Mechanisms Underlying Dopaminergic Regulation of Nicotine-Induced Kinetic Tremor

Nicotine induces kinetic tremor, which resembles pharmacological features of essential tremors, via activating the inferior olive (IO) neurons. Since nicotine is known to enhance dopamine release by stimulating α4β2 and/or α6 nACh receptors, we examined the effects of various dopamine receptor ligands on nicotine-induced tremor to clarify the role of the dopaminergic system in modulating nicotine tremor. A tremorgenic dose of nicotine increased the dopamine level in the pons and medulla oblongata (P/MO), and the levels of dopamine metabolites in the hippocampus, P/MO, and striatum. Treatment of animals with the D1/5 agonist SKF-38393 inhibited the induction of nicotine tremor, whereas the D3 agonist PD-128,907 facilitated nicotine-induced tremor. The D2 agonist sumanirole showed no effect. In addition, nicotine tremor was significantly enhanced by the D1/5 antagonist SCH-23390 and inhibited by the D3 antagonist U-99194. Neither the D2 (L-741,626) nor D4 (L-745,870) antagonist affected the generation of nicotine tremor. Furthermore, microinjection of U-99194 into the cerebellum significantly inhibited nicotine-induced tremor, whereas its injection into IO or the striatum did not affect tremor generation. Although intrastriatal injection of SCH-23390 showed no effects, its injection into IO tended to enhance nicotine-induced tremor. The present study suggests that dopamine D3 and D1/5 receptors regulate the induction of nicotine tremor in an opposite way, D3 receptors facilitately and D1/5 receptors inhibitorily. In addition, the cerebellar D3 receptors may play an important role in modulating the induction of nicotine tremor mediated by the olivo-cerebellar system.

Tremor is an involuntary and rhythmic movement disorder which appears in different parts of the body. According to the clinical manifestation, tremor can be classified into several types: 1) resting tremor occurring in the resting state of the limb, 2) kinetic tremor which appears during movement of parts of the body, 3) postural tremor which occurs when the body part is voluntarily maintained against gravity (holding a specific posture) (Puschmann and Wszolek, 2011;Kosmowska and Wardas, 2021). Resting tremor is one of the typical symptoms of Parkinson's disease, while kinetic and postural tremors are frequently manifested in patients with essential tremor. In addition, several drugs, discretionary products (e.g., coffee and cigarettes), and toxins are known to induce tremor.
Previous studies reported that cigarette smoking and oral nicotine induce hand tremor (Lippold et al., 1980;Shiffman et al., 1983;Louis, 2007;Lin et al., 2012) and worsen essential tremor (Marshall and Schnieden, 1966). We previously showed that nicotine at relatively low doses (0.5-1 mg/kg, i.p.) evokes kinetic tremor in rodents Kunisawa et al., 2017;Kunisawa et al., 2018). Immunohistochemical analysis of Fos protein expression, a biological marker of neural excitation , revealed that tremorgenic doses of nicotine region-specifically increased the activities of the inferior olive (IO) neurons  similarly to the rat model of essential tremor (Ohno et al., 2015a). In addition, electrical lesion of IO suppressed the induction of nicotine tremor, illustrating that nicotine induces kinetic tremor by activating IO neurons. Since excitation of the olivo-cerebellar neural pathway is reportedly involved in generation of essential tremor in humans (Puschmann and Wszolek, 2011;Kosmowska and Wardas, 2021;Welton et al., 2021), our findings suggest that nicotine-induced tremor has the same neural basis as essential tremor. In fact, nicotine tremor was significantly alleviated by drugs effective for human essential tremor (e.g., propranolol, diazepam, and phenobarbital), but was unaffected by medications for Parkinson's disease tremor (i.e., trihexyphenidyl) (Kunisawa et al., 2018). Thus, nicotineinduced tremor may serve as an animal model of essential tremor. Interestingly, we found several factors which can modulate the intensity of nicotine tremor, including 5-HT 1A and 5-HT 2 receptors, adrenergic β receptors, Na v1 channels, Ca v3 channels, and GABA A receptors. (Ohno et al., 2015a;Kunisawa et al., 2017;Kunisawa et al., 2018).
In the present study, we examined the effects of various dopamine receptor ligands on nicotine-induced tremor to explore the role and mechanism of the dopaminergic system in the generation of nicotine tremor. Since blockade of D 3 receptors or stimulation of D 1/5 receptors was found to reduce nicotine-induced tremor, D 3 and D 1/5 receptors may be involved in regulation of the induction of kinetic tremor.

Animals
Male ddY mice and SD rats (Japan SLC, Shizuoka, Japan) at 7-11 weeks of age were used. The animals were given food and water ad libitum and kept in air-conditioned rooms (24 ± 2°C and 55 ± 10% relative humidity) under a 12 h light/dark cycle (light on at 8:00 a.m.). The animal care and housing methods complied with the Guide for the Care and Use of Laboratory Animals of the Ministry of Education, Science, Sports and Culture of Japan. The experimental protocols were approved by the Experimental Animal Research Committee at Osaka Medical and Pharmaceutical University.

Evaluation of Nicotine-Induced Tremor in Mice
Nicotine-induced tremor was evaluated as reported previously (Kunisawa et al., 2018). Briefly, mice were treated with a tremorgenic dose (1 mg/kg, i.p.) of nicotine and individually placed in an observation cage (16 × 24 × 12 cm). The intensity and duration of nicotine-induced tremor were measured in a time-sampling manner over 1-2, 3-4, 5-6, 7-8, and 9-10 min (each for 1-min measurement) after the nicotine injection. The tremor intensity was measured using 4-point ranked scoring (0: no tremor, 1: mild tremor in head and tail, and straub tail, 2: moderate tremor in upper trunk, 3: marked tremor in whole body). The total tremor score and tremor duration were calculated as a sum of the tremor score or duration time per point, respectively.
After a recovery period of about 1 week, microinjection experiments with dopamine antagonists were performed. Under freely moving conditions, injection cannulae filled with SCH-23390 (3 μg/μl), U-99194 (30 μg/μl), or saline solution (control) were inserted into IO, the striatum, or cerebellum via guide cannulae, and 1 μl/site of the drug solution was gradually injected into the target site at a flow rate of 0.25 μl/min for 4 min using a microinfusion pump (KDS220, KD scientific, Holliston, MA, United States). Fifteen minutes after finishing the microinjection, the rats were treated with nicotine (1 mg/kg, i.p.) and behavioral observations of nicotine-induced tremor were performed during 5-16 min after the nicotine treatment, as described previously. The microinjection dosage of each dopamine antagonist was set to effectively act on each dopamine receptor according to previous reports (SCH-23390; Pardey et al., 2013, U-99194;Shimizu et al., 2014).

Statistical Analyses
Data are presented as the mean ± S.E.M. Significant differences on two-group comparisons were determined by Student's t-test (parametric analysis) or the Mann-Whitney's U-test (nonparametric analysis). For multigroup comparisons, the Kruskal-Wallis test followed by Steel-Dwass post-hoc multiple comparison test was performed. When the p-value was less than 0.05, it was considered significant.

Effects of Tremorgenic Nicotine on Brain Dopamine and its Metabolite Levels
To investigate the changes in brain dopamine and its metabolite levels by nicotine, we treated mice with a tremorgenic dose (1 mg/kg, i.p.) of nicotine and measured the levels of dopamine, DOPAC, and HVA in 8 brain regions. Nicotine significantly increased the dopamine level in P/MO as compared with control animals (t = 5.1374, df = 12, p = 0.0002) ( Table 1). The dopamine level also tended to increase with nicotine in the striatum and hippocampus ( Table 1). The level of DOPAC was significantly elevated in the hippocampus (t = 3.9544, df = 12, p = 0.0019) and P/MO (t = 2.3037, df = 12, p = 0.0399) ( Table 1). Moreover, the HVA level was significantly increased in the striatum (t = 3.7921, df = 12, p = 0.0026) ( Table 1).

Microinjection Studies With Dopamine Receptor Antagonists
To explore the regions of D 1/5 and D 3 receptors for regulation of nicotine-induced tremor, we conducted microinjection experiments Frontiers in Pharmacology | www.frontiersin.org June 2022 | Volume 13 | Article 938175 using D 1/5 (SCH-23390) and D 3 (U-99194) antagonists in rats. Since IO and the striatum are important sites for the induction of nicotine tremor  and parkinsonian tremor (Ohno et al., 2013;2015b), respectively, SCH-23390 or U-99194 was injected into these two regions. In addition, as D 3 receptors are highly expressed in the cerebellum and known to regulate locomotion and extrapyramidal movement disorders de Beaurepaire, 1996, 2005;Kolasiewicz et al., 2008;Shimizu et al., 2014), U-99194 was also injected into the cerebellum (lobe IX). Microinjection of SCH-23390 (3 μg/injection site) into IO tended to enhance nicotine-induced tremor, although the changes did not reach significance (total tremor score: z = 1.7400, p = 0.0819; total tremor duration: z = 1.6963, p = 0.0898) ( Figure 4A). Microinjection of SCH-23390 (3 μg/ injection site) into the striatum did not affect the induction of nicotine tremor ( Figure 4B). Furthermore, although microinjection of U-99194 into IO or the striatum failed to affect nicotine-induced tremor, its injection into the cerebellum markedly inhibited the induction of nicotine tremor (total tremor score: z = 2.4488, p = 0.0143; total tremor duration: z = 1.9393, p = 0.0525) ( Figure 5).

DISCUSSION
Nicotine increases dopamine release and enhances dopaminergic neurotransmission in several brain regions (e.g., striatum, nucleus accumbens, and ventral tegmental area), which are suggested to be mediated by α4β2, α6β2, and partly α7 nACh receptors (Damsma et al., 1988;Haikala and Ahtee, 1988;Wonnacott, 1997;Wonnacott et al., 2000;Exley et al., 2008;Quik et al., 2011;Besson et al., 2012;Harrington et al., 2016). Previous studies showed that nicotine weakly increase the level of dopamine metabolites (e.g., DOPAC) by about 20-30% without affecting DA levels (Tani et al., 1997;Suemaru et al., 2006). The present study confirmed that nicotine at a tremorgenic dose increased dopamine and its metabolite levels in several parts of the brain (e.g., striatum, hippocampus, and P/MO), suggesting activation of the dopaminergic system by nicotine in these regions. Our results of nicotine actions on the striatal and hippocampal dopamine metabolism are similar to those in above previous studies. Meanwhile, in P/MO, nicotine elevated both dopamine and DOPAC levels by about 40-50%. Although the reason why nicotine elevated the DA level in P/MO remains uncertain, the effects of nicotine on dopamine neurons may involve its interactions with the serotonergic and/or noradrenergic systems (Gros et al., 2021;Pierucci et al., 2022). Although we did not specifically measure the dopamine level in IO, it is of interest that nicotine increased the dopaminergic tone in the P/MO region containing IO that forms the olivo-cerebellar neural circuit regulating tremor induction (Puschmann and Wszolek, 2011;Ohno et al., 2015a;Kosmowska and Wardas, 2021).
The present study using selective dopamine receptor ligands revealed that activation of D 3 receptors facilitates the induction of nicotine tremor, whereas D 1/5 receptor stimulation negatively regulates nicotine-induced tremor. Although D 3 receptors belong to the D 2 receptor family (Prieto, 2017;Kosmowska and Wardas, 2021), selective stimulation of D 2 receptors by sumanirole showed no effects. Furthermore, since D 1/5 and D 3 antagonists per se enhanced and inhibited nicotine-induced tremor, respectively, activation of these receptors by endogenous dopamine may be involved in the regulation of nicotine tremor induction. It was confirmed that selective block of D 2 or D 4 receptors did not affect the induction of nicotine tremor, indicating that only D 3 receptors in the D 2 receptor family actively modulate the induction of kinetic tremor. Although we cannot completely deny the possibility that multiple actions of dopamine ligands used (e.g., interaction of SCH-23390 with 5-HT 2 receptors and partial antagonism of SKF-38393 at D 1/5 receptors) (Hoyer et al., 1989;Desai et al., 2005) also affected nicotine-induced tremor, our results suggest that We previously demonstrated that electrical lesion of IO suppressed nicotine-induced tremor . Consistent with the fact that IO is closely involved in the pathogenesis of essential tremor in humans (Puschmann and Wszolek, 2011;Kosmowska and Wardas, 2021;Welton et al., 2021), our results suggest that the IO-cerebellar system plays a crucial role in the generation of nicotine-induced kinetic tremor. IO receives dopaminergic innervation from the ventral tegmental and prerubral parafascicular areas (Fallon et al., 1984;Toonen et al., 1998;Kitahama et al., 2000), suggesting a potential role of dopaminergic neurons in modulating tremor induction. Although microinjection of D 1/5 and D 3 antagonists into IO did not significantly affect nicotine tremor, the D 1/5 antagonist markedly increased the intensity and duration of nicotine tremor. Our results suggest that D 1/5 receptors in IO may be partly involved in modulation of nicotine tremor induction. Other D 1/5 receptor regions involved in regulation of nicotine tremor remain to be clarified.
It is well-known that striatal dopamine receptors, particularly D 2 receptors, play a crucial role in regulation of extrapyramidal motor functions and induction of parkinsonian symptoms, including resting tremor (Ohno et al., 2013;Ohno et al., 2015b). In the present study, however, the selective D 2 receptor agonist sumanirole did not affect the generation of nicotine-induced kinetic tremor. In addition, neither microinjection of D 1/5 nor D 3 antagonist into the striatum altered the induction of nicotine tremor. Thus, our results indicate that, unlike parkinsonian resting tremor, striatal dopamine receptors are not involved in regulation of nicotine-induced kinetic tremor.
In the D 2 receptor family, D 3 receptors are highly expressed in the cerebellum in addition to cerebral cortex, limbic regions, and striatum (Sokoloff et al., 1990;Bouthenet et al., 1991;Lévesque et al., 1992;Diaz et al., 2000;Kim et al., 2009). The cerebellum receives dopaminergic neurons from the red nucleus, substantia nigra, and ventral tegmental area (Kizer et al., 1976;Kosmowska and Wardas, 2021), and contains a high density of D 3 receptors whereas the levels of D 1 , D 2 , D 4 , and D 5 receptors are low (Diaz et al., 2000;Kosmowska and Wardas, 2021). We and other groups showed that cerebellar D 3 receptors regulated spontaneous locomotor activity and the induction of extrapyramidal Frontiers in Pharmacology | www.frontiersin.org June 2022 | Volume 13 | Article 938175 8 movement disorders (i.e., catalepsy) ( de Beaurepaire, 1996, 2005;Kolasiewicz et al., 2008;Shimizu et al., 2014). Interestingly, the present study demonstrated that microinjection of the D 3 antagonist U-99194 suppressed nicotine-induced tremor generation. Our results strongly suggest that cerebellar D 3 receptors positively control the induction of nicotine tremor mediated by the olivo-cerebellar neural pathway.
We previously showed that nicotine-induced tremor was of IO origin and alleviated by medications for essential tremor (e.g., propranolol, diazepam, and phenobarbital) 2018), suggesting that nicotine-induced tremor is useful as a model of essential tremor. Essential tremor is one of the common neurological disorders affecting approximately 1% of people worldwide (Puschmann and Wszolek, 2011;Kosmowska and Wardas, 2021). Although several drugs are applicable in its treatment, there are no specific drugs for essential tremor, and approximately one third of patients are treatment-resistant. The present study suggests that the selective antagonists for D 3 receptors or agonists for D 1/5 receptors may have a therapeutic potential. Interestingly, linkage analysis of genes associated with essential tremor identified four loci: chromosome 3q13 (ETM1) (Gulcher et al., 1997), 2p22-p25 (ETM2) (Higgins et al., 1997), 6p23 (ETM3) (Shatunov et al., 2006), and 5q35 (Hicks et al., 2016), among which ETM1 contains the DRD3 gene encoding D 3 receptors (Gulcher et al., 1997). In addition, DRD3-Gly (Ser9Gly variant), which shows a greater affinity for dopamine than the non-mutated form, is reportedly associated with the risk of essential tremor (Jeanneteau et al., 2006). Thus, dopamine D 3 receptors may be involved in the modulation of essential tremor. Taken together with the present results, dopamine D 3 receptor antagonists may be useful for the treatment of essential tremor. Further studies are required to validate this hypothesis and clarify the role of dopaminergic neurons in the pathogenesis of essential tremor.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusion of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The animal study was reviewed and approved by Osaka Medical and Pharmaceutical University Experimental Animal Research Committee.

AUTHOR CONTRIBUTIONS
YO designed the research. MK, NK, SS, HI, and YO performed experiments, analyzed data, and wrote the article.