Chronic pre-treatment with nicotine enhances nicotine-evoked striatal dopamine release and α₆ and β₃ nicotinic acetylcholine receptor subunit mRNA in the substantia nigra pars compacta of the rat

Abstract

Whilst local intrastriatal infusion of nicotine consistently elicits striatal dopamine release, systemic administration often fails to do so. Since chronic nicotine administration is known to result in desensitisation-induced upregulation of nicotinic acetylcholine receptors (nAChRs), the present study investigated whether chronic pre-treatment could enhance the response to systemic nicotine and, if so, whether increases in specific nAChR subunit mRNA levels in the substantia nigra pars compacta (SNc) may underlie this effect. In vivo microdialysis studies in male Sprague–Dawley rats revealed that following 4 days pre-treatment with nicotine (0.8 mg kg⁻¹ s.c.), local intrastriatal nicotine infusion (3 mM) elicited significantly higher dopamine efflux compared to vehicle pre-treated controls (peak release: 1273 ± 199% basal versus 731 ± 113% basal), whereas systemic nicotine challenge (0.8 mg kg⁻¹ s.c.) elicited no response. In contrast, following 8 days pre-treatment with nicotine (0.8 mg kg⁻¹ s.c.), systemic nicotine challenge (0.8 mg kg⁻¹ s.c.) now produced significantly higher dopamine efflux than that of vehicle pre-treated controls (147 ± 30% basal versus 91 ± 5% basal). Eight days pre-treatment with nicotine also significantly elevated the levels of α6 (∼55%) and β3 (∼43%) nAChR subunit mRNA in the SNc, suggesting that up-regulation of these nAChR subunit genes in the nigrostriatal tract may contribute to the enhanced nicotine-evoked striatal dopamine release.

Introduction

Parkinson's disease (PD) is characterised pathologically by the progressive degeneration of pigmented dopaminergic neurones in the substantia nigra pars compacta (SNc), resulting in reduced levels of extracellular dopamine in the terminal region of the nigrostriatal pathway, the striatum. This deficit in striatal dopamine underlies the characteristic motor symptoms of PD, namely bradykinesia, postural instability and tremor and forms the rationale behind the use of dopamine-replacement therapies such as levodopa in the treatment of PD. Nicotine administration has been shown experimentally to increase the firing rate of SNc neurones (Lichtensteiger et al., 1982) and to evoke subsequent striatal dopamine release (Balfour and Fagerstrom, 1996). Therefore, agonists acting at neuronal nicotinic acetylcholine receptors (nAChRs) may provide symptom relief in PD. Consistent with this, administration of nicotine in many forms (e.g. cigarette smoking, nicotine patches and gum) has been shown to transiently relieve motor symptoms in PD patients (Ishikawa and Miyatake, 1993, Fagerstrom et al., 1994) and indeed may also have the additional benefit of altering disease progression (O'Neill et al., 2002, Quik, 2004). Given the reported 50–70% decline in nicotine binding in the parkinsonian striatum and SNc (Aubert et al., 1992, Perry et al., 1995), the suggested mechanism underlying this relief of motor symptoms in PD is nicotine-mediated stimulation of nAChRs on remaining viable nigrostriatal tract neurons (Giorguieff-Chesslet et al., 1979) in order to evoke dopamine release and thereby restore the striatal dopamine levels.

Experimentally, local infusion of nicotine into the striatum of naïve rats does invariably induce dopamine release (Toth et al., 1992, Marshall et al., 1997, Visanji et al., 2002). In contrast, systemic administration, as would be required therapeutically, often fails to do so (Brazell et al., 1990; Birrell and Balfour, 1995, Visanji et al., 2002) unless studies are performed under artificially elevated extracellular CaCl₂ levels (Di Chiara and Imperato, 1988) or with the inclusion of nomifensine in the dialysate to prevent the normally rapid reuptake of striatal dopamine following its release (Abercrombie et al., 1989, Benwell and Balfour, 1997). The variability in response to systemic nicotine administration in the literature may be due to the variation in doses of nicotine and the routes and dosing protocols employed. This does pose problems for the likely clinical efficacy of nicotine or nAChR agonists in the symptomatic treatment of PD. However, evidence suggests that chronic activation of nAChRs, as would be provided therapeutically, might beneficially alter nAChR expression in the brain to circumvent some of these problems. For example, it is well established that chronic treatment of rats with nicotine or nAChR agonists results in an up-regulation of nicotinic agonist binding sites (Collins et al., 1990, Wonnacott et al., 1990), and a similar increase has been demonstrated in the brains of smokers (Benwell et al., 1988). Furthermore, the up-regulated receptors are believed to be functional since the increase in agonist binding is paralleled by an increase in local intrastriatal nicotine-induced striatal dopamine release (Wonnacott et al., 1990). Thus the chronic administration of nicotine or other nAChR agonists may lead to enhanced levels of dopamine release via desensitisation-induced receptor up-regulation.

