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Effects of riluzole on harmaline induced tremor and ataxia in rats: Biochemical, histological and behavioral studies
European Journal of Pharmacology 695 (2012) 40–47
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Neuropharmacology and analgesia
Effects of riluzole on harmaline induced tremor and ataxia in rats: Biochemical, histological and behavioral studies Fatemeh Rahimi Shourmasti a, Iran Goudarzi a,n, Taghi Lashkarbolouki a, Kataneh Abrari a, Mahmoud Elahdadi Salmani a, Afsaneh Goudarzi a a
School of Biology, Damghan university, Damghan, Iran
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 March 2012 Received in revised form 26 August 2012 Accepted 27 August 2012 Available online 5 September 2012
Essential tremor (ET) is one of the most common and most disabling movement disorders among adults. The drug treatment of essential tremor remains unsatisfactory. Additional therapies are required for patients with inadequate response or intolerable side effects. Thus, we aimed to investigate the therapeutic effects of riluzole on harmaline-induced tremor and ataxia in rat, and determining whether riluzole exerts its effect through modulation of glutamate levels in cerebellum. The study included 5 groups of Wistar rats weighing 80–100 g, injected with harmaline (50 mg/kg i.p.) for inducing experimental tremors and ataxia. The rats in group 1 served as control (saline induced) and group 2 received harmaline alone, whereas the animals in groups 3, 4 and 5, were also given riluzole intraperitoneally at doses of 2, 4 and 8 mg/kg 10 min after harmaline administration, respectively. The intensity and duration of tremor were recorded. Rotarod test, distance traveled and number of crossings were used to evaluate motor performance. Results of this study demonstrated that riluzole dose dependently attenuated duration and intensity of harmaline-induced tremors. Also, riluzole significantly improves time to fall, distance traveled and number of crossings in combined riluzole and harmaline treated rats. Histological analysis indicated that harmaline could cause vermis Purkinje cell (PC) loss and riluzole prevented this toxic effect. Harmaline also could increase glutamate levels in vermis and treatment with riluzole restored glutamate levels. In conclusion, riluzole has relatively protective effects on harmaline-induced tremor, probably related to its inhibitory effect on glutamatergic neurotransmission. & 2012 Elsevier B.V. All rights reserved.
Keywords: Harmaline Tremor Riluzole Cerebellum (Rat)
1. Introduction Tremors are an involuntary rhythmical movement of body parts among which essential tremor is one of the most common movement disorders with prevalence estimates from 0.4% to 5%. Individuals with essential tremor often manifest a combination of bilateral postural and kinetic tremors that involve, in nearly 90% of individuals, the hands and forearms. To a lesser extent, the head, neck, jaw or vocal cords can be affected. While essential tremor is often referred to as benign since it does not alter life expectancy, it can result in significant social and functional disability (Louis, 2005; Pahwa and Lyons, 2003). Essential tremors result from both physiologic and pathologic processes in the nervous system, and always involve the interaction of central and peripheral nervous systems.
n Correspondence to: School of Biology, Damghan University, Postal Code: 3671641167, Damghan, Iran. Tel./fax: þ 98 232 5247146. E-mail address: [email protected] (I. Goudarzi).
0014-2999/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2012.08.014
Administration of harmaline produces abnormal motor behavior that includes a high frequency tremor associated with ataxia in rats (O0 Hearn and Molliver, 1993; Zetler et al., 1974). Harmaline has powerful central nervous system (CNS) excitatory effects manifested by a markedly increased firing rate of neurons within the inferior olivary nucleus (De Montigny and Lamarre, 1973, 1974; Llinas and Volkind, 1973). The mechanisms by which beta-carboline compounds produce increased inferior olive activity are not fully characterized. In accord with the excitotoxic hypothesis (Olney, 1978; Choi, 1988), harmaline should produce a sustained increase in neuronal firing in the inferior olive, leading to release of excessive glutamate from climbing fiber terminals that synapse on longitudinal arrays of Purkinje cells. The repetitive release of an excitatory neurotransmitter, sustained over many hours, is likely to produce irreversible, excitotoxic damage to Purkinje cells, followed by their degeneration. Owing to a lack of understanding of the basic mechanism and origin of tremors, it has been difficult to develop pharmacological agents with selective and specific antitremor activity. The current drug treatment, when recommended, does not cure nor prevent
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disease progression and is only symptomatic. Thus, an effective and well tolerated drug is still lacking. Riluzole (2-amino-6-trifluoromethoxybenzothiazole) can slow the progression of disease in patients with amyotrophic lateral sclerosis (ALS) and has accordingly been approved for the treatment of this disorder in several countries (Bensimon et al., 1994; Lacomblez et al., 1996). It was also reported that riluzole has neuroprotective effect in 3-acetylpyridine (3-AP) induced ataxia in rat (Janahmadi et al., 2009). The neuroprotective mechanism of riluzole was initially attributed to its inhibitory effect on glutamatergic neurotransmission (Martin et al., 1993; MacIver et al., 1996) and the resultant excitotoxic neuronal injury (Estevez et al., 1995; Mary et al., 1995; Doble, 1999). Also, riluzole has been reported to inhibit multiple ion channels such as glutamate-gated channels (Debono et al., 1993; Hubert et al., 1994), voltage-gated channels (Benoit and Escande, 1991; Hebert et al., 1994; Song et al., 1997), and volume-sensitive chloride channels (Bausch and Roy, 1996). The beneficial neuroprotective action of riluzole on the motor diseases, its modulatory effects on ion channels and its inhibitory effect on glutamatergic neurotransmission prompted us to examine therapeutic effects of riluzole and its mechanism of action in harmaline-induced termor and ataxia in rats.
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Ataxia was defined as impairment in the ability to execute coordinated motor functions and was scored as follows: (0) normal; (1) mud walking; (2) tremor while walking; (3) motor deficits of hind limbs; (4) no weight bearing by hind limbs (Watanabe et al., 1997).
2.4. Assessment of motor activity 2.4.1. Accelerating rotarod assay: a motor performance test Rotarod test, which measures balance, coordination, and motor control, was used to evaluate motor performance as previously described (Janahmadi et al., 2009). Briefly, each rat was placed on a rod (8.9 cm long and 3.8 cm in diameter) covered with rubber to evaluate rotarod performance. Rats were left for 5 min on the rod for habituation. The accelerating rotarod was set to accelerate gradually from 4 to 40 rpm over the course of 5 min. The starting speed was 4 rpm, and the total time of each trial was 300 s. The interval between placing the rat on the rod and falling from the rod were measured three times with a maximum of 300 s. The sum of the intervals of each rat was determined. Data were collected from rat rotarod performance before the beginning of treatment, 2 h after treatment (day of treatment), 1 day after treatment, 3 days and 7 days later.
