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. 2015 Oct 13;22(1):53–62. doi: 10.1111/cns.12467

Pramipexole at a Low Dose Induces Beneficial Effect in the Harmaline‐induced Model of Essential Tremor in Rats

Barbara Kosmowska 1, Jadwiga Wardas 1, Urszula Głowacka 1, Subramaniam Ananthan 2, Krystyna Ossowska 1,✉
PMCID: PMC6492867  PMID: 26459182

Summary

Aims

The aim of the study was to examine the effects of preferential agonists of dopamine D3 receptors: pramipexole and 7‐OH‐DPAT on the harmaline‐induced tremor in rats (a model of essential tremor, ET). To study receptor mechanisms of these drugs, rats were pretreated with dopamine D3 receptor antagonists—SB‐277011‐A and SR‐21502, an antagonist of presynaptic D2/D3 receptors—amisulpride, or a nonselective antagonist of D2‐like receptors, haloperidol, at a postsynaptic dose.

Methods

For tremor measurement, fully automated force plate actimeters were used and data were analyzed using fast Fourier transform.

Results

Harmaline (15 mg/kg ip)‐triggered tremor was manifested by an increase in the power within 9–15 Hz band (AP2). Pramipexole administered at a low (0.1 mg/kg sc), but not higher doses (0.3 and 1 mg/kg sc), and 7‐OH‐DPAT (0.1, 0.3, and 1 mg/kg sc) reversed the harmaline‐increased AP2. None of the examined dopamine antagonists: SB‐277011‐A (10 mg/kg ip), SR‐21502 (15 mg/kg ip), haloperidol (0.5 mg/kg ip), or amisulpride (1 mg/kg ip) influenced the above effect of dopamine agonists.

Conclusion

The present study indicates that pramipexole reduces the harmaline‐induced tremor, which may suggest its beneficial effects in ET patients. However, mechanisms underlying its action are still unclear and need further examination.

Keywords: Cerebellum, Dopamine receptors, Essential tremor, Harmaline‐induced tremor, Pramipexole

Introduction

Essential tremor (ET) is one of the most common motor disorders in humans, current medications for which are still limited and only partially effective. Pathophysiological mechanisms underlying ET are complex and poorly understood. However, based on clinical data and animal models, hyperactivity of the olivocerebellar circuit has been proposed to be crucial 1.

Harmaline, a β‐carboline derivative induces tremor similar to ET which is generally accepted as a model of this disorder in animals. The harmaline tremor is kinetic/postural in different animal species with peak frequency of 10–12 Hz in rats 2. It has been suggested that its primary cause is synchronous activation of glutamatergic climbing fibers arising from the inferior olive which project to Purkinje cells (PCs) of the cerebellar cortex 2, increase in glutamate release in the cerebellum 3, and enhancement of complex spike activity of PCs 4.

Several studies have indicated that cerebellar functions may be modulated by dopaminergic transmission. First, the cerebellar cortex receives sparse dopaminergic input from the mesencephalon 5. Moreover, although levels of dopamine D1, D2, D4, and D5 receptors are low in the cerebellum, D3 receptors are abundant in this structure, especially in PCs and their dendrites in lobules 9 and 10 6. Additionally, PCs, which are GABA‐ergic neurons, express also tyrosine hydroxylase 7, dopamine transporter (DAT) 8, and release dopamine, which excites them via dopamine D3 autoreceptors 8. Modulatory influence of dopamine on cerebellar neurons has been supported by an increase in c‐fos expression induced by systemic injections of D3 preferring agonist, 7‐OH‐DPAT 9. Moreover, lesions of dopaminergic nigrostriatal pathway have been found to induce persistent activation of PCs 10. The above effects may result directly from alterations of dopaminergic transmission in the cerebellum 11, or be secondary to those in cerebral structures, for example, basal ganglia, which are relayed to the cerebellum via polysynaptic pathways 12.

In addition to its stimulatory action on the olivo‐cerebellar climbing fibers, harmaline is a monoamine oxidase‐A (MAO‐A) inhibitor 13. Potential significance of increased dopaminergic transmission for the harmaline‐induced tremor seems to be supported by our recent study which showed an enhancement of this phenomenon by systemic injections of a nonselective dopamine receptor agonist, apomorphine, in rats 14.

