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l-dopa-induced dyskinesias (LIDs) are a side effect of Parkinson's disease therapy that is thought to arise, at least in part, because of excessive dopaminergic activity. Thus, drugs that regulate dopaminergic tone may provide an approach to manage LIDs. Our previous studies showed that nicotine treatment reduced LIDs in Parkinsonian animal models. This study investigates whether nicotine may exert its beneficial effects by modulating pre-synaptic dopaminergic function. Rats were unilaterally lesioned by injection of 6-hydroxydopamine (6-OHDA) (2 × 3 ug per site) into the medial forebrain bundle to yield moderate Parkinsonism. They were then implanted with minipumps containing vehicle or nicotine (2.0 mg/kg/d) and rendered dyskinetic with l-dopa (8 mg/kg plus 15 mg/kg benserazide). Lesioning alone decreased the striatal dopamine transporter, nicotinic receptor (nAChR) levels, and nAChR-mediated 3H-dopamine release, consistent with previous results. Nicotine administration reduced l-dopa-induced abnormal involuntary movements throughout the course of the study (4 months). Nicotine treatment led to declines in the striatal dopamine transporter, α6β2* nAChRs and various components of α6β2* and α4β2* nAChR-mediated release. l-dopa treatment had no effect. These data suggest that nicotine may improve LIDs in Parkinsonian animal models by dampening striatal dopaminergic activity.
Parkinson's disease symptoms are greatly improved by dopamine replacement therapy with l-dopa, but within a few years of treatment unwanted side effects such as abnormal involuntary movements or dyskinesias develop, which may be mild to severely incapacitating (Rascol et al. 2011; Schapira and Jenner 2011). Current treatments to reduce l-dopa-induced dyskinesias (LIDs) are of limited success and include reductions in l-dopa dose, adjunct therapy with amantadine or surgical intervention (Rascol et al. 2011; Schapira and Jenner 2011). Further studies to identify the mechanisms that contribute to LIDs are thus important as they may yield better therapeutic strategies.
Extensive evidence indicates that both pre- and post-synaptic dopaminergic factors contribute to the development of LIDs. Pre-synaptic mechanisms of particular relevance include the nigrostriatal dopaminergic neuronal loss that results in a decreased dopamine buffering capacity and the increased extracellular dopamine levels that arise with l-dopa treatment (Cenci 2007; Lindgren et al. 2010; Carta and Bezard 2011; Fisone and Bezard 2011). Typical oral l-dopa doses produce large transient increases in the levels of dopamine in striatum of patients affected by LIDs (Pavese et al. 2006). Animal models also reveal excessive extracellular striatal dopamine concentrations after l-dopa treatment (Meissner et al. 2006; Lundblad et al. 2009; Carta and Bezard 2011). The loss of pre-synaptic dopamine transporters responsible for the clearance of dopamine from the synaptic cleft, coupled with increased dopamine synthesis, is proposed to contribute to the enhanced dopaminergic activity with l-dopa use (Lundblad et al. 2009; Carta and Bezard 2011; Murer and Moratalla 2011). Thus, drugs that normalize striatal dopaminergic tone may reduce LIDs. As nicotinic acetylcholine receptors (nAChRs) are significant modulators of striatal dopamine release, balancing dopaminergic tone via nAChR regulation may provide an approach to attenuate LIDs.
Evidence for a role for nAChRs is based on studies showing that both nicotine and nAChR agonists reduce LIDs (Quik et al. 2007, 2013, b; Bordia et al. 2008, 2010; Huang et al. 2011a, b). Notably, a nAChR antagonist mecamylamine also reduced LIDs (Bordia et al. 2010). A similar effect of both nAChR agonists and antagonists suggests that agonists may induce their effects by nAChR desensitization, which would effectively result in a functional blockade. This interpretation is consistent with recent studies suggesting that nAChR desensitization may represent a mechanism whereby nicotine and nAChR drugs modulate alcoholism, addiction, and anxiety (Dopico and Lovinger 2009; Anderson and Brunzell 2012).
Numerous nAChRs are expressed on different neuronal elements in the nigrostriatal pathway. Within the large family of striatal nAChRs, the majority contain the β2 subunit. The α6β2* nAChRs are exclusively expressed on dopaminergic neurons while α4β2* receptors are located on both dopaminergic and non-dopaminergic neurons (Zoli et al. 2002; Gotti et al. 2010; Grady et al. 2010b; Quik and Wonnacott 2011). The asterisk indicates the possible presence of other subunits in the receptor complex. Both α6β2* and α4β2* receptors are decreased with nigrostriatal degeneration (Quik et al. 2003; Perez et al. 2009) and play an important role in nicotine-mediated reduction in l-dopa-induced dyskinesias (Huang et al. 2011b; Quik et al. 2012b).
The objective of this study was to understand the mechanisms whereby nicotine treatment may reduce LIDs. To approach this, 6-OHDA-lesioned rats, treated with or without nicotine, were rendered dyskinetic using l-dopa. They were then killed and striatal dopaminergic function, specifically striatal 3H-dopamine release, was assessed. Long-term nicotine treatment led to significant decreases in various dopaminergic measures, nAChRs and nAChR-mediated dopamine release in striatum. Overall, these data suggest that the antidyskinetic effect of nicotine in moderate Parkinson's disease may be because of its ability to reduce dopaminergic tone via an interaction at nAChRs.
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- Materials and methods
Aberrant pre-synaptic dopaminergic activity is thought to have a major impact in the development of LIDs (Bezard et al. 2001; Fisone et al. 2007; Jenner 2008; Cenci and Konradi 2010; Brotchie and Jenner 2011; Carta and Bezard 2011). The nigrostriatal dopamine terminal loss that occurs in Parkinson's disease and the excessive dopaminergic stimulation in response to intermittent l-dopa dosing both appear to play a critical role. These findings suggest that treatments that modulate striatal dopaminergic tone may improve LIDs.
Our work showed that nicotine and nAChR agonists reduce LIDs in Parkinsonian rats, mice, and monkeys by acting, in part, at nAChRs on nigrostriatal dopamine terminals (Quik et al. 2007, 2013, b; Bordia et al. 2008, 2010; Huang et al. 2011a, b). This finding was initially unexpected as acute nAChR stimulation enhances dopamine release (Gotti et al. 2010; Quik and Wonnacott 2011) and thus might be expected to worsen LIDs. However, long-term nicotine administration has been shown to elicit a very different response, with nAChR desensitization and/or down-regulation and corresponding declines in dopaminergic function (Picciotto et al. 2008; Buccafusco et al. 2009). The goal of these experiments was to investigate whether a long-term decrease in striatal nAChR-mediated dopaminergic function may represent a mechanism that underlies the nicotine-mediated decline in LIDs.
To approach this, we measured nAChR-evoked 3H-dopamine release from synaptosomes, a well-established technique used to assess striatal dopamine function in vitro (Rapier et al. 1990; Grady et al. 1992, 2002; Wonnacott et al. 2000; Quik et al. 2003). Our results show that chronic nicotine administration decreased nAChR-mediated dopamine release from striatum, providing support for the idea that nicotine may reduce l-dopa-induced AIMs by dampening dopaminergic tone, as depicted schematically in Fig. 9.
Figure 9. Schematic representation of the effect of long-term nicotine treatment on dopamine (DA) release from nigrostriatal terminals. The nicotine-induced decline in striatal dopamine release may counter-balance the effect of excess dopamine production (due to l-dopa) that is not cleared because of the dopamine nerve terminal loss.