2. Materials and methods 2.1. Drugs and chemicals Harmaline HCl, riluzole were purchased from Sigma-Aldrich, Germany. Two compounds were prepared freshly on the day of the experiment. Harmaline HCl and riluzole dissolved in normal saline and solution of 0.1 N HCl, respectively. 2.2. Animals The experimental protocol was approved by the Research and Ethics Committee of Damghan University. Male Wistar rats (weighing 80–100 g) were purchased from Pastur Institute of Iran. Animals were kept under standard laboratory conditions with a 12-h light/dark cycle and ad libitum food and water throughout the experiments. 2.3. Drug-preparation and administration On the day of experimentation, animals were transferred to the testing room and left for 1 h for acclimatization. They were randomly assigned to 5 experimental groups. Experimental tremors were produced in animals by a single injection of harmaline (50 mg/kg) intraperitoneally as previously described (Biary et al., 2000). The rats in group 1 served as control (saline induced) and group 2 received harmaline alone, whereas the animals in groups 3, 4 and 5 were also given riluzole intraperitoneally at doses of 2, 4 and 8 mg/kg 10 min after harmaline administration, respectively. Riluzole dosage was selected on the basis of earlier reports that demonstrated its neuroprotective effects in ataxic rat (Janahmadi et al., 2009). The occurrence of tremors was rated by an observer blinded to treatment protocol. The duration of tremors was recorded as the time between onset (appearance of the first symptoms of tremors) and complete disappearance of tremors. The intensity of tremors was first recorded at tremor onset and then every 30 min post harmaline administration over a period of 430 min. The clinical grading of tremor intensity was done according to Arshaduddin et al. (2004) as follows: 0: no tremor, 1: mild tremor, 2: moderate intermittent tremor, 3: moderate persistent tremor and 4: pronounced severe tremor.
2.4.2. Open field test The horizontal activity of the rat from each group was recorded for a period of 5 min and analyzed using Ethovision software (version 3.1), a video tracking system for automation of behavioral experiments (Noldus Information Technology, the Netherlands). Rats were individually placed at the center of a clean open field apparatus (40 cm 40 cm 15 cm, divided into nine squares). Prior to the evaluation, animals were habituated to the box for 1 min within the box. The behavioral parameters include total distance moved (TDM, cm) and number of squares crossed (locomotor activity) were recorded for 5 min (Burger et al., 2005).
2.5. Cerebellum histological analysis After completion of behavioral measurements (7 days after treatment), rats from control and treatment groups were sacrificed under ketamine-xylazine anesthesia. Transcardial perfusion was performed with physiological saline followed by fixing with 10% paraformaldehyde solution dissolved in 0.1% phosphate buffer solution. The animals were decapitated and the cerebellums were removed and immersed in 10% buffered paraformaldehyde for a week. Then, the block of cerebellum was immersed in 30% sucrose-buffer solution overnight. Coronal sections (40 mm thick) at the vermal level were cut using cryostat (Leica, Germany). The sections were subsequently stained with cresyl violet (0.1%) and examined for histological changes.
2.6. Biochemical study 2.6.1. Determination of glutamate levels Concentrations of glutamate in the cerebellums were measured using a glutamate assay kit (abcam83389). Briefly, cerebellar vermis were homogenized in buffer, and then centrifuged at 15,000 g for 20 min. Supernatants were incubated with reaction mix solution for 30 min at 37 1C. Absorption determined at 450 nm using a spectrophotometer and amounts of glutamate determined based on of standard curve.
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2.7. Data analysis Statistical analysis of data was performed using one-way and twoway ANOVA. Individual comparisons were performed by Tukey’s or Dunnett’s multiple comparisons tests where appropriate. Differences with P-valueo0.05 were considered significant.
3. Results 3.1. Behavioral studies 3.1.1. Effect of riluzole on intensity and duration of tremor in harmaline -treated rats Harmaline administration to rats induced the characteristic pattern of tremors starting within 2.12 70.125 min following administration and lasted for more than 420 min. Furthermore, this compound decreased the number of crossings, and induced some signs of ataxia characterized by abnormal, awkward walking, stepping with one or both feet drawing aside, and balance disturbances. The tremor intensity at 10 min following harmaline administration was 3.6370.26. The intensity of the tremor at 30, 60, 120 and 180 min following harmaline remained unchanged throughout this period. Treatment with riluzole in the doses of 2, 4 and 8 mg/kg resulted in a significant reduction in the intensity of the tremor at 30 min (270.22, 2.670.22, 1.670.2; Po0.001, dose 2, 4 and 8 mg/kg and respectively), 60 min (2.970.14, 2.370.21, 1.770.18; Po0.001, dose 2, 4 and 8 mg/kg and respectively), 90 min (370.26, 270.22; Po0.001, dose 4 and
8 mg/kg, respectively), 120 min (2.970.14; Po 0.05, dose 8 mg/ kg), 150 min (2.170.14; Po0.001, dose 8 mg/kg), 180 min (2.870.14, 2.570.22, 1.1 70.20; Po0.001, dose 2, 4 and 8 mg/ kg, respectively), 210 min (1.970.14; P o0.001, doses 2 mg/kg), 240 min (1.370.18; Po0.001, doses 2 mg/kg), 270 min (1.770.17; Po0.001, doses 2 mg/kg) (Fig. 1A). The total duration of tremors was significantly reduced by riluzole (Fig. 1B). In harmaline-alone-treated rats the mean duration of the tremor was 427 70.14 min, whereas treatment of animals with riluzole in the doses of 2, 4, and 8 mg/kg body weight reduced the duration to 26772.34, 23977.46 and 18871.89 min, respectively (Po0.001). Rats showed score 3 of ataxia in harmaline group and after riluzole treatment ataxia score reduced to 1.
3.1.2. Effect of riluzole on latencies to fall from the rotarod in harmaline -treated rats The rotarod test was used to assess balance and motor coordination in each group. The impaired motor function following harmaline injection was evidenced by poorer performance on a rotarod 2 h after treatment (day of treatment), 3 and 7 days later. Harmaline treated rats spent significantly less time (Po 0.001, n ¼9) walking on top of the rotating rod than control group indicating the severe motor impairment in harmaline treated rats (Fig. 2A). However, co-treatment with riluzole improved motor performance on rotarod when compared with harmaline treated rats (Po 0.01, 2 h after riluzole treatment in dose 4 and 8 mg/kg;
Fig. 1. Effect of riluzole on duration (A) and intensity (B) of harmaline induced tremor. Values are mean 7 S.E.M of nine rats, *Po 0.05, **P o0.001 and ***Po 0.001 as compared with harmaline alone treated animals by ANOVA followed by post-hoc comparison using Dunnett’s test. HR, harmaline; Ril, riluzole 2, 4 and 8 mg/kg. Times in xline indicate time after harmaline treatment.
