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Nicotine reduces dopaminergic deficits in parkinsonian animals when administered before nigrostriatal damage. Here we tested whether nicotine is also beneficial when given to rats and monkeys with pre-existing nigrostriatal damage. Rats were administered nicotine before and after a unilateral 6-hydroxydopamine (6-OHDA) lesion of the medial forebrain bundle, and the results compared to those in which rats received nicotine only after lesioning. Nicotine pretreatment attenuated behavioral deficits and lessened lesion-induced losses of the striatal dopamine transporter, and α6β2* and α4β2* nicotinic receptors (nAChRs). In contrast, nicotine administered two weeks after lesioning, when 6-OHDA-induced neurodegenerative effects are essentially complete, did not improve these same measures. Similar results were observed in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned monkeys. Nicotine did not enhance striatal markers when administered to monkeys with pre-existing nigrostriatal damage, in contrast to previous data that showed improvements when nicotine was given to monkeys before lesioning. These combined findings in two animal models suggest that nicotine is neuroprotective rather than neurorestorative against nigrostriatal damage. Receptor studies with 125I-α-conotoxinMII (α-CtxMII) and the α-CtxMII analog E11A were next done to determine whether nicotine treatment pre- or post-lesioning differentially affected expression of α6α4β2* and α6(nonα4)β2* nAChR subtypes in striatum. The observations suggest that protection against nigrostriatal damage may be linked to striatal α6α4β2* nAChRs.
Extensive evidence now indicates there is a decreased incidence of Parkinson's disease with smoking (Gorell et al., 2004; Ritz et al., 2007; Thacker et al., 2007). Although the compound(s) in smoke responsible for this inverse association is uncertain, a growing body of evidence suggests that nicotine may play a role, at least in part. This possibility stems from studies using experimental animal models, which show that nicotine pre-treatment improves toxin-induced nigrostriatal damage (O'Neill et al., 2002; Quik et al., 2007b; Picciotto and Zoli, 2008). Nicotine administered via several different routes enhanced different dopaminergic measures in both rodent and nonhuman primate models, with improvement dependent on nicotine dose and lesion size (Ryan et al., 2001; Bordia et al., 2006; Quik et al., 2006a; Visanji et al., 2006)
In the above studies, nicotine treatment was given before and during the neurodegenerative process. The mechanism of nicotine's action could thus be through protection against the toxic insult and/or restoration of existing nigrostriatal damage. To evaluate the relative contribution of these two mechanisms, experiments have also been done in which nicotine was given after nigrostriatal damage. Meshul and coworkers showed that nicotine improved glutamatergic measure and motor function when nicotine was given 2 weeks after 6-hydroxydopamine (6-OHDA) lesioning (Meshul et al., 2002), a time at which the neurodegenerative process is essentially complete. However, there was no improvement in dopaminergic measures in another study in which nicotine was administrated post-lesioning (Costa et al., 2001). It is thus unclear whether nicotine restores integrity/function of damaged dopaminergic neurons.
Accumulating work indicates that nicotine most likely exerts its effect on the nigrostriatal system through an interaction at nicotinic receptors (nAChRs) (Gotti et al., 2007; Grady et al., 2007; Quik et al., 2007a). Several populations of nAChRs have been identified in rodent and monkey striatum, including the α4β2* and α6β2* subtypes. The asterisk indicates the possible presence of other subunits in the receptor complex. The primary α4β2* nAChRs consist of the α4β2 and α4α5β2 or α4α2β2 populations, whereas the predominant α6β2* subtypes are α6α4β2* and α6(nonα4)β2* nAChRs (Salminen et al., 2004; Quik et al., 2005). Nigrostriatal damage results in losses of both α4β2* and α6β2* nAChRs, with the latter population decreased to a greater extent after lesioning. This observation suggests that the α6β2* nAChRs are primarily located on dopaminergic nigrostriatal terminals while α4β2* nAChRs are present both on dopaminergic terminals and other striatal neurons (Quik et al., 2001; Quik et al., 2003).
As mentioned above, there appear to exist two major α6β2* subtypes, the α6α4β2* and α6(nonα4)β2* nAChRs. Interestingly, our recent data show that these two populations exhibit a differential susceptibility to nigrostriatal damage, with the α6α4β2* primarily reduced with mild to moderate degeneration, and the α6(nonα4)β2* subtype decreased only with more severe lesioning (Bordia et al., 2007). In addition to a selective vulnerability to nigrostriatal damage, the α6α4β2* and α6(nonα4)β2* are differentially regulated with chronic nicotine treatment (Perez et al., 2008). These combined studies suggest there is a complex regulation of nAChR subtype expression under varying experimental conditions.
The present studies were done to determine whether the ability of nicotine to enhance striatal dopaminergic measures is due to a neuroprotective and/or neurorestorative effect, and whether nicotine's beneficial effects are linked to alterations in α6β2* nAChR subtypes. The results suggest that the primary role of nicotine may be in neuroprotection and that the presence of the α6α4β2* nAChRs may be important for this effect.
Male Sprague-Dawley rats weighing ~100 g (for nicotine pre-treatment) or ~ 275 g (for nicotine post-treatment) were purchased from Charles River (Wilmington, MA). The initial body weight of rats in the nicotine pre-treatment group was lower than that of the rats in the post-treatment group. This was necessary to ensure that the weight and thus the brain of the rats in the two groups were of a similar size at the time of lesioning. Consequently, nicotine dosing in the nicotine-pretreatment studies were initiated during an adolescent phase and continued into adulthood, while the dosing for rats in the post-treatment studies was done only during the adult stage. Rats were housed two per cage under a 13/11-h light-dark cycle, with free access to food chow and drinking water.
For the receptor expression studies, nicotine (free base) was given to rats in drinking water at final doses of 12.5, 25, 50 and 100 μg/ml, as follows. Animals were given nicotine in a 1% saccharin solution to mask nicotine's bitter taste. After two days of saccharin only, nicotine treatment was initiated at either 12.5 or 25 μg/ml and the rats maintained at those doses for 2 weeks. For the rats at the higher doses of nicotine, the concentration was increased to 50 μg/ml after 2 days and maintained at that dose, or increased to 100 μg/ml after another 2 days. Rats were kept at the final doses of nicotine for 2 weeks, with the nicotine removed 18 hours before the rats were killed to minimize residual effects of nicotine in the receptor assays.
Based on the results of the receptor studies, the 50 μg/ml dose of nicotine was selected for the neuroprotection/neurorestoration studies. All rats were first administered 1% saccharin water for 2 days after which nicotine was added to the drinking solution of the nicotine-treated groups, starting at a 25 μg/ml and increased to 50 μg/ml after 2 days. The rats were maintained at this final concentration throughout the study.
As an index of nicotine consumption, we assayed plasma cotinine levels, a primary nicotine metabolite, because of its long half-life (~18 h) (Matta et al., 2007). Blood (~0.5 ml) from the lateral saphenous vein was drawn under isofluorane anesthesia after 10 days on the final dose of nicotine. Cotinine was measured using an EIA kit according to the manufacturer's instