (s.c.) about 14 days
after the toxin infusion. Only those animals which showed over 100 full contralateral turns in a 1 h test session were used. One week after apomorphine testing, the actual experiments were started. 2.2.2. Peripheral administration of histamine H3- and adrenergic a 2-ligands Graded doses (s.c.) of H3- and a2-agonists and antagonists (R-a-methylhistamine and thioperamide: 2.5, 5 or 10 mg/ kg; dexmedetomidine: 0.005, 0.01 and 0.02 mg/kg and atipamezole: 0.15, 0.3 and 0.6 mg/kg) were given with levodopa (10 mg/kg, i.p.) and carbidopa (30 mg/kg, i.p.). The combinations of both agonists and both antagonists were also tested (with levodopa/carbidopa) to study whether they could enhance each other. The drugs were also given with amphetamine (2.5 mg/kg, s.c.). H3- and a2-agonists and antagonists were given at the same time as carbidopa (when used), and levodopa or amphetamine 15 min after these injections. The monitoring of the circling was started immediately after levodopa or amphetamine injection. Within each series, the studies were organized in a cross-over manner. On each experimental day, all different treatments were given at least to one rat, and on the following experimental day, each rat was given a new treatment in a randomized order. Some rats were discarded: those who lost their vests in the middle of the turning session, and those who did not respond to levodopa after any drug treatment. 2.3. Statistics Arithmetic means, SEMs and SDs were calculated. The results were analyzed with one-way (treatment) analysis of variance (ANOVA). Newman–Keuls analysis was used as a post-hoc test. 3. Results 3.1. Success of nigral lesions The dopamine content in the healthy side was 7:9 ^ 0:78 mg=g (mean ^ SEM, n 14 and in the lesion side 0:001 ^ 0:0001 mg=g n 14 demonstrating the highly successful nature of the lesions. 3.2. Effect of graded doses of R-a-methylhistamine and thioperamide alone Neither drug induced any turning behavior at any dose (2.5, 5 or 10 mg/kg) (data not shown). 3.3. Effect of graded doses of R-a-methylhistamine, thioperamide, dexmedetomidine and atipamezole with levodopa (10 mg/kg i.p.) and carbidopa (30 mg/kg i.p.) on turning behavior In the R-a-methylhistamine group, the levodopa/carbidopa
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Fig. 1. Effects of graded doses (s.c.) of R-a-CH3-histamine (Fig. 1A and B) and thioperamide (Fig. 1C and D) on levodopa/carbidopa-induced turning behavior. All rats received carbidopa (30 mg/kg) and saline or R-a-CH3-histamine (Fig. 1A) 2.5 mg/kg (A), 5 mg/kg (W), 10 mg/kg (K), thioperamide (Fig. 1C) 2.5 mg/ kg (A), 5 mg/kg (W), 10 mg/kg (K) 15 min before levodopa (10 mg/kg, B, Fig. 1A and C). The rats were monitored 240 min after levodopa injection. Averages and SEMs of contralateral turns were calculated every 10 min (Fig. 1A and C) and total amount after 180 min (Fig. 1B and D). n 10–21; *p ⬍ 0:05 vs. the effect of levodopa/carbidopa alone in Newman–Keuls test.
treatment alone caused 1143 ^ 117 (mean ^ SEM, n 18 full contralateral turns in 3 h. R-a-methylhistamine pretreatment reduced F 2:881; p ⬍ 0:05 contralateral turning behavior, 10 mg/kg being the most effective dose 707 ^ 104; n 20; p ⬍ 0:05 (Fig. 1A and B). In this part of the study, thioperamide had no effect on levodopa/carbidopa 1531 ^ 185; n 11 induced turning behavior (Fig. 1C and D). Dexmedetomidine pretreatment decreased the number of contralateral turns (3.460, p ⬍ 0:05 having its maximum effect at 20 mg/kg 575 ^ 84; n 18; p ⬍ 0:05 vs. levodopa/carbidopa-treatment: 1086 ^ 134; n 14 (Fig. 2A and B). Atipamezole pretreatment increased the number of contralateral turns 1722 ^ 225; n 13 from those of levodopa/carbidopa alone 1345 ^ 211; n 12 but the effect did not reach statistical significance (Fig. 2C and D).