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Fig. 2. Effect of riluzole on motor function in harmaline treated rats. Motor dysfunction was tested by the rotarod test (A), which measures balance, coordination, and motor control. Comparison of the rotarod performances between control and harmaline treated rats showed a significant motor impairment in harmaline treated group. In the open field test, harmaline treated rats also revealed that the distance traveled (B) and number of crossings (C) were significantly decreased as compared to control animals. Combined riluzole and harmaline improved motor performance on the rotarod as reflected in a longer time spent walking on rotating drum before falling (A). Riluzole treatment also increased the distance traveled (B), number of crossings in treated rats. Values are mean 7S.E.M of nine rats. * Po 0.05, ** Po 0.01 and *** Po 0.001 compared with control group. y P o 0.05, yy P o 0.01 and yyy P o0.001 compared with harmaline group.
Po0.001, 3 and 7 days after riluzole treatment, respectively) (Fig. 2A, n ¼9). There were no significant differences between harmaline and riluzole treated rats in rotarod performance in the next day after treatment.
3.1.3. Effect of riluzole on open field test in harmaline-treated rats Analysis of the total distance moved showed that harmaline injection alone produced locomotor hypoactivity as compared with control rats. In the days following treatment, ataxic rats showed significant decrease in the horizontal distance traveled (Po0.001).
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However, harmalineþriluzole treated animals showed significantly higher distance traveled compared to harmaline treated rats (Fig. 2B). Distance moved 2 h and 7 days after combined treatment with riluzole and harmaline were significantly increased as compared with harmaline injection alone (Po0.01, Po0.05; n¼9). In the open field test, harmaline significantly reduced the number of crossings in harmaline group than control group (Po0.001, n¼9), indicating a decreased locomotor spontaneous activity. Administration of riluzole improved the performance of the riluzoleþharmaline treated rats in the open-field task (2 h and 3 days after treatment, Po0.001, Po0.01 and Po0.05; n¼9) (Fig. 2C). 3.2. Histological assessment Purkinje cell bodies formed a continuous, uninterrupted layer in Nissl-stained sections from control rats (Fig. 3A). After treatment with harmaline, the Purkinje cell layer exhibited discontinuities
marked by absent Purkinje cell bodies (Fig. 3B) adjacent to zones with normal appearing cell bodies. However, in riluzole treated rats as in the control, Purkinje cell somas were arranged in a monolayer and formed a continuous, uninterrupted layer in Nissl-stained sections from control rats (compare Fig. 3C–E with A and B).
3.3. Measurements of cerebellar glutamate levels We measured levels of glutamate on 25 min and 2 h after treatment in vermis of cerebellum. The results showed that glutamate level significantly increased in harmaline treated rats as compared with control group in 25 min after treatment (Fig. 4A, Po0.05, n¼6). Also, there were no significant differences in riluzole treated rats than harmaline treated group. However, 2 h after treatment, glutamate level in riluzole treated rats (riluzole 4 and
Fig. 3. Loss of Purkinje cells after harmaline treatment and restoration following riluzole (dose 2, 4 and 8 mg/kg) treatment. After 7 days of treatment and termination of behavioral tests, cerebellar vermis areas were collected and coronal sections of cerebellar vermis were stained with cresyl violet (0.1%) for observation Purkinje cell under a light microscope. (A) Vermis from control rats; (B) harmaline treatment group. (C) Combined riluzole (2 mg/kg) and harmaline treatment. (D) Combined riluzole (4 mg/kg) and harmaline treatment. (E) Combined riluzole (8 mg/kg) and harmaline treatment. Purkinje cell layers showed a disconnected pattern, with a wide gap between the cells in harmaline treated rats than control group that relatively restored in riluzole treatment rats. Scale bars ¼ 50 mm.
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Fig. 4. Concentrations of glutamate in the cerebellar vermis of control, harmaline and riluzole treated rats. Measurement of glutamate content was done 25 min (A) and 2 h (B) after treatment. The concentration of glutamate increased significantly in harmaline treated rats compared to those of control rats 25 min and 2 h after the treatment. However, riluzole treated group (4 and 8 mg/kg) showed significant reduction in glutamate concentration in 2 h after treatment, but there was not significant decrease of the glutamate concentration in riluzole treated rats than harmaline group. Values are mean 7 S.E.M of six rats. P-value less than 0.05 (P o0.05) was considered to be statistically significant. * P o0.05 and ** P o 0.01 compared with control group. y Po 0.05 and yy P o 0.01 compared with harmaline group.
8 mg/kg) showed significant decrease in comparison to harmaline treated rats (Fig. 4B, Po0.01 and Po0.05, n¼6).