There have been several recent studies investigating the subunit composition of pentameric nAChRs resident on the dopaminergic terminals of the rodent striatum (Zoli et al., 2002, Salminen et al., 2004, Luetje, 2004). Studies by Zoli and colleagues (2002) suggested that dopaminergic terminals expressed α4α5β2, α4α6β2(β3) and α6β2(β3) nicotinic sub-types. This was extended in a series of experiments that examined nicotine-induced dopamine release in striatal synaptosomes and recorded ACh mediated currents in dopaminergic neurones of α4, α6 and β2 knock-out mice (Champtiaux et al., 2003). These authors concluded that a combination of α6β2^∗ and α4β2^∗ nAChR may mediate dopamine release at the terminals. Interestingly, the results also suggested that (non α6)α4β2^∗ nAChRs represented the majority of functional heteromeric nAChRs in DA neuronal soma (Champtiaux et al., 2003).

Whilst chronic nicotine treatment has been shown to increase nAChR binding in the striatum and SNc of rats (e.g. Marks et al., 1992), the effects on individual subunits that make up the nAChRs in the nigrostriatal pathway is largely unknown. Due to a lack of subunit selective tools, investigations of this nature have largely been confined to measurements of gene expression using in situ hybridisation studies. Of those subunit genes known to be expressed within the SNc (α2-α7 and β2-4; reviewed in Quik and Kulak, 2002), studies to date have found that chronic infusion of nicotine for 10 days has no effect on α2, α3, α4, α5 or β2 nAChR subunit mRNA in the SNc (Marks et al., 1992), but does increase α7 nAChR subunit mRNA levels in this region (Ryan and Loiacono, 2001), thereby implying differential nicotine-induced regulation of individual nAChR subunit genes. Whether similar changes occur in those subunits showing a relatively restricted distribution in the SNc (α6 and β3) remains to be established. The discrete localization of α6 and β3 make receptors comprising these subunits ideal drug targets, but currently there are no subtype-selective agonists available for these receptors as difficulty in the expression of functional α6 receptors has hampered screening efforts (Evans et al., 2003). However, a recent report that the expression of nigrostriatal α6 containing nAChRs is reduced (but not eliminated) by β3 subunit gene deletion suggests that there is a link between these two receptor subunits (Gotti et al., 2005). Further evidence that the β3 receptor subunit is important have come from mice with a null mutation of the β3 gene (Cui et al., 2003). Studies using this transgenic revealed a β3-dependent conotoxin MII-sensitive site that modulated striatal dopamine release. The mice also showed changes in dopamine-mediated behaviours (locomotor activity and prepulse inhibition of acoustic startle). While transgenic mice have provided valuable insights, one must consider that they may have developed mechanisms that compensate for the loss of a particular subunit. Additionally many of the studies cited utilise ex vivo tissue, which may not truly reflect the intact nigrostrital pathway.

In light of the variable responses noted following systemic injection of nicotine and the fact that striatal terminals likely express a variety of nAChR populations, the aims of the present study were two-fold. The initial aim was to investigate whether chronic intermittent nicotine pre-treatment, as would be desired therapeutically in PD, could enhance striatal dopamine release evoked by systemic nicotine challenge. Secondly, studies were performed to establish whether changes in expression of the relatively localised α6 and β3 nAChR subunit genes in the SNc might contribute to any observed changes in nicotine-evoked striatal dopamine release brought about by this dosing regimen.