3.4. Effect of a combination of histamine H3- and adrenergic a 2-ligands on levodopa/carbidopa-induced turning behavior Levodopa/carbidopa treatment alone caused 1113 ^ 162 (mean ^ SEM, n 18 full contralateral turns in 4 h. As above, both agonists, a histamine H3-agonist R-amethylhistamine (5 mg/kg, s.c., 889 ^ 138; n 13 and an adrenergic a2-agonist dexmedetomidine (10 mg/kg, s.c., 710 ^ 101; n 13 tended to decrease the number of contralateral turns. When given together they seemed to enhance each others’ action. 568 ^ 76; n 14 None of these effects were statistically significant (Fig. 3). The antagonists thioperamide (5 mg/kg, s.c.) and atipamezole (0.3 mg/kg, s.c.) both enhanced the number of contralateral turns. The effect of atipamezole 2200 ^ 573; n 7 was significant p ⬍ 0:05 and the effect of
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Fig. 2. Effects of graded doses (s.c.) of dexmedetomidine (Fig. 2A and B) and atipamezole (Fig. 2C and D) on levodopa/carbidopa-induced turning behavior. All rats received carbidopa (30 mg/kg) and saline or dexmedetomidine (Fig. 2A) 0.005 mg/kg (A), 0.01 mg/kg (W), 0.02 mg/kg (K) or atipamezole (Fig. 2C) 0.15 mg/kg (A), 0.30 mg/kg (W), 0.60 mg/kg (K) 15 min before levodopa (10 mg/kg, B). The rats were monitored 240 min after levodopa injection. Averages and SEMs of contralateral turns were calculated every 10 min (Fig. 2A and C) and total amount after 180 min (Fig. 2B and D). n 13–21; *p ⬍ 0:05 vs. the effect of levodopa/carbidopa alone in Newman–Keuls test.
thioperamide 1834 ^ 411; n 8 was also very close to being statistically significant p 0:055: The effect of thioperamide, but not that of atipamezole, was potentiated p ⬍ 0:05 when the drugs were given together 2771 ^ 495; n 9 (Fig. 3).
slight decrease of amphetamine-induced behavior (to 222 ^ 23; n 16 evoked by dexmedetomidine (10 mg/kg) did not reach statistical significance.
4. Discussion 3.5. Effect of histamine H3- and adrenergic a 2-ligands on the amphetamine-induced ipsilateral turning Amphetamine (2.5 mg/kg) alone caused 489 ^ 92 n 19 full ipsilateral turns in 4 h. The turning behavior was not significantly altered by 5 mg/kg of R-a-methylhistamine 470 ^ 84; n 10 or 5 mg/kg of thioperamide 499 ^ 49; n 9: In studies with a2-ligands, amphetamine itself caused 308 ^ 53 n 28 ipsilateral turns. Atipamezole (0.3 mg/ kg) increased p ⬍ 0:001 the amphetamine-induced ipsilateral turning behavior 780 ^ 129; n 19: In contrast, the
At first glance, the effects of histamine H3-receptor and adrenergic a2-receptor ligands on levodopa-induced turning behavior were quite similar and the results fit well with the findings that H3-receptors [4,22] and a2-receptors [23,24] are presynaptic inhibitory heteroreceptors on several different neurons. With respect to dexmedetomidine and atipamezole, our results partially agree with previous preclinical and clinical findings obtained using other a2-ligands [16,25]. These are the first results reported about the effect of histamine H3-ligands, on levodopa- and amphetamineinduced turning behavior. Considering the high density of
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Fig. 3. Effect of a combination of H3- and a2-ligands with levodopa/carbidopa-induced turning behavior. The rats received either antagonist or their combination or either agonist or their combination together with carbidopa (30 mg/kg) 15 min before levodopa (10 mg/kg) injection. Doses (s.c.) of the H3- and a2-ligands were as follows: R-a-CH3-histamine and thioperamide, 5 mg/kg; dexmedetomidine, 0.01 mg/kg and atipamezole, 0.30 mg/kg. n 7–18; F 9:682 (one-way [treatment] analysis of variance), *p ⬍ 0:05 and ***p ⬍ 0:001 vs. levodopa/carbidopa treatment alone and ⫹ p ⬍ 0:05 vs. combination of the antagonists in Newman–Keuls test.
histamine H3-receptors in the striatum [5–9], which are known to increase in number after 6-OHDA lesions [13,14], we propose that in this lesion model, H3-receptors modify those pathways participating in the striatal outflow (e.g. acetylcholine, GABA, 5-HT). Evidently some functional nigrostriatal dopaminergic neurons still exist even after extensive 6-OHDA lesions which decrease striatal dopamine levels to nearly zero, but their contribution may be minor. Some of these neurons may have lost their function or phenotype but they can be restored by growth factors as has been demonstrated in the rat and primate models of Parkinson’s disease [26–29]. Although we obtained some evidence that the histamine H3-antagonist, thioperamide may potentiate the effect of levodopa/carbidopa, it should be emphasized that this effect was rather marginal and not a constant finding. In this respect, the effects of adrenergic a2-antagonists such as atipamezole [16,25], idazoxan and efaroxan [16], have been more consistent. Considering the high turning activity caused by levodopa alone in both of the dose-response experiments, we may have missed the effect of either antagonist. It is also possible that there is no or only little tonic activation of both a2- and H3-receptors. We were more fortunate in the combination study where both a2- and H3-antagonists moderately enhanced the effect of levodopa, and even supplemented each others’ effect. However, the therapeutical prospects for either
approach may be limited. In the case of a2-antagonists, the improvement of adrenergic activity, which is also defective in Parkinson’s disease, may be the principal reason for their clinical effectiveness [30]. Since the noradrenergic innervation of the striatum is sparse and of unknown origin [31,32], other possibilities should be discussed. In particular, the substantia nigra pars compacta has a high density of a2-receptors with a well-defined origin from the locus coeruleus [31,33,34]. Noradrenaline–dopamine interactions may also occur in the ventral tegmental area, the nucleus accumbens, the septum and the prefrontal cortex [31,35,36]. Other neurotransmitters (e.g. acetylcholine, glutamate, 5HT, GABA) can modulate directly or indirectly the dopamine-induced locomotor activity and circling behavior, and these neuron tracts are known to be influenced by a2-ligands [37]. None of these possibilities have been studied with histamine H3-ligands, though there is an abundance of histamine H3-receptors in the substantia nigra [5–9]. Also, H3-heteroreceptors are known to regulate the functioning of many different neurons [2]. These facts point to some resemblance in the mechanism of action of both a2-ligands and histamine H3-ligands. However, the failure of histamine H3-ligands to influence the amphetamine-induced ipsilateral turning behavior, in contrast to a2-adrenergic ligands, does not support the concept that these two presynaptic receptors are
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identical in their functional properties or in their neuronal distribution.
[18]
Acknowledgements
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The excellent technical help of Ms Pirjo Ha¨nninen is greatly appreciated. We are also grateful to Dr Ewen MacDonald for linguistic advice.
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Effects of histamine H3-ligands on the levodopa-induced turning behavior of hemiparkinsonian rats M. Huotari, K. Kukkonen, N. Liikka, T. Potasev, A. Raasmaja, P.T. Ma¨nnisto¨* University of Kuopio, Department of Pharmacology and Toxicology, P.O. Box 1627, FIN-70211 Kuopio, Finland
Abstract Histamine H3-receptors act as heteroreceptors on many neurons. The effects of H3-ligands (an agonist, R-a-methylhistamine and an antagonist, thioperamide) on levodopa-induced turning behavior in a rat model of Parkinson’s disease were quite similar to those seen with a2-adrenoceptor ligands (dexmedetomidine and atipamezole). R-a-methylhistamine clearly reduced contralateral turning behavior but the increase of turning behavior after thioperamide was less clear. The lack of effect of H3-ligands, in contrast to a2-ligands, on the amphetamineinduced ipsilateral turning behavior points to different roles or neuronal distribution of these two presynaptic receptors. We propose that in this lesion model, H3-receptors modify those pathways participating striatal outflow. 䉷 2000 Elsevier Science Ltd. All rights reserved. Keywords: Histamine H3-receptors; Thioperamide; R-a-methylhistamine; Adrenergic a2-receptors; Dexmedetomidine; Atipamezole; Turning behavior; Rat Parkinson model
1. Introduction Since the early 1980s, histamine has been reported to have important actions on central neurotransmission [1,2]. At about the same time a new receptor subtype, the histamine H3-receptor, was characterized [3] and novel ligands for this receptor were synthesized [4]. Recently the histamine H3-receptor has been cloned and functionally expressed [5]. In addition to functioning as an autoreceptor in histaminergic neurons, histamine H3-receptors can also act as inhibitory presynaptic heteroreceptors on a number of other neurons [2]. However, very little is known about the functional role of this histamine receptor subtype [2]. Histamine H3-receptors are found in several brain regions, notably in the striatum and substantia nigra [5–9]. It is also known that activation of histamine H3-receptors inhibits dopamine release in the mouse striatum [10]. Asymmetric lesioning of the dopaminergic nigrostriatal pathways by 6-OH-dopamine (6-OHDA) is an established rat model of unilateral Parkinson’s disease [11]. After a unilateral infusion of 6-OHDA into the substantia nigra, nigral dopamine cell bodies are destroyed, and supersensitivity of the postsynaptic, striatal dopamine receptors on the lesioned side develops. The rats rotate in a direction away from the lesion (contralaterally) when direct dopamine * Corresponding author. Tel.: ⫹ 358-17-162-401; fax: ⫹ 358-17-162424. E-mail address: [email protected] (P.T. Ma¨nnisto¨).
agonists (e.g. apomorphine or bromocriptine) or the dopamine precursor, levodopa, are given. Indirectly acting dopamine-releasing compounds (e.g. amphetamine) cause rotation in a direction towards the lesioned side (ipsilateral turnings) [11,12]. This model is a very sensitive way of testing changes in the dopaminergic tone in the brain. Recently it was demonstrated that after 6-OHDA lesions, the numbers of H3-receptors were greatly increased in the ipsilateral striatum and substantia nigra [13,14]. These findings indicate that histamine H3-receptors may be targets for the drug treatment of Parkinson’s disease. However, clinical findings have shown that central histaminergic neurons are preserved in Parkinson’s disease [15]. In this study we wanted to examine whether a histamine H3receptor agonist, R-a-methylhistamine, or an antagonist, thioperamide, would modify the turning behavior induced by either levodopa or amphetamine in the unilaterally lesioned rats. An established a2-adrenergic agonist, dexmedetomidine, and an a2-adrenergic antagonist, atipamezole, were used as control treatments reflecting the modification of presynaptic receptors. The effects of some other a2-adrenergic ligands in the rat turning model were recently reported [16].
2. Material and methods 2.1. Animals
1353-8020/00/$ - see front matter 䉷 2000 Elsevier Science Ltd. All rights reserved. PII: S1353-802 0(00)00007-9
Male albino rats (Han/Kuo), weighing 230–300 g, were
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obtained from the National Laboratory Animal Center, Kuopio, Finland. The rats were kept under standard laboratory conditions (temperature 20 ^ 3⬚C in groups (3–5 rats/ group) in a 12 h dark/light cycle (lights on 7.00 a.m.). The rats had free and continuous access to fresh tap water and mice/rat food pellets. All animal procedures were reviewed by the Animal Ethics Committee of the University of Kuopio and approved by the local Provincial Government. 2.1.1. Lesion on the medial forebrain bundle The procedure was performed according to Ungerstedt [11] with some modifications [17,18]. The details are given below briefly. 6-OHDA was dissolved in isotonic NaCl solution containing 0.2% of ascorbic acid (2.5 mg of 6-OHDA in 1 ml). For administration of 6-OHDA into the right substantia nigra, the rats were anesthetized with chloral hydrate anesthesia (350 mg/kg, i.p.). A stainless steel guide cannula (made from a 23 G needle) was lowered 2.0 mm above the target. An infusion needle (made from a 30 G needle) was lowered 2.0 mm below the lower tip of the guide cannula. The coordinates for the final infusion site near substantia nigra were AP 4.4, L 1.2, DV ⫺8.3 [19]. The volume of the infusion was 4 ml, and it was given during 8 min. We used Hamilton syringes (1 ml) connected to PE 10 plastic tubing for infusion. The syringes were run by a motor-driven slow-motion syringe pump (CMA/102, Carnegie Medicin, Sweden). The needle was retained in position for an extra 4 min after the infusion of 6-OHDA. During the 1 week recovery period from anesthesia, the animals were housed singly and after that in groups of 3–5 rats. 2.1.2. Testing of turning behavior The rats were placed in individual hemispherical plastic bowls (diameter 35 cm). They were clothed with vests having two holes for the front legs. The vest was connected to an automatic eight-channel rotameter (Coulbourn Instruments, Inc., Allentown, PA). The rotameter registered right and left full turns separately. The rats were allowed to adapt to the bowls for about 30 min before the drug treatments. 2.1.3. Analysis of the striatal dopamine levels To confirm the success of the unilateral lesion, dopamine levels in both striata were analyzed by HPLC using electrochemical detection as earlier described [20]. The rats were decapitated at about 7 days after the last turning experiment. The brains were exposed within 30 s, cooled in liquid nitrogen and both striata were dissected according to Glowinski and Iversen [21]. The dissected tissues were frozen in liquid nitrogen and stored at ⫺80⬚C until analyzed. 2.2. Treatments 2.2.1. Apomorphine test To test the success of the lesioning, apomorphine (0.1 mg/kg) was given subcutaneously (s.c.) about 14 days
after the toxin infusion. Only those animals which showed over 100 full contralateral turns in a 1 h test session were used. One week after apomorphine testing, the actual experiments were started. 2.2.2. Peripheral administration of histamine H3- and adrenergic a 2-ligands Graded doses (s.c.) of H3- and a2-agonists and antagonists (R-a-methylhistamine and thioperamide: 2.5, 5 or 10 mg/ kg; dexmedetomidine: 0.005, 0.01 and 0.02 mg/kg and atipamezole: 0.15, 0.3 and 0.6 mg/kg) were given with levodopa (10 mg/kg, i.p.) and carbidopa (30 mg/kg, i.p.). The combinations of both agonists and both antagonists were also tested (with levodopa/carbidopa) to study whether they could enhance each other. The drugs were also given with amphetamine (2.5 mg/kg, s.c.). H3- and a2-agonists and antagonists were given at the same time as carbidopa (when used), and levodopa or amphetamine 15 min after these injections. The monitoring of the circling was started immediately after levodopa or amphetamine injection. Within each series, the studies were organized in a cross-over manner. On each experimental day, all different treatments were given at least to one rat, and on the following experimental day, each rat was given a new treatment in a randomized order. Some rats were discarded: those who lost their vests in the middle of the turning session, and those who did not respond to levodopa after any drug treatment. 2.3. Statistics Arithmetic means, SEMs and SDs were calculated. The results were analyzed with one-way (treatment) analysis of variance (ANOVA). Newman–Keuls analysis was used as a post-hoc test. 3. Results 3.1. Success of nigral lesions The dopamine content in the healthy side was 7:9 ^ 0:78 mg=g (mean ^ SEM, n 14 and in the lesion side 0:001 ^ 0:0001 mg=g n 14 demonstrating the highly successful nature of the lesions. 3.2. Effect of graded doses of R-a-methylhistamine and thioperamide alone Neither drug induced any turning behavior at any dose (2.5, 5 or 10 mg/kg) (data not shown). 3.3. Effect of graded doses of R-a-methylhistamine, thioperamide, dexmedetomidine and atipamezole with levodopa (10 mg/kg i.p.) and carbidopa (30 mg/kg i.p.) on turning behavior In the R-a-methylhistamine group, the levodopa/carbidopa
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Fig. 1. Effects of graded doses (s.c.) of R-a-CH3-histamine (Fig. 1A and B) and thioperamide (Fig. 1C and D) on levodopa/carbidopa-induced turning behavior. All rats received carbidopa (30 mg/kg) and saline or R-a-CH3-histamine (Fig. 1A) 2.5 mg/kg (A), 5 mg/kg (W), 10 mg/kg (K), thioperamide (Fig. 1C) 2.5 mg/ kg (A), 5 mg/kg (W), 10 mg/kg (K) 15 min before levodopa (10 mg/kg, B, Fig. 1A and C). The rats were monitored 240 min after levodopa injection. Averages and SEMs of contralateral turns were calculated every 10 min (Fig. 1A and C) and total amount after 180 min (Fig. 1B and D). n 10–21; *p ⬍ 0:05 vs. the effect of levodopa/carbidopa alone in Newman–Keuls test.