4. Discussion The results of this study showed that riluzole decreased the harmaline-induced action/postural tremor as well as ataxia and hypolocomotion. Reduction of harmaline-induced tremors was evident from decreased duration and severity of tremors in the rats treated with riluzole (Fig. 1A and B). Administration of harmaline to rats induced severe tremor as previously reported beginning within a few minutes, and lasted for at least 7 h (Biary et al., 2000). Harmaline produces tremors in the 8–12 Hz frequency range that is believed to be due to an enhancement of neuronal synchrony and rhythmicity in the inferior olive (Wilms et al., 1999). The harmaline model is of particular interest since it presents behavioral, metabolic imaging and pharmacological similarities to essential tremor. Furthermore, beta-carbolines related to harmaline, are detected in patients suffering from essential tremor and induce severe tremors in humans (Pennes and Hoch, 1957). Abnormal synchronous activation of climbing glutamatergic fibers, arising from the inferior olive and projecting to Purkinje
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cells is generally accepted to be a primary cause of the harmalineinduced tremor. The mechanisms by which harmaline and related beta-carbolines cause tremor have been investigated extensively. Harmaline induces rhythmic bursts of activity in inferior olivary neurons, which excite Purkinje cells via climbing fiber synapses leading to repetitive burst firing of Purkinje cells (De Montigny and Lamarre, 1973; Lamarre and Mercier, 1971; Llinas and Volkind, 1973). Harmaline increases and sustains rhythmic complex spike activity of these neurons (Lamarre et al., 1971; Beitz and Saxon, 2004), which leads to a suppression of their simple spike activity, triggered by parallel fibers (Lamarre et al., 1971). In consequence, rhythmic bursts appear in vestibulo-spinal and reticulo-spinal systems, as well as in a- and g-motoneurons (Lamarre et al., 1971; Lamarre and Weiss, 1973). We observed that tremor and ataxia gradually resolved over 6–8 h and spontaneous activity increased. Locomotion, motor activity and motor coordination appeared normal the next day after harmaline treatment but were abnormal 3 and 7 days after treatment. O0 Hearn and Molliver (1993) reported that tremor, ataxia and locomotion disappeared the next day after ibogaine and harmaline treatment. The acute tremor and ataxia that were transiently present minutes to hours after drug administration are likely due to a physiologic effect of excessive olivo-cerebellar stimulation rather than resulting from loss of Purkinje cells. Hence, the initial, transient expression of acute tremor and ataxia appears to reflect abnormally increased activity of Purkinje cells. In fact, any disturbance along cerebellar module is bound to result in similar symptoms due to a deficit in movement coordination. A decrease in Purkinje cell activity will decrease the amount of inhibition of the deep cerebellar nuclei (CN), which in turn will increase the inhibition of the olivary neurons, preventing the generation of the requested pattern. Likewise, increasing Purkinje cell activity will strongly inhibit the cerebellar nuclei neurons, leading to uncontrollable rhythmic firing of olivary neurons which might end up in tremor-like symptoms. In fact, one can predict that specifically activating the inhibitory projection neurons of the cerebellar nuclei will have similar results as reducing Purkinje cell activity. Furthermore, direct inhibition of olivary activity should also result in similar behavioral deficits. It is interesting to know that alcohol has an inhibitory effect on olivary activity (Lampl and Yarom, 1997). Indeed, uncoordinated movements under alcohol intoxication are reminiscent of ataxia. The reduction in Purkinje cell firing observed after alcohol administration (Van Skike et al., 2010) can, by removal of inhibition of the cerebellar nuclei neurons, also increase the inhibition of olivary activity. Biochemical and histological results of the present study indicated that harmaline significantly increased glutamate levels in the rat cerebellar vermis and caused Purkinje cells loss (Figs. 3 and 4A and B). These results are in accord with those of earlier reports that also demonstrated the Purkinje cell loss and enhancement glutamate levels induced after harmaline in the rat brain (Kim et al., 2003; Beitz and Saxon, 2004). We have assessed abnormal motor function in harmaline treatment group to monitor behavioral manifestations that are known to be associated with cerebellar neuronal damage (Breton et al., 1998; Ogura et al., 1980). Kim et al. (2003) demonstrated that a subset of Purkinje cell in the vermis and paravermis degenerated after harmaline treatment, but harmaline produced little or no Purkinje cell degeneration after inferior olivary ablation. They suggested that harmaline induced activation of inferior olivary neurons may lead to release of glutamate from climbing fiber synaptic terminal distributed over the Purkinje cells, and may lead to cytotoxic degeneration of Purkinje cells. The closely related alkaloid, ibogaine produce tremor and ataxia, which is quite similar to those induced by harmaline, following i.p. administration (O0 Hearn and
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Molliver, 1993). Purkinje cell degeneration was observed only at a later time point, well after the tremor has resolved, as also described for ibogaine (O0 Hearn and Molliver, 1997, 1999). Therefore, the reduction of motor activity and motor coordination recorded on 3 and 7 days after treatment are probably due to Purkinje cell loss. The present study highlighted the neurobehavioral evidence of harmaline administration is associated with a loss of cerebellar Purkinje cells and a related behavioral deficit in a manner that is prevented by riluzole. Riluzole reduced the tremogenic and the neurotoxic effects of harmaline. Experimental and clinical evidences implicated the role of glutamatergic system in essential tremor (Eblen et al., 1996; Ma´lly et al., 1996; Du et al., 1997). Current evidence is most consistent with an action of riluzole which causes a presynaptic reduction of transmitter release, rather than direct suppression of postsynaptic neurotransmitter receptor responses. Reduction of glutamate levels in vermis could be due to the enhancement of glutamate transporter uptake in Bergmann glia by riluzole. Glutamate uptake by rat spinal cord synaptosomes was increased by low concentrations (0.1–1 mM) of riluzole through a pertussis toxinsensitive mechanism (Azbill et al., 2000). As shown in Fig. 2, riluzole significantly reduced locomotion and number of crossings at dose 8 mg/kg. There is evidence that riluzole also may inhibit acetylcholine release in muscular endplate (Jehle et al., 2000), which may explain diminishing its beneficial effects on motor function. Therefore, in addition to glutamate neurotransmission, riluzole may affect other neurotransmitter system. Also, ataxia and hypolocomotion in harmaline treated rats are not a direct consequence of the tremor. In our study, tremor ended at 7 h after harmaline treatment in rats whereas ataxia remained in next days after harmaline treatment. Therefore, antagonism of ataxia and normalization of locomotion in harmaline treated rat is not a direct result from the antitremor effect of riluzole. Our results demonstrated that riluzole could decrease Glutamate levels in vermis of cerebellum. Also, vermis in riluzole treated rats (4 and 8 mg/kg) was normal, displaying an aligned Purkinje cell layer that was indistinguishable from control. Presumably, antiglutamatergic action of riluzole may counteract the neurotoxic glutamatergic effect of harmaline, and thus may prevent tremor and cerebellar neuronal death. Riluzoleinduced neuroprotection were observed in various animal models of injury (Malgouris et al., 1989; Stutzmann et al., 1997) and in neurodegenerative pathologies, such as Amyotrophic lateral sclerosis and Parkinson’s disease (Doble, 1999; Jacquin and Gruol, 1999; Noh et al., 2000). Janahmadi et al. (2009) reported that combined riluzole (4 mg/kg) and 3-acetylpyridine treatment preserved the Purkinje cells normal appearance and alignment, prevented the cell dispersion and promoted the survival of rat cerebellar Purkinje cells at the vermal level of central lobules when compared to 3-acetylpyridine treatment alone. It also preserved the most intrinsic electrophysiological properties of Purkinje cells and partially improved motor performance. In conclusion, we suggest that riluzole might be a potentially useful choice in the treatment of essential tremor and ataxia. Moreover, our data suggest that the role of the glutamatergic system in harmaline-induced tremor and also protective effect of riluzole may be related to its inhibitory effect on glutamatergic neurotransmission. Therefore, role of the glutamatergic system in harmaline-induced tremor should be further investigated in more details.