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As nicotine elicits striatal dopamine release by acting at α4β2* and/or α6β2* nAChRs (Quik and Wonnacott 2011), we next evaluated the contribution of these two subtypes to the nicotine-mediated decline in striatal 3H-dopamine release. To study this, we used the α6β2* nAChR-directed neurotoxin α-CtxMII, which selectively blocks α6β2* but not α4β2* nAChR-mediated function (McIntosh et al. 2004). The results showed that long-term nicotine treatment decreased α6β2* nAChR-mediated 3H-dopamine release in response to 1 μM, but not 10.0 μM, application of nicotine to the synaptosomal preparation from either the intact or lesioned striatum. These two concentrations were selected as the former leads to submaximal and the later to maximal dopamine release (Salminen et al. 2004). The differential response at these two nicotine concentrations can be explained by the presence of least two α6β2* nAChRs in striatum, the α6α4β2β3 and α6β2β3 subtypes, with the former being more sensitive to nicotine (Salminen et al. 2004; Bordia et al. 2007; Gotti et al. 2010). Thus, 1 μM nicotine primarily elicits dopamine release mediated by the α6α4β2β3 subtype, while 10 μM nicotine predominantly evokes release at the α6β2β3 receptor subtype. A decline in release only at the 1.0 μM nicotine concentration may relate to the fact that α6α4β2β3, but not α6β2β3 receptors are down-regulated with long-term nicotine treatment (Perez et al. 2008). Low nicotine (1.0 μM) application would elicit less release because the remaining α6β2β3 require a higher nicotine concentration to be activated. These data are consistent with the 125I-α-CtxMII-binding studies, which show that α6β2* nAChRs are decreased with long-term nicotine treatment although it is not known whether these represent α6α4β2β3 or α6β2β3 receptor subtypes. Interestingly, accumulating studies suggest that the α6α4β2β3 nAChR may be the one critical for a variety of different nAChR-mediated behaviors including locomotor activity and addiction (Exley et al. 2011; Zhao-Shea et al. 2011). These studies suggest that a decline in α6α4β2β3 nAChRs is linked to the nicotine-mediated decline in l-dopa-induced AIMs.
We also investigated the effect of long-term nicotine treatment on α4β2* nAChR-mediated dopamine release from striatal synaptosomes. In this case, long-term nicotine administration significantly decreased 3H-dopamine release elicited by the 10 μM, but not the 1.0 μM nicotine concentration. This varying responsiveness at the two concentrations may be because of differential effects at striatal α4β2* nAChR subtypes, with the two main populations being the lower affinity α4β2 and higher affinity α4α5β2 receptor (Gotti et al. 2010; Grady et al. 2010b). If chronic nicotine treatment selectively down-regulates the lower affinity α4β2 subtype, one might expect a decrease in 3H-dopamine release only at the 10 μM nicotine concentration. Release at the 1.0 μM dose of nicotine would be unchanged as α4β2 receptors are not reduced by long-term nicotine treatment.
α4β2* nAChR binding studies were done to evaluate the effect of chronic nicotine treatment on receptor levels. However, correlation of these results with α4β2* nAChR-mediated dopamine release is complicated by the fact that only 20% of the α4β2* nAChRs in the striatum are present on the dopamine terminals and the remaining 80% on other neurons in the striatum (Zoli et al. 2002; Quik and Wonnacott 2011). This finding is supported by the current data which show that a ~50% dopaminergic nerve terminal loss with 6-OHDA lesioning leads to only a ~10% decline in α4β2* nAChR binding. This small population (20%) of α4β2* nAChRs on the dopamine terminals may be controlled in a similar manner as those on striatal neurons, that is, up-regulated by nicotine. The release studies, however, demonstrate a decline in α4β2* nAChR function. This is most likely because of α4β2* nAChR desensitization (Picciotto et al. 2008; Buccafusco et al. 2009). Long-term α4β2* receptor desensitization or inactivation would dampen dopaminergic tone. It is also possible that α4β2* nAChR desensitization leads to changes in downstream signaling mechanisms that improve LIDs. The molecular and cellular mechanisms that underlie the nicotine-mediated reduction are long-term events as several weeks of nicotine treatment are required to decrease LIDs (Quik et al. 2007; Bordia et al. 2010; Huang et al. 2011a, b).