treatment alone caused 1143 ^ 117 (mean ^ SEM, n 18 full contralateral turns in 3 h. R-a-methylhistamine pretreatment reduced F 2:881; p ⬍ 0:05 contralateral turning behavior, 10 mg/kg being the most effective dose 707 ^ 104; n 20; p ⬍ 0:05 (Fig. 1A and B). In this part of the study, thioperamide had no effect on levodopa/carbidopa 1531 ^ 185; n 11 induced turning behavior (Fig. 1C and D). Dexmedetomidine pretreatment decreased the number of contralateral turns (3.460, p ⬍ 0:05 having its maximum effect at 20 mg/kg 575 ^ 84; n 18; p ⬍ 0:05 vs. levodopa/carbidopa-treatment: 1086 ^ 134; n 14 (Fig. 2A and B). Atipamezole pretreatment increased the number of contralateral turns 1722 ^ 225; n 13 from those of levodopa/carbidopa alone 1345 ^ 211; n 12 but the effect did not reach statistical significance (Fig. 2C and D).
3.4. Effect of a combination of histamine H3- and adrenergic a 2-ligands on levodopa/carbidopa-induced turning behavior Levodopa/carbidopa treatment alone caused 1113 ^ 162 (mean ^ SEM, n 18 full contralateral turns in 4 h. As above, both agonists, a histamine H3-agonist R-amethylhistamine (5 mg/kg, s.c., 889 ^ 138; n 13 and an adrenergic a2-agonist dexmedetomidine (10 mg/kg, s.c., 710 ^ 101; n 13 tended to decrease the number of contralateral turns. When given together they seemed to enhance each others’ action. 568 ^ 76; n 14 None of these effects were statistically significant (Fig. 3). The antagonists thioperamide (5 mg/kg, s.c.) and atipamezole (0.3 mg/kg, s.c.) both enhanced the number of contralateral turns. The effect of atipamezole 2200 ^ 573; n 7 was significant p ⬍ 0:05 and the effect of
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Fig. 2. Effects of graded doses (s.c.) of dexmedetomidine (Fig. 2A and B) and atipamezole (Fig. 2C and D) on levodopa/carbidopa-induced turning behavior. All rats received carbidopa (30 mg/kg) and saline or dexmedetomidine (Fig. 2A) 0.005 mg/kg (A), 0.01 mg/kg (W), 0.02 mg/kg (K) or atipamezole (Fig. 2C) 0.15 mg/kg (A), 0.30 mg/kg (W), 0.60 mg/kg (K) 15 min before levodopa (10 mg/kg, B). The rats were monitored 240 min after levodopa injection. Averages and SEMs of contralateral turns were calculated every 10 min (Fig. 2A and C) and total amount after 180 min (Fig. 2B and D). n 13–21; *p ⬍ 0:05 vs. the effect of levodopa/carbidopa alone in Newman–Keuls test.
thioperamide 1834 ^ 411; n 8 was also very close to being statistically significant p 0:055: The effect of thioperamide, but not that of atipamezole, was potentiated p ⬍ 0:05 when the drugs were given together 2771 ^ 495; n 9 (Fig. 3).
slight decrease of amphetamine-induced behavior (to 222 ^ 23; n 16 evoked by dexmedetomidine (10 mg/kg) did not reach statistical significance.