Although not confirmed by all studies, the Ser9Gly polymorphism of the D3 dopamine receptor gene, which increases dopamine affinity to this receptor, has been suggested to be a risk factor of ET 15. A potential role of D3 receptors in ET has been supported by a study which showed beneficial effect of a preferential D3 receptor agonist—pramipexole 16, 17, 18, 19 in a small number of ET patients in an open‐label study 20. Taking into account a potential therapeutic significance of D3 receptors in ET, this study was undertaken to examine the influence of pramipexole, and another preferential agonist of these receptors, 7‐OH‐DPAT 17, 21, 22, 23, 24, on the harmaline‐induced tremor in rats. Furthermore, in order to gain insight into receptor mechanisms that contribute to the pharmacological actions of these compounds, we investigated the effects of administration of different dopamine D2 and D3 receptor antagonists.

Methods

Animals

The experiments were carried out in compliance with the Animal Experiments Bill of January 21, 2005 (published in Journal of Laws no. 33/2005 item 289, Poland), and according to the EC Directive 86/609/EEC on the protection of animals used for scientific purposes. Additionally, the experiments were approved by the Local Ethics Committee. All efforts were made to minimize the number and suffering of animals used. Male Wistar rats weighing 240–350 g prior to the experiments were kept under a 12/12‐h light/dark cycle (the light on from 7 am to 7 pm) with free access to food and water. All experiments were carried out during the light period. A total of 281 rats were used.

Drugs

Harmaline hydrochloride dihydrate (Sigma‐Aldrich, St. Louis, MO, USA) was administered at a dose of 15 mg/kg ip. immediately before tremor measurements which lasted 60 min. Pramipexole dihydrochloride (Abcam, Cambridge, UK; 0.1, 0.3 or 1 mg/kg sc) or 7‐OH‐DPAT [(±)‐7‐Hydroxy‐2‐(di‐n‐propylamino)tetralin] hydrobromide (Sigma‐Aldrich; 0.1, 0.3 or 1 mg/kg sc) was injected either alone or with harmaline. Pramipexole and 7‐OH‐DPAT have been reported to be present in the brain 25, 26 and to occupy dopamine D3 receptors 27, 28 between 20 (or 30) and 120 min after their systemic administration. Based on these reports, pramipexole and 7‐OH‐DPAT were injected 30 min before harmaline. Moreover, sc route was chosen for the administration of both drugs as was carried out in some earlier studies [e.g., 25, 28], to minimize suffering of animals by multiple injections at the same place.

Two selective antagonists of dopamine D3 receptors: (1) SB‐277011‐A [trans‐N‐[4‐[2‐(6‐cyano‐1,2,3,4‐tetrahydroisoquinolin‐2‐yl)ethyl]cyclohexyl]‐4‐quinolinicarboxamide] dihydrochloride (Abcam; 10 mg/kg ip) 27, 29, 30 and (2) SR‐21502 [N‐(4‐(4‐(2‐(tert‐butyl)‐6‐(trifluoromethyl)pyrimidin‐4‐yl)piperazin‐1‐yl)butyl)imidazol[1,2‐α]pyridine‐2‐carboxamide] dihydrochloride (Southern Research Institute, Birmingham, AL, USA; 15 mg/kg ip) 31, 32, 33, a nonselective antagonist of dopamine D2‐like receptors, haloperidol 34 (Warszawskie Zakłady Farmaceutyczne, Polfa, Warszawa, Poland, ampules a 5 mg/1 mL aqua pro injectione; 0.5 mg/kg ip), and an antagonist of presynaptic D2/D3 autoreceptors, amisulpride (Tocris Bioscience, Bristol, UK; 1 mg/kg ip) 34, 35, were used. Physiological saline was used for all control injections.

Harmaline, pramipexole, 7‐OH‐DPAT, and SR‐21502 were dissolved in redistilled water. SB‐277011‐A was dissolved in 25% w/v solution of 2‐hydroxypropyl‐β‐cyclodextrin (Sigma‐Aldrich). Amisulpride was dissolved in redistilled water with addition of a drop of HCl 0.1N.

SR‐21502 was administered 10 min, SB‐277011‐A or haloperidol 60 min, and amisulpride 30 min, before pramipexole or 7‐OH‐DPAT.

The doses of the above antagonists, their route of administration (ip), and the lapse of time between their injections and those of dopamine agonists were chosen according to occupation of dopamine D3 receptors ex vivo (SB‐277011‐A) 27, 30, induction of catalepsy as a measure of the blockade of postsynaptic dopamine D2 receptors (haloperidol, our own results), inhibition of pharmacological effects of pramipexole (amisulpride) 36, or cocaine (SR‐21502) 31, 32.

Force Plate Actimeters

Professor Stephen C. Fowler and Troy Zarcone of the University of Kansas (Lawrence, KS) developed the force plate actimeters (BASi). The concept and applications of this instrument for measurement of tremor and locomotor activity in rodents were described in the USA and pending international patents (USA Patent No. 6,601,010), as well as in published research 37.

Immediately after harmaline injections, rats were placed in force plate actimeters. An animal was placed on a 44 cm × 44 cm plate covered by a Plexiglas enclosure (33 cm height) and put into a ventilated sound‐attenuating chamber. The force plate actimeters tracked the rat movements across a plate. Four force transducers below the corners of the plate recorded its position on a Cartesian plane and measured the force exerted on the plate at each time point. Data were collected and stored during time units of 10.24 s (“frames”) with the sampling frequency of 100 points per second and accompanying software analyzed specific behaviors of interest.

Tremor was analyzed using fast Fourier transform (FFT) on each frame of the experiment. Then, the resulting power spectra were log base 10‐transformed and averaged over 2 consecutive 180‐frame series [two time periods of ca. 30 min each (30.72 min)] to give the following parameters: AP1, averaged power in frequency band I (0–8 Hz) and AP2, averaged power in frequency band II (9–15 Hz) (Figure 1). The total distance traveled during two consecutive 180‐frame series in millimeters was used as a measure of locomotor activity. Because vibration noise causes the measured position of the animal to fluctuate, this parameter could be artificially increased.

Figure 1.

Figure 1

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Effect of pramipexole (A) and 7‐OH‐DPAT (B) on the power spectrum related to the harmaline‐induced tremor. The power spectrum within a range of 0–25 Hz averaged for the first period of measurement (0–30 min) for all animals is shown. AP1, power in the 0–8 Hz band; AP2, power in the 9–15 Hz band; SAL, controls; HARM, harmaline 15 mg/kg; PRA 0.1, 0.3, and 1.0, pramipexole 0.1, 0.3, and 1.0 mg/kg; 7‐OH‐DPAT 0.1, 0.3, and 1.0, 7‐OH‐DPAT 0.1, 0.3, and 1.0 mg/kg. Number of rats: (A) SAL, n = 28; HARM, n = 44; PRA 0.1 + HARM, n = 41; PRA 0.3 + HARM, n = 10; PRA 1.0 + HARM, n = 8; (B) SAL, n = 28; HARM, n = 35; 7‐OH‐DPAT 0.1 + HARM, n = 23; 7‐OH‐DPAT 0.3 + HARM, n = 10; 7‐OH‐DPAT 1.0 + HARM, n = 10.

Statistics

Statistical analyses were carried out by ANOVA for repeated measures followed by LSD post hoc test using the software Statistica v.10 (StatSoft Inc., Tulsa, OK, USA).

Results

The Harmaline‐induced Tremor in Rats

The dose of harmaline (15 mg/kg ip) and time of tremor measurement were chosen according to our previous studies 14, 38. Harmaline at this dose induces generalized tremor which appears already a few minutes after administration and is evidenced by a quick increase (within first 10 min) in power within frequency band of 9–15 Hz (AP2), level of which is relatively stable for at least 90 min 14, 38. In agreement with the above studies, the present results showed that harmaline increased AP2 and lowered the averaged power within frequency band of 0–8 Hz (AP1) (Figures 1, 2, 3, 4, 5, 6).

Figure 2.

Figure 2

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Effect of pramipexole on the distance traveled (A) and the harmaline‐altered AP1 (B) and AP2 (C). AP1, power in the 0–8 Hz band; AP2, power in the 9–15 Hz band; SAL, controls; HARM, harmaline 15 mg/kg; PRA 0.1, 0.3, and 1.0, pramipexole 0.1, 0.3, and 1.0 mg/kg. Number of animals in (A) SAL, n = 10; PRA 0.1, n = 10; SAL, n = 10; PRA 0.3, n = 11; SAL, n = 8; PRA 1.0, n = 9. Number of animals in (B and C) SAL, n = 10; HARM, n = 10; PRA 0.1 + HARM, n = 10; SAL, n = 10; HARM, n = 10; PRA 0.3 + HARM, n = 10; SAL, n = 8; HARM, n = 12; PRA 1.0 + HARM, n = 8. Statistics: ANOVA for repeated measures + LSD post hoc test. **P ≤ 0.01, ***P ≤ 0.001, a0.05 < P < 0.1 versus SAL; ## P ≤ 0.01, b0.05 < P < 0.1 versus HARM.

Figure 3.

Figure 3

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Influence of antagonists of dopamine D3 receptors: SB‐277011‐A (A) and SR‐21502 (B) on the tremorlytic effects of pramipexole. AP1, power in the 0–8 Hz band; AP2, power in the 9–15 Hz band; SAL, controls; HARM, harmaline; PRA, pramipexole (0.1 mg/kg); SB, SB‐277011‐A (10 mg/kg); SR, SR‐21502 (15 mg/kg). Number of animals in (A) SAL, n = 8; HARM, n = 12; PRA + HARM, n = 10; SB + PRA + HARM, n = 8. Number of animals in (B) SAL, n = 10; HARM, n = 14; PRA + HARM, n = 13; SR + PRA + HARM, n = 10. Statistics: ANOVA for repeated measures + LSD post hoc test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 versus SAL; ## P ≤ 0.01, ### P ≤ 0.001 versus HARM.

Figure 4.

Figure 4

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Influence of antagonists of dopamine D2‐like receptors: haloperidol (A) and amisulpride (B) on the tremorlytic effects of pramipexole. AP1, power in the 0–8 Hz band; AP2, power in the 9–15 Hz band; SAL, controls; HARM, harmaline; PRA, pramipexole (0.1 mg/kg); HALO, haloperidol (0.5 mg/kg); AMI, amisulpride (1 mg/kg). Number of animals in (A) SAL, n = 10; HARM, n = 14; PRA + HARM, n = 13; HALO + PRA + HARM, n = 8. Number of animals in (B) SAL, n = 10; HARM, n = 8; PRA + HARM, n = 8; AMI + PRA + HARM, n = 8. Statistics: ANOVA for repeated measures + LSD post hoc test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, a0.05 < P < 0.1 versus SAL; # P ≤ 0.05, ## P ≤ 0.01, ### P ≤ 0.001 versus HARM; & P ≤ 0.05 versus PRA + HARM.

Figure 5.

Figure 5

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Effect of 7‐OH‐DPAT on the harmaline‐altered AP1 (A) and AP2 (B). AP1, – power in the 0–8 Hz band; AP2, power in the 9–15 Hz band; SAL, controls; HARM, harmaline 15 mg/kg; 7‐OH‐DPAT 0.1, 0.3, and 1.0, 7‐OH‐DPAT 0.1, 0.3, and 1.0 mg/kg. Number of animals: SAL, n = 8; HARM, n = 12, 7‐OH‐DPAT 0.1 + HARM, n = 10; SAL, n = 10; HARM, n = 9, 7‐OH‐DPAT 0.3 + HARM, n = 10; SAL, n = 10; HARM, n = 9, 7‐OH‐DPAT 1.0 + HARM, n = 10. Statistics: ANOVA for repeated measures + LSD post hoc test. **P ≤ 0.01, *** P ≤ 0.001, versus SAL; # P ≤ 0.05, ## P ≤ 0.01, ### P ≤ 0.001, versus HARM.

Figure 6.

Figure 6

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Effect of antagonists of dopamine receptors: SR‐21502 (A) and haloperidol (B) on the 7‐OH‐DPAT‐induced tremorlytic effect. AP1, power in the 0–8 Hz band; AP2, power in the 9–15 Hz band; SAL, controls; HARM, harmaline; 7‐OH‐DPAT, 7‐OH‐DPAT (0.1 mg/kg); SR, SR‐21502 (15 mg/kg); HALO, haloperidol (0.5 mg/kg). Number of animals in (A) SAL, n = 10; HARM, n = 14; 7‐OH‐DPAT + HARM, n = 13; SR + 7‐OH‐DPAT + HARM, n = 10. Number of animals in (B) SAL, n = 10; HARM, n = 14; 7‐OH‐DPAT + HARM, n = 13; HALO + 7‐OH‐DPAT + HARM, n = 8. Statistics: ANOVA for repeated measures + LSD post hoc test. test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 versus SAL; # P ≤ 0.05, ## P ≤ 0.01, b0.05 < P < 0.1 versus HARM.

Effect of Pramipexole on the Locomotor Activity of Rats

Measurements of rats' locomotion started 30 min after pramipexole injections. Pramipexole (0.1 mg/kg) decreased the total distance traveled during the first 30 min of the measurement, but between 30 and 60 min, it lost its effectiveness (treatment effect F(1,18) = 51.2, P = 0.000; treatment × time interaction F(1,18) = 44.5, P = 0.000). Moreover, a significant treatment effect (F(1,15) = 6.9, P = 0.019) and treatment × time interaction (F(1,15) = 54.0, P = 0.000) for pramipexole (1 mg/kg), as well as treatment × time interaction (F(1,19) = 55.7, P = 0.000) for pramipexole (0.3 mg/kg), were found. These effects of both doses (0.3 and 1 mg/kg) were related to significant increases in the distance traveled (LSD post hoc) during 30–60 min of measurement. The dose of 0.3 mg/kg tended (0.05 < P < 0.1, LSD post hoc) additionally to lower locomotion of rats during first 30 min, while the dose of 1 mg/kg was ineffective within this period of time (Figure 2A).

Effect of Pramipexole on the Harmaline‐induced Tremor

Pramipexole was administered 30 min before harmaline and the power within the frequency band of 9–15 Hz (AP2) was analyzed immediately after the latter agent injection. ANOVA for repeated measures indicated significant treatment effect (F(2,27) = 5.0–8.1, F(2,25) = 10.0; P = 0.001–0.01) and time effect (F(2,27) = 4.4–9.8, F(2,25) = 7.3; P = 0.004–0.05). Additional individual comparisons by LSD post hoc test showed that harmaline increased AP2 during the whole measurement period (0–60 min). Pramipexole at the dose of 0.1 mg/kg significantly reversed the harmaline effect during the first 30 min of measurement and tended (0.05 < P < 0.1) to do that also between 30 and 60 min. In contrast, higher doses (0.3 and 1 mg/kg) of pramipexole were ineffective in this respect (Figure 2C).

Significant treatment effect (F(2,27) = 9.6–10.9, F(2,25) = 10.1; P = 0.000–0.001), time effect (F(2,27) = 40.9–49.0, F(2,25) = 36.1; P = 0.000), and treatment × time interaction (F(2,27) = 22.6–22.8, F(2,25) = 28.5; P = 0.000) were also observed in the power within 0–8 Hz frequency band (AP1). Harmaline decreased AP1 during the first 30 min which was not significantly affected by pramipexole (Figure 2B).

Effect of Selective Dopamine D3 and Nonselective D2‐like Antagonists on Tremorlytic Effects of Pramipexole

As only the lowest dose of pramipexole (0.1 mg/kg) decreased the power within frequency band related to the harmaline‐induced tremor (AP2), receptor mechanisms of its tremorlytic efficiency were examined further.

ANOVA for repeated measures showed significant treatment effect (F(3,34) = 8.8–40.7, F(3,43) = 3.2–31.9, F(3,41) = 3.7–26.0, F(3,30) = 19.1; P = 0.000–0.03), and/or treatment × time interaction (F(3,34) = 6.3–50.0, F(3,43) = 3.5–113.5, F(3,41) = 3.4–88.5, F(3,30) = 5.8–72.3, P = 0.000–0.03) with regard to both AP2 and AP1. LSD post hoc test revealed significant harmaline‐induced increases in AP2 and decreases in AP1. Pramipexole reversed the harmaline‐induced enhancement of AP2 during the first 30‐min period of measurement and deepened the harmaline‐induced loss of AP1, but in one experiment (with SB‐277011‐A), only. Neither D3 (SB‐277011‐A, SR‐21502) nor D2‐like (haloperidol, amisulpride) receptor antagonists influenced the pramipexole‐induced effects in the harmaline‐treated rats (Figures 3 and 4). However, in the second (30–60 min) period, joint treatments with pramipexole and haloperidol or pramipexole and amisulpride reversed the harmaline‐increased AP2 (Figure 4).

Effect of 7‐OH‐DPAT on the Harmaline‐induced Tremor

7‐OH‐DPAT was administered, like pramipexole at three doses (0.1, 0.3, and 1 mg/kg), 30 min before harmaline. ANOVA for repeated measures indicated significant treatment (F(2,27) = 16.43, F(2,26) = 5.71–5.79, P = 0.000–0.009), and time effect (F(1,27) = 14.84, F(1,26) = 11.22–11.31, P = 0.001–0.002) with regard to AP2 and LSD post hoc analysis showed increases in AP2 by harmaline (Figure 5B) and reversal of this effect by all three doses of 7‐OH‐DPAT (Figure 5B) during the first 30 min of measurement. The effect of the lowest dose of 7‐OH‐DPAT lasted longer and was visible also during the second period of measurement (30–60 min).

Significant treatment effect (F(2,27) = 34.53, F(2,26) = 28.52–29.24, P = 0.000) and time effect (F(1,27) = 106.75, F(1,26) = 79.78–81.99, P = 0.000) were noted for AP1. Harmaline lowered this parameter and 7‐OH‐DPAT at the lowest dose deepened this harmaline‐induced effect (Figure 5A).

Effect of SR‐21502 and Haloperidol on Tremorlytic Effects of 7‐OH‐DPAT

Like in the aforementioned results, significant treatment effect was discovered for both AP1 and AP2 (F(3,43) = 3.1–28.3, F(3,41) = 2.7–27.8; P = 0.000–0.05), and harmaline increased AP2 and decreased AP1 (Figure 6). Moreover, LSD post hoc test revealed that 7‐OH‐DPAT administered at a dose of 0.1 mg/kg sc 30 min before harmaline reversed the enhancement of AP2 induced by the latter compound during the first 30 min of the measurement (Figure 6). Neither SR‐21502 nor haloperidol significantly influenced the above effects of 7‐OH‐DPAT in harmaline‐treated rats (Figure 6).

Discussion

The present study shows that a low (0.1 mg/kg sc) dose of pramipexole reduced the harmaline‐induced tremor in rats. Pramipexole significantly decreased the power in the frequency band 9–15 Hz (AP2), characteristic for tremor induced by this agent. Similar effect was observed by us earlier after administration of propranolol 14 [the first‐line treatment of ET 1. The pramipexole tremorlytic effect was repeatable from one experiment to another. However, it was not present after higher doses (0.3 and 1 mg/kg sc) of this drug.

Pramipexole is a well‐known antiparkinsonian drug, which among other symptoms, inhibits tremor at rest 39. This drug at a dose of 1–4 mg/kg inhibited also tremulous jaw movements induced by pilocarpine in rats which was supposed to model parkinsonian tremor 40. Moreover, a recent study has reported that pramipexole reduced tremor in a small number of ET patients 20. As the harmaline‐induced tremor has long been accepted to be an animal model of ET 2, the present study seems to substantiate potential usefulness of pramipexole to treat ET, and encourage continuation of clinical trials with this drug.

However, mechanisms underlying tremorlytic action of a low dose of pramipexole are still unclear. Pramipexole is a dopamine D3 receptor preferring agonist which binds to these receptors in low nanomolar concentrations (Ki or KD = 0.2–10 nM; 16, 17, 18, 19. Depending on binding conditions, its affinity to D3 versus D2 receptors was reported to be 6–95 times higher 16, 17, 19. As D3 receptors are abundant in the cerebellum 6, the structure crucial for the harmaline‐induced tremor 4, and regulate excitability of Purkinje cells 8, we initially assumed that tremorlytic pramipexole action may result from its agonistic effect on these receptors. This hypothesis seemed to be supported further by the finding that the same dose of this drug administered in rats occupied D3 receptors in lobules 9 and 10 of the cerebellum, but not dopamine D2 receptors in the basal ganglia 27. Moreover, the present study shows that 7‐OH‐DPAT, the agonist similar to pramipexole in affinity to D3 receptors (Ki or IC50 = 0.2–4 nM) and preference to D3 versus D2 receptors (5–100 times higher) 17, 21, 22, 24, also reduced tremor induced by harmaline (lowered AP2). The contribution of D3 receptors to this effect of 7‐OH‐DPAT also seemed probable, because this drug was administered at low doses which occupy D3 receptors in vivo 28.