Acknowledgment We acknowledge Damghan University for supporting this work.
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Contents lists available at SciVerse ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Neuropharmacology and analgesia
Effects of riluzole on harmaline induced tremor and ataxia in rats: Biochemical, histological and behavioral studies Fatemeh Rahimi Shourmasti a, Iran Goudarzi a,n, Taghi Lashkarbolouki a, Kataneh Abrari a, Mahmoud Elahdadi Salmani a, Afsaneh Goudarzi a a
School of Biology, Damghan university, Damghan, Iran
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 March 2012 Received in revised form 26 August 2012 Accepted 27 August 2012 Available online 5 September 2012
Essential tremor (ET) is one of the most common and most disabling movement disorders among adults. The drug treatment of essential tremor remains unsatisfactory. Additional therapies are required for patients with inadequate response or intolerable side effects. Thus, we aimed to investigate the therapeutic effects of riluzole on harmaline-induced tremor and ataxia in rat, and determining whether riluzole exerts its effect through modulation of glutamate levels in cerebellum. The study included 5 groups of Wistar rats weighing 80–100 g, injected with harmaline (50 mg/kg i.p.) for inducing experimental tremors and ataxia. The rats in group 1 served as control (saline induced) and group 2 received harmaline alone, whereas the animals in groups 3, 4 and 5, were also given riluzole intraperitoneally at doses of 2, 4 and 8 mg/kg 10 min after harmaline administration, respectively. The intensity and duration of tremor were recorded. Rotarod test, distance traveled and number of crossings were used to evaluate motor performance. Results of this study demonstrated that riluzole dose dependently attenuated duration and intensity of harmaline-induced tremors. Also, riluzole significantly improves time to fall, distance traveled and number of crossings in combined riluzole and harmaline treated rats. Histological analysis indicated that harmaline could cause vermis Purkinje cell (PC) loss and riluzole prevented this toxic effect. Harmaline also could increase glutamate levels in vermis and treatment with riluzole restored glutamate levels. In conclusion, riluzole has relatively protective effects on harmaline-induced tremor, probably related to its inhibitory effect on glutamatergic neurotransmission. & 2012 Elsevier B.V. All rights reserved.
Keywords: Harmaline Tremor Riluzole Cerebellum (Rat)
1. Introduction Tremors are an involuntary rhythmical movement of body parts among which essential tremor is one of the most common movement disorders with prevalence estimates from 0.4% to 5%. Individuals with essential tremor often manifest a combination of bilateral postural and kinetic tremors that involve, in nearly 90% of individuals, the hands and forearms. To a lesser extent, the head, neck, jaw or vocal cords can be affected. While essential tremor is often referred to as benign since it does not alter life expectancy, it can result in significant social and functional disability (Louis, 2005; Pahwa and Lyons, 2003). Essential tremors result from both physiologic and pathologic processes in the nervous system, and always involve the interaction of central and peripheral nervous systems.
n Correspondence to: School of Biology, Damghan University, Postal Code: 3671641167, Damghan, Iran. Tel./fax: þ 98 232 5247146. E-mail address: [email protected] (I. Goudarzi).
0014-2999/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2012.08.014
Administration of harmaline produces abnormal motor behavior that includes a high frequency tremor associated with ataxia in rats (O0 Hearn and Molliver, 1993; Zetler et al., 1974). Harmaline has powerful central nervous system (CNS) excitatory effects manifested by a markedly increased firing rate of neurons within the inferior olivary nucleus (De Montigny and Lamarre, 1973, 1974; Llinas and Volkind, 1973). The mechanisms by which beta-carboline compounds produce increased inferior olive activity are not fully characterized. In accord with the excitotoxic hypothesis (Olney, 1978; Choi, 1988), harmaline should produce a sustained increase in neuronal firing in the inferior olive, leading to release of excessive glutamate from climbing fiber terminals that synapse on longitudinal arrays of Purkinje cells. The repetitive release of an excitatory neurotransmitter, sustained over many hours, is likely to produce irreversible, excitotoxic damage to Purkinje cells, followed by their degeneration. Owing to a lack of understanding of the basic mechanism and origin of tremors, it has been difficult to develop pharmacological agents with selective and specific antitremor activity. The current drug treatment, when recommended, does not cure nor prevent
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disease progression and is only symptomatic. Thus, an effective and well tolerated drug is still lacking. Riluzole (2-amino-6-trifluoromethoxybenzothiazole) can slow the progression of disease in patients with amyotrophic lateral sclerosis (ALS) and has accordingly been approved for the treatment of this disorder in several countries (Bensimon et al., 1994; Lacomblez et al., 1996). It was also reported that riluzole has neuroprotective effect in 3-acetylpyridine (3-AP) induced ataxia in rat (Janahmadi et al., 2009). The neuroprotective mechanism of riluzole was initially attributed to its inhibitory effect on glutamatergic neurotransmission (Martin et al., 1993; MacIver et al., 1996) and the resultant excitotoxic neuronal injury (Estevez et al., 1995; Mary et al., 1995; Doble, 1999). Also, riluzole has been reported to inhibit multiple ion channels such as glutamate-gated channels (Debono et al., 1993; Hubert et al., 1994), voltage-gated channels (Benoit and Escande, 1991; Hebert et al., 1994; Song et al., 1997), and volume-sensitive chloride channels (Bausch and Roy, 1996). The beneficial neuroprotective action of riluzole on the motor diseases, its modulatory effects on ion channels and its inhibitory effect on glutamatergic neurotransmission prompted us to examine therapeutic effects of riluzole and its mechanism of action in harmaline-induced termor and ataxia in rats.
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Ataxia was defined as impairment in the ability to execute coordinated motor functions and was scored as follows: (0) normal; (1) mud walking; (2) tremor while walking; (3) motor deficits of hind limbs; (4) no weight bearing by hind limbs (Watanabe et al., 1997).
2.4. Assessment of motor activity 2.4.1. Accelerating rotarod assay: a motor performance test Rotarod test, which measures balance, coordination, and motor control, was used to evaluate motor performance as previously described (Janahmadi et al., 2009). Briefly, each rat was placed on a rod (8.9 cm long and 3.8 cm in diameter) covered with rubber to evaluate rotarod performance. Rats were left for 5 min on the rod for habituation. The accelerating rotarod was set to accelerate gradually from 4 to 40 rpm over the course of 5 min. The starting speed was 4 rpm, and the total time of each trial was 300 s. The interval between placing the rat on the rod and falling from the rod were measured three times with a maximum of 300 s. The sum of the intervals of each rat was determined. Data were collected from rat rotarod performance before the beginning of treatment, 2 h after treatment (day of treatment), 1 day after treatment, 3 days and 7 days later.
2. Materials and methods 2.1. Drugs and chemicals Harmaline HCl, riluzole were purchased from Sigma-Aldrich, Germany. Two compounds were prepared freshly on the day of the experiment. Harmaline HCl and riluzole dissolved in normal saline and solution of 0.1 N HCl, respectively. 2.2. Animals The experimental protocol was approved by the Research and Ethics Committee of Damghan University. Male Wistar rats (weighing 80–100 g) were purchased from Pastur Institute of Iran. Animals were kept under standard laboratory conditions with a 12-h light/dark cycle and ad libitum food and water throughout the experiments. 2.3. Drug-preparation and administration On the day of experimentation, animals were transferred to the testing room and left for 1 h for acclimatization. They were randomly assigned to 5 experimental groups. Experimental tremors were produced in animals by a single injection of harmaline (50 mg/kg) intraperitoneally as previously described (Biary et al., 2000). The rats in group 1 served as control (saline induced) and group 2 received harmaline alone, whereas the animals in groups 3, 4 and 5 were also given riluzole intraperitoneally at doses of 2, 4 and 8 mg/kg 10 min after harmaline administration, respectively. Riluzole dosage was selected on the basis of earlier reports that demonstrated its neuroprotective effects in ataxic rat (Janahmadi et al., 2009). The occurrence of tremors was rated by an observer blinded to treatment protocol. The duration of tremors was recorded as the time between onset (appearance of the first symptoms of tremors) and complete disappearance of tremors. The intensity of tremors was first recorded at tremor onset and then every 30 min post harmaline administration over a period of 430 min. The clinical grading of tremor intensity was done according to Arshaduddin et al. (2004) as follows: 0: no tremor, 1: mild tremor, 2: moderate intermittent tremor, 3: moderate persistent tremor and 4: pronounced severe tremor.
2.4.2. Open field test The horizontal activity of the rat from each group was recorded for a period of 5 min and analyzed using Ethovision software (version 3.1), a video tracking system for automation of behavioral experiments (Noldus Information Technology, the Netherlands). Rats were individually placed at the center of a clean open field apparatus (40 cm 40 cm 15 cm, divided into nine squares). Prior to the evaluation, animals were habituated to the box for 1 min within the box. The behavioral parameters include total distance moved (TDM, cm) and number of squares crossed (locomotor activity) were recorded for 5 min (Burger et al., 2005).
2.5. Cerebellum histological analysis After completion of behavioral measurements (7 days after treatment), rats from control and treatment groups were sacrificed under ketamine-xylazine anesthesia. Transcardial perfusion was performed with physiological saline followed by fixing with 10% paraformaldehyde solution dissolved in 0.1% phosphate buffer solution. The animals were decapitated and the cerebellums were removed and immersed in 10% buffered paraformaldehyde for a week. Then, the block of cerebellum was immersed in 30% sucrose-buffer solution overnight. Coronal sections (40 mm thick) at the vermal level were cut using cryostat (Leica, Germany). The sections were subsequently stained with cresyl violet (0.1%) and examined for histological changes.
2.6. Biochemical study 2.6.1. Determination of glutamate levels Concentrations of glutamate in the cerebellums were measured using a glutamate assay kit (abcam83389). Briefly, cerebellar vermis were homogenized in buffer, and then centrifuged at 15,000 g for 20 min. Supernatants were incubated with reaction mix solution for 30 min at 37 1C. Absorption determined at 450 nm using a spectrophotometer and amounts of glutamate determined based on of standard curve.
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2.7. Data analysis Statistical analysis of data was performed using one-way and twoway ANOVA. Individual comparisons were performed by Tukey’s or Dunnett’s multiple comparisons tests where appropriate. Differences with P-valueo0.05 were considered significant.
3. Results 3.1. Behavioral studies 3.1.1. Effect of riluzole on intensity and duration of tremor in harmaline -treated rats Harmaline administration to rats induced the characteristic pattern of tremors starting within 2.12 70.125 min following administration and lasted for more than 420 min. Furthermore, this compound decreased the number of crossings, and induced some signs of ataxia characterized by abnormal, awkward walking, stepping with one or both feet drawing aside, and balance disturbances. The tremor intensity at 10 min following harmaline administration was 3.6370.26. The intensity of the tremor at 30, 60, 120 and 180 min following harmaline remained unchanged throughout this period. Treatment with riluzole in the doses of 2, 4 and 8 mg/kg resulted in a significant reduction in the intensity of the tremor at 30 min (270.22, 2.670.22, 1.670.2; Po0.001, dose 2, 4 and 8 mg/kg and respectively), 60 min (2.970.14, 2.370.21, 1.770.18; Po0.001, dose 2, 4 and 8 mg/kg and respectively), 90 min (370.26, 270.22; Po0.001, dose 4 and
8 mg/kg, respectively), 120 min (2.970.14; Po 0.05, dose 8 mg/ kg), 150 min (2.170.14; Po0.001, dose 8 mg/kg), 180 min (2.870.14, 2.570.22, 1.1 70.20; Po0.001, dose 2, 4 and 8 mg/ kg, respectively), 210 min (1.970.14; P o0.001, doses 2 mg/kg), 240 min (1.370.18; Po0.001, doses 2 mg/kg), 270 min (1.770.17; Po0.001, doses 2 mg/kg) (Fig. 1A). The total duration of tremors was significantly reduced by riluzole (Fig. 1B). In harmaline-alone-treated rats the mean duration of the tremor was 427 70.14 min, whereas treatment of animals with riluzole in the doses of 2, 4, and 8 mg/kg body weight reduced the duration to 26772.34, 23977.46 and 18871.89 min, respectively (Po0.001). Rats showed score 3 of ataxia in harmaline group and after riluzole treatment ataxia score reduced to 1.
3.1.2. Effect of riluzole on latencies to fall from the rotarod in harmaline -treated rats The rotarod test was used to assess balance and motor coordination in each group. The impaired motor function following harmaline injection was evidenced by poorer performance on a rotarod 2 h after treatment (day of treatment), 3 and 7 days later. Harmaline treated rats spent significantly less time (Po 0.001, n ¼9) walking on top of the rotating rod than control group indicating the severe motor impairment in harmaline treated rats (Fig. 2A). However, co-treatment with riluzole improved motor performance on rotarod when compared with harmaline treated rats (Po 0.01, 2 h after riluzole treatment in dose 4 and 8 mg/kg;
Fig. 1. Effect of riluzole on duration (A) and intensity (B) of harmaline induced tremor. Values are mean 7 S.E.M of nine rats, *Po 0.05, **P o0.001 and ***Po 0.001 as compared with harmaline alone treated animals by ANOVA followed by post-hoc comparison using Dunnett’s test. HR, harmaline; Ril, riluzole 2, 4 and 8 mg/kg. Times in xline indicate time after harmaline treatment.