The current data show that nicotine treatment decreased dopamine release in both intact and lesioned striatum. This observation raises questions about the relevance of these findings to the nicotine-mediated decline in LIDs, which is generally associated with nigrostriatal damage. One possibility is that the decrease in release on the intact side does not lead to behavioral consequences under non-pathological conditions. On the other hand, the reduction in dopamine release on the lesioned side may contribute to the decline in LIDs because there are fewer terminals to clear the excess dopamine that arises with l-dopa administration. Another point of note is that the level of nAChR-evoked 3H-dopamine release in nicotine-treated rats is greater on the intact as compared with the lesioned striatum. Thus, there may be a reserve of striatal dopamine on the intact side such that overall behavior is not affected under control conditions.
Although long-term nicotine treatment decreased nicotine-evoked 3H-dopamine release, l-dopa administration did not affect the release of radioactive dopamine from striatal synaptosomes. This finding was not unexpected as the current studies evaluate release of exogenously added 3H-dopamine loaded into a synaptosomal preparation. In contrast, release of endogenous dopamine from synaptosomes is most likely increased in striatum of l-dopa-treated rats, especially as striata are harvested an h after l-dopa administration. Evidence for such an assumption is based on the studies showing that striatal dopamine levels are elevated after l-dopa treatment. Interestingly, the increase was most prominent in striatum of dyskinetic compared with non-dyskinetic rats, suggesting that enhanced dopamine levels may underlie the development of dyskinesias (Carta et al. 2006).
These studies in rats show that chronic nicotine treatment reduces various components of both striatal α6β2* and α4β2* nAChR-mediated dopamine release. However, we found no decrease in striatal 3H-dopamine release in a recent study in Parkinsonian monkeys treated with nicotine (Quik et al. 2013). These discrepant results may be because of differences in the treatment regimen as the rats were implanted with nicotine minipumps, which provides a constant level of nicotine exposure while the monkeys were given nicotine in the drinking water, which yields intermittent nicotine exposure. Minipump nicotine administration has been reported to yield more pronounced changes in striatal nAChRs (Moretti et al. 2010). Differential effects may also arise because of species differences in nAChR regulation. Long-term nicotine treatment resulted in 30–50% declines in α6β2* nAChR levels in rodents, but only a small or no change in α6β2* nAChR levels in monkeys (Lai et al. 2005; McCallum et al. 2006). The increased number of animals in this study (9–13 rats/group) also improves the ability to discriminate differences between treatment groups.
In addition to pre-synaptic dopaminergic mechanisms, l-dopa treatment also results in a host of post-synaptic changes that contribute to LIDs. These may arise as a result of enhanced stimulation of dopamine D1, and possibly D2, receptors, and the consequent activation of downstream signaling mechanisms by dopamine derived from exogenous l-dopa (Berthet and Bezard 2009; Cenci and Konradi 2010). An accumulating literature also indicates that dysregulated release of dopamine from serotonergic neurons may exacerbate LIDs that arise with therapeutic doses of l-dopa (Carta et al. 2007; Cenci 2007; Munoz et al. 2008; Nahimi et al. 2012). Studies to determine whether nicotine modulates post-synaptic mechanisms remain to be done.
In summary, our previous work had shown that chronic nicotine treatment improved l-dopa-induced AIMs in several Parkinsonian animal models. The present results provide a mechanistic basis for these findings by showing that long-term nicotine treatment decreased both α6β2* and α4β2* nAChR-mediated 3H-dopamine release from striatal synaptosomes. Long-term nicotine treatment also reduced 125I-α-CtxMII-binding sites, suggesting that receptor down-regulation may underlie the α6β2* nAChR-mediated decrease in release. By contrast, α4β2* nAChR desensitization may contribute to the α4β2* nAChR-mediated decline in striatal 3H-dopamine release. Overall, these data suggest that nicotine may improve LIDs in moderate Parkinson's disease by decreasing α6β2* and α4β2* nAChR striatal dopaminergic function.