However, administration of two different selective antagonists of D3 receptors, SB‐277011‐A and SR‐21502, did not influence the aforementioned tremorlytic effects of pramipexole and 7‐OH‐DPAT. Both these compounds bind to dopamine D3 receptors with low nanomolar affinities ~ 100 times higher than to D2 receptors 29, 31, 33. The doses of SB‐277011‐A and of SR‐21502, as well as times of their injections before tremor measurements, were chosen according to previous studies which showed that they occupied >90% D3, but no D2 receptors in vivo 27, 30, and/or induced several pharmacological effects in rats 30, 31, 32, 41. The lack of any influence of these antagonists on tremorlytic effect of pramipexole and 7‐OH‐DPAT seemed to negate any contribution of dopamine D3 receptors to it.

The present study, as well as earlier papers 27, 36, 42, 43, 44, 45, showed that pramipexole and 7‐OH‐DPAT administered at the dose of 0.1 mg/kg inhibited locomotor activity of rats (decreased the distance traveled). The role of D3 receptors in hypomotility, as well as in other effects (e.g., inhibition of operant behavior, a decrease in body temperature), induced by low doses of the above drugs has been questioned by others. They were not antagonized by SB‐277011‐A, and other D3 receptor antagonists in rats and mice, or did not disappear in D3 knockout mice 27, 36, 43, 44, 46. Therefore, the role of D3 receptors in different behaviors induced by low doses of pramipexole and 7‐OH‐DPAT (including their tremorlytic effect) seems obscure.

Earlier studies have suggested that hypomotility and yawning induced by low doses of D3/D2 agonists may result from its action on D3/D2 autoreceptors 35, 36, 42, 47. This suggestion was supported by the finding that both pramipexole and 7‐OH‐DPAT inhibited firing of dopamine neurons, dopamine synthesis, metabolism and release, or DOPA accumulation 17, 45, 48.

The role of stimulation of dopamine autoreceptors in tremorlytic action of pramipexole and 7‐OH‐DPAT seemed likely because our recent study showed an opposite effect, that is, enhancement of the harmaline‐induced tremor after administration of postsynaptic doses of a nonselective agonist, apomorphine 14. However, data concerning pre‐ versus postsynaptic doses of D3 receptor antagonists have not been available, so far. Although SB‐277011‐A was reported to inhibit yawning induced by another D3/D2 receptor agonist (PD‐128,907) 47, its effective doses (32 and 56 mg/kg) were much higher than could be accepted as presynaptic. Therefore, we tried to antagonize the tremorlytic effect of pramipexole with amisulpride. Amisulpride binds to dopamine D2 and D3 receptors with similar nanomolar affinity 34 and is considered to be an antagonist of D2 and D3 autoreceptors in vivo which inhibits both biochemical and behavioral effects of presynaptic doses of dopamine agonists 34, 35. Among other behaviors, amisulpride inhibited hypomotility induced by a low dose of pramipexole in mice 36. In the present study, however, amisulpride did not antagonize the tremorlytic effect of pramipexole. Moreover, a joint treatment with this drug and pramipexole lowered AP2 enhanced by harmaline in the second time period of measurement (30–60 min), when pramipexole given alone lost already its effectiveness. The latter result suggests synergistic, rather than antagonistic, effect of both drugs on the harmaline‐induced tremor.

We examined additionally the influence of haloperidol, a nonselective antagonist of D2‐like receptors 34, administered at a postsynaptic dose (0.5 mg/kg), proven by an appearance of catalepsy before tremor measurements (data not shown), on the pramipexole and 7‐OH‐DPAT‐induced tremorlytic effects, and this treatment was again ineffective.

Surprisingly, pramipexole lost its effectiveness against the harmaline‐induced tremor when administered at higher doses (0.3 and 1 mg/kg). However, the dose of 1 mg/kg of this agonist has been reported to occupy in vivo not only D3, but ~ 80% of D2 dopamine receptors, as well 27. Moreover, the present study, as well as earlier papers 42, shows that in accordance with their agonistic action on postsynaptic dopamine D2 receptors, the above higher doses of pramipexole stimulated locomotor activity of rats. Because, as mentioned above, our previous study has shown that a nonselective agonist of dopamine D1/ D2‐like receptors, apomorphine, in postsynaptic doses inducing locomotor hyperactivity, increased the tremor induced by harmaline 14, it may be speculated that D2 agonistic component of higher doses of pramipexole may counteract its potential tremorlytic effect. As a result, both these higher doses were ineffective. This conclusion seems to be supported by the present finding showing that in contrast to pramipexole, 7‐OH‐DPAT at higher doses (0.3 and 1 mg/kg) also decreased the harmaline‐elevated AP2. However, these doses have been reported not to increase locomotor activity of rats 45 and only the highest one (1 mg/kg) occupied dopamine D2 receptors in vivo, but no more than 15% of them 49.

Taking into account the lack of any influence of the aforementioned pre‐ and postsynaptic D3 and D2 antagonists on the effect of pramipexole and 7‐OH‐DPAT in the harmaline model, its underlying mechanism remains unclear. Both agonists administered at low doses in animals 25, 26, 50 and at clinical doses in humans 51 appear in plasma or brain extracellular fluid in nanomolar concentrations. In vitro studies have shown that either pramipexole does not bind to D1, D5, α1, β1,2, α2A,B,, 5‐HT1A,B,D, 5‐HT2A,B,C, and H1 receptors, or binds in high nanomolar or micromolar concentrations 17, 19, which do not seem to be achieved in vivo. Similarly, 7‐OH‐DPAT does not bind in nanomolar concentrations to dopamine D1, α1, α2 receptors, but in contrast to pramipexole, it has relatively high (nanomolar) affinity to 5‐HT1A and sigma receptors 17, 23. As, however, pramipexole and 7‐OH‐DPAT tremorlytic effects were similar, they should share similar mechanisms, as well. Therefore, contribution of all the above receptors to tremorlytic effects of the above drugs seems doubtful. Contrariwise, binding of pramipexole and 7‐OH‐DPAT with nanomolar affinity to dopamine D4 receptors 17, 19 may be another, still not examined, mechanism involved in this effect. Moreover, in vitro studies have indicated that dopamine receptors belonging to the D2‐like family may interact with each other forming D2‐D3 and D2‐D4 heteromers 52. Although functional in vivo importance of these heteromers is unknown, they exhibit different pharmacological characteristics in comparison with their constituent monomers. While haloperidol shows comparative actions at D2‐D3 heteromers versus D2 receptors, pramipexole acts with amplified potency at these heteromers in comparison with D2 or D3 and increase their pool, and partial agonists lose their intrinsic activity and transforms to antagonists 52. Whether the existence of the above heteromers may explain insensitivity of some behavioral actions of dopamine preferential agonists (including tremorlytic effects) to dopamine antagonists is unknown, and answering this question may be a matter of future investigations. As, however, nondopaminergic (mitochondrial) mechanisms have already been proposed to contribute to some pramipexole effects (neuroprotection) 53, 54, it cannot be excluded that the tremorlytic actions of this drug and 7‐OH‐DPAT were not related to their agonistic action on dopamine receptors, either.

Summing up, the present study shows that the dopamine agonist pramipexole reduces the harmaline‐induced tremor, which may suggest its beneficial effects in ET patients. However, mechanisms underlying its action are still unclear and need further examination.

Conflict of Interest

The authors declare no conflict of interest.

Acknowledgment

The study was supported by Statutory Funds of the Department of Neuro‐Psychopharmacology, Institute of Pharmacology, Polish Academy of Sciences, Kraków, Poland, and partly by the grant of the National Science Centre OPUS 6, 2013/11/B/NZ4/04565. We also wish to gratefully acknowledge the support from the National Institute on Drug Abuse (NIDA), National Institutes of Health (NIH), USA, by a Grant R01DA024675 to S.A. for the synthesis of SR‐21502. Barbara Kosmowska is a holder of scholarship from the KNOW sponsored by Ministry of Science and Higher Education, Republic of Poland.

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