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Fig. 2. Effect of riluzole on motor function in harmaline treated rats. Motor dysfunction was tested by the rotarod test (A), which measures balance, coordination, and motor control. Comparison of the rotarod performances between control and harmaline treated rats showed a significant motor impairment in harmaline treated group. In the open field test, harmaline treated rats also revealed that the distance traveled (B) and number of crossings (C) were significantly decreased as compared to control animals. Combined riluzole and harmaline improved motor performance on the rotarod as reflected in a longer time spent walking on rotating drum before falling (A). Riluzole treatment also increased the distance traveled (B), number of crossings in treated rats. Values are mean 7S.E.M of nine rats. * Po 0.05, ** Po 0.01 and *** Po 0.001 compared with control group. y P o 0.05, yy P o 0.01 and yyy P o0.001 compared with harmaline group.
Po0.001, 3 and 7 days after riluzole treatment, respectively) (Fig. 2A, n ¼9). There were no significant differences between harmaline and riluzole treated rats in rotarod performance in the next day after treatment.
3.1.3. Effect of riluzole on open field test in harmaline-treated rats Analysis of the total distance moved showed that harmaline injection alone produced locomotor hypoactivity as compared with control rats. In the days following treatment, ataxic rats showed significant decrease in the horizontal distance traveled (Po0.001).
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However, harmalineþriluzole treated animals showed significantly higher distance traveled compared to harmaline treated rats (Fig. 2B). Distance moved 2 h and 7 days after combined treatment with riluzole and harmaline were significantly increased as compared with harmaline injection alone (Po0.01, Po0.05; n¼9). In the open field test, harmaline significantly reduced the number of crossings in harmaline group than control group (Po0.001, n¼9), indicating a decreased locomotor spontaneous activity. Administration of riluzole improved the performance of the riluzoleþharmaline treated rats in the open-field task (2 h and 3 days after treatment, Po0.001, Po0.01 and Po0.05; n¼9) (Fig. 2C). 3.2. Histological assessment Purkinje cell bodies formed a continuous, uninterrupted layer in Nissl-stained sections from control rats (Fig. 3A). After treatment with harmaline, the Purkinje cell layer exhibited discontinuities
marked by absent Purkinje cell bodies (Fig. 3B) adjacent to zones with normal appearing cell bodies. However, in riluzole treated rats as in the control, Purkinje cell somas were arranged in a monolayer and formed a continuous, uninterrupted layer in Nissl-stained sections from control rats (compare Fig. 3C–E with A and B).
3.3. Measurements of cerebellar glutamate levels We measured levels of glutamate on 25 min and 2 h after treatment in vermis of cerebellum. The results showed that glutamate level significantly increased in harmaline treated rats as compared with control group in 25 min after treatment (Fig. 4A, Po0.05, n¼6). Also, there were no significant differences in riluzole treated rats than harmaline treated group. However, 2 h after treatment, glutamate level in riluzole treated rats (riluzole 4 and
Fig. 3. Loss of Purkinje cells after harmaline treatment and restoration following riluzole (dose 2, 4 and 8 mg/kg) treatment. After 7 days of treatment and termination of behavioral tests, cerebellar vermis areas were collected and coronal sections of cerebellar vermis were stained with cresyl violet (0.1%) for observation Purkinje cell under a light microscope. (A) Vermis from control rats; (B) harmaline treatment group. (C) Combined riluzole (2 mg/kg) and harmaline treatment. (D) Combined riluzole (4 mg/kg) and harmaline treatment. (E) Combined riluzole (8 mg/kg) and harmaline treatment. Purkinje cell layers showed a disconnected pattern, with a wide gap between the cells in harmaline treated rats than control group that relatively restored in riluzole treatment rats. Scale bars ¼ 50 mm.
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Fig. 4. Concentrations of glutamate in the cerebellar vermis of control, harmaline and riluzole treated rats. Measurement of glutamate content was done 25 min (A) and 2 h (B) after treatment. The concentration of glutamate increased significantly in harmaline treated rats compared to those of control rats 25 min and 2 h after the treatment. However, riluzole treated group (4 and 8 mg/kg) showed significant reduction in glutamate concentration in 2 h after treatment, but there was not significant decrease of the glutamate concentration in riluzole treated rats than harmaline group. Values are mean 7 S.E.M of six rats. P-value less than 0.05 (P o0.05) was considered to be statistically significant. * P o0.05 and ** P o 0.01 compared with control group. y Po 0.05 and yy P o 0.01 compared with harmaline group.
8 mg/kg) showed significant decrease in comparison to harmaline treated rats (Fig. 4B, Po0.01 and Po0.05, n¼6).