4. Discussion 3.5. Effect of histamine H3- and adrenergic a 2-ligands on the amphetamine-induced ipsilateral turning Amphetamine (2.5 mg/kg) alone caused 489 ^ 92 n 19 full ipsilateral turns in 4 h. The turning behavior was not significantly altered by 5 mg/kg of R-a-methylhistamine 470 ^ 84; n 10 or 5 mg/kg of thioperamide 499 ^ 49; n 9: In studies with a2-ligands, amphetamine itself caused 308 ^ 53 n 28 ipsilateral turns. Atipamezole (0.3 mg/ kg) increased p ⬍ 0:001 the amphetamine-induced ipsilateral turning behavior 780 ^ 129; n 19: In contrast, the
At first glance, the effects of histamine H3-receptor and adrenergic a2-receptor ligands on levodopa-induced turning behavior were quite similar and the results fit well with the findings that H3-receptors [4,22] and a2-receptors [23,24] are presynaptic inhibitory heteroreceptors on several different neurons. With respect to dexmedetomidine and atipamezole, our results partially agree with previous preclinical and clinical findings obtained using other a2-ligands [16,25]. These are the first results reported about the effect of histamine H3-ligands, on levodopa- and amphetamineinduced turning behavior. Considering the high density of
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Fig. 3. Effect of a combination of H3- and a2-ligands with levodopa/carbidopa-induced turning behavior. The rats received either antagonist or their combination or either agonist or their combination together with carbidopa (30 mg/kg) 15 min before levodopa (10 mg/kg) injection. Doses (s.c.) of the H3- and a2-ligands were as follows: R-a-CH3-histamine and thioperamide, 5 mg/kg; dexmedetomidine, 0.01 mg/kg and atipamezole, 0.30 mg/kg. n 7–18; F 9:682 (one-way [treatment] analysis of variance), *p ⬍ 0:05 and ***p ⬍ 0:001 vs. levodopa/carbidopa treatment alone and ⫹ p ⬍ 0:05 vs. combination of the antagonists in Newman–Keuls test.
histamine H3-receptors in the striatum [5–9], which are known to increase in number after 6-OHDA lesions [13,14], we propose that in this lesion model, H3-receptors modify those pathways participating in the striatal outflow (e.g. acetylcholine, GABA, 5-HT). Evidently some functional nigrostriatal dopaminergic neurons still exist even after extensive 6-OHDA lesions which decrease striatal dopamine levels to nearly zero, but their contribution may be minor. Some of these neurons may have lost their function or phenotype but they can be restored by growth factors as has been demonstrated in the rat and primate models of Parkinson’s disease [26–29]. Although we obtained some evidence that the histamine H3-antagonist, thioperamide may potentiate the effect of levodopa/carbidopa, it should be emphasized that this effect was rather marginal and not a constant finding. In this respect, the effects of adrenergic a2-antagonists such as atipamezole [16,25], idazoxan and efaroxan [16], have been more consistent. Considering the high turning activity caused by levodopa alone in both of the dose-response experiments, we may have missed the effect of either antagonist. It is also possible that there is no or only little tonic activation of both a2- and H3-receptors. We were more fortunate in the combination study where both a2- and H3-antagonists moderately enhanced the effect of levodopa, and even supplemented each others’ effect. However, the therapeutical prospects for either
approach may be limited. In the case of a2-antagonists, the improvement of adrenergic activity, which is also defective in Parkinson’s disease, may be the principal reason for their clinical effectiveness [30]. Since the noradrenergic innervation of the striatum is sparse and of unknown origin [31,32], other possibilities should be discussed. In particular, the substantia nigra pars compacta has a high density of a2-receptors with a well-defined origin from the locus coeruleus [31,33,34]. Noradrenaline–dopamine interactions may also occur in the ventral tegmental area, the nucleus accumbens, the septum and the prefrontal cortex [31,35,36]. Other neurotransmitters (e.g. acetylcholine, glutamate, 5HT, GABA) can modulate directly or indirectly the dopamine-induced locomotor activity and circling behavior, and these neuron tracts are known to be influenced by a2-ligands [37]. None of these possibilities have been studied with histamine H3-ligands, though there is an abundance of histamine H3-receptors in the substantia nigra [5–9]. Also, H3-heteroreceptors are known to regulate the functioning of many different neurons [2]. These facts point to some resemblance in the mechanism of action of both a2-ligands and histamine H3-ligands. However, the failure of histamine H3-ligands to influence the amphetamine-induced ipsilateral turning behavior, in contrast to a2-adrenergic ligands, does not support the concept that these two presynaptic receptors are
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identical in their functional properties or in their neuronal distribution.
[18]
Acknowledgements
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The excellent technical help of Ms Pirjo Ha¨nninen is greatly appreciated. We are also grateful to Dr Ewen MacDonald for linguistic advice.
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