4. Discussion The results of this study showed that riluzole decreased the harmaline-induced action/postural tremor as well as ataxia and hypolocomotion. Reduction of harmaline-induced tremors was evident from decreased duration and severity of tremors in the rats treated with riluzole (Fig. 1A and B). Administration of harmaline to rats induced severe tremor as previously reported beginning within a few minutes, and lasted for at least 7 h (Biary et al., 2000). Harmaline produces tremors in the 8–12 Hz frequency range that is believed to be due to an enhancement of neuronal synchrony and rhythmicity in the inferior olive (Wilms et al., 1999). The harmaline model is of particular interest since it presents behavioral, metabolic imaging and pharmacological similarities to essential tremor. Furthermore, beta-carbolines related to harmaline, are detected in patients suffering from essential tremor and induce severe tremors in humans (Pennes and Hoch, 1957). Abnormal synchronous activation of climbing glutamatergic fibers, arising from the inferior olive and projecting to Purkinje
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cells is generally accepted to be a primary cause of the harmalineinduced tremor. The mechanisms by which harmaline and related beta-carbolines cause tremor have been investigated extensively. Harmaline induces rhythmic bursts of activity in inferior olivary neurons, which excite Purkinje cells via climbing fiber synapses leading to repetitive burst firing of Purkinje cells (De Montigny and Lamarre, 1973; Lamarre and Mercier, 1971; Llinas and Volkind, 1973). Harmaline increases and sustains rhythmic complex spike activity of these neurons (Lamarre et al., 1971; Beitz and Saxon, 2004), which leads to a suppression of their simple spike activity, triggered by parallel fibers (Lamarre et al., 1971). In consequence, rhythmic bursts appear in vestibulo-spinal and reticulo-spinal systems, as well as in a- and g-motoneurons (Lamarre et al., 1971; Lamarre and Weiss, 1973). We observed that tremor and ataxia gradually resolved over 6–8 h and spontaneous activity increased. Locomotion, motor activity and motor coordination appeared normal the next day after harmaline treatment but were abnormal 3 and 7 days after treatment. O0 Hearn and Molliver (1993) reported that tremor, ataxia and locomotion disappeared the next day after ibogaine and harmaline treatment. The acute tremor and ataxia that were transiently present minutes to hours after drug administration are likely due to a physiologic effect of excessive olivo-cerebellar stimulation rather than resulting from loss of Purkinje cells. Hence, the initial, transient expression of acute tremor and ataxia appears to reflect abnormally increased activity of Purkinje cells. In fact, any disturbance along cerebellar module is bound to result in similar symptoms due to a deficit in movement coordination. A decrease in Purkinje cell activity will decrease the amount of inhibition of the deep cerebellar nuclei (CN), which in turn will increase the inhibition of the olivary neurons, preventing the generation of the requested pattern. Likewise, increasing Purkinje cell activity will strongly inhibit the cerebellar nuclei neurons, leading to uncontrollable rhythmic firing of olivary neurons which might end up in tremor-like symptoms. In fact, one can predict that specifically activating the inhibitory projection neurons of the cerebellar nuclei will have similar results as reducing Purkinje cell activity. Furthermore, direct inhibition of olivary activity should also result in similar behavioral deficits. It is interesting to know that alcohol has an inhibitory effect on olivary activity (Lampl and Yarom, 1997). Indeed, uncoordinated movements under alcohol intoxication are reminiscent of ataxia. The reduction in Purkinje cell firing observed after alcohol administration (Van Skike et al., 2010) can, by removal of inhibition of the cerebellar nuclei neurons, also increase the inhibition of olivary activity. Biochemical and histological results of the present study indicated that harmaline significantly increased glutamate levels in the rat cerebellar vermis and caused Purkinje cells loss (Figs. 3 and 4A and B). These results are in accord with those of earlier reports that also demonstrated the Purkinje cell loss and enhancement glutamate levels induced after harmaline in the rat brain (Kim et al., 2003; Beitz and Saxon, 2004). We have assessed abnormal motor function in harmaline treatment group to monitor behavioral manifestations that are known to be associated with cerebellar neuronal damage (Breton et al., 1998; Ogura et al., 1980). Kim et al. (2003) demonstrated that a subset of Purkinje cell in the vermis and paravermis degenerated after harmaline treatment, but harmaline produced little or no Purkinje cell degeneration after inferior olivary ablation. They suggested that harmaline induced activation of inferior olivary neurons may lead to release of glutamate from climbing fiber synaptic terminal distributed over the Purkinje cells, and may lead to cytotoxic degeneration of Purkinje cells. The closely related alkaloid, ibogaine produce tremor and ataxia, which is quite similar to those induced by harmaline, following i.p. administration (O0 Hearn and
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Molliver, 1993). Purkinje cell degeneration was observed only at a later time point, well after the tremor has resolved, as also described for ibogaine (O0 Hearn and Molliver, 1997, 1999). Therefore, the reduction of motor activity and motor coordination recorded on 3 and 7 days after treatment are probably due to Purkinje cell loss. The present study highlighted the neurobehavioral evidence of harmaline administration is associated with a loss of cerebellar Purkinje cells and a related behavioral deficit in a manner that is prevented by riluzole. Riluzole reduced the tremogenic and the neurotoxic effects of harmaline. Experimental and clinical evidences implicated the role of glutamatergic system in essential tremor (Eblen et al., 1996; Ma´lly et al., 1996; Du et al., 1997). Current evidence is most consistent with an action of riluzole which causes a presynaptic reduction of transmitter release, rather than direct suppression of postsynaptic neurotransmitter receptor responses. Reduction of glutamate levels in vermis could be due to the enhancement of glutamate transporter uptake in Bergmann glia by riluzole. Glutamate uptake by rat spinal cord synaptosomes was increased by low concentrations (0.1–1 mM) of riluzole through a pertussis toxinsensitive mechanism (Azbill et al., 2000). As shown in Fig. 2, riluzole significantly reduced locomotion and number of crossings at dose 8 mg/kg. There is evidence that riluzole also may inhibit acetylcholine release in muscular endplate (Jehle et al., 2000), which may explain diminishing its beneficial effects on motor function. Therefore, in addition to glutamate neurotransmission, riluzole may affect other neurotransmitter system. Also, ataxia and hypolocomotion in harmaline treated rats are not a direct consequence of the tremor. In our study, tremor ended at 7 h after harmaline treatment in rats whereas ataxia remained in next days after harmaline treatment. Therefore, antagonism of ataxia and normalization of locomotion in harmaline treated rat is not a direct result from the antitremor effect of riluzole. Our results demonstrated that riluzole could decrease Glutamate levels in vermis of cerebellum. Also, vermis in riluzole treated rats (4 and 8 mg/kg) was normal, displaying an aligned Purkinje cell layer that was indistinguishable from control. Presumably, antiglutamatergic action of riluzole may counteract the neurotoxic glutamatergic effect of harmaline, and thus may prevent tremor and cerebellar neuronal death. Riluzoleinduced neuroprotection were observed in various animal models of injury (Malgouris et al., 1989; Stutzmann et al., 1997) and in neurodegenerative pathologies, such as Amyotrophic lateral sclerosis and Parkinson’s disease (Doble, 1999; Jacquin and Gruol, 1999; Noh et al., 2000). Janahmadi et al. (2009) reported that combined riluzole (4 mg/kg) and 3-acetylpyridine treatment preserved the Purkinje cells normal appearance and alignment, prevented the cell dispersion and promoted the survival of rat cerebellar Purkinje cells at the vermal level of central lobules when compared to 3-acetylpyridine treatment alone. It also preserved the most intrinsic electrophysiological properties of Purkinje cells and partially improved motor performance. In conclusion, we suggest that riluzole might be a potentially useful choice in the treatment of essential tremor and ataxia. Moreover, our data suggest that the role of the glutamatergic system in harmaline-induced tremor and also protective effect of riluzole may be related to its inhibitory effect on glutamatergic neurotransmission. Therefore, role of the glutamatergic system in harmaline-induced tremor should be further investigated in more details.
Acknowledgment We acknowledge Damghan University for supporting this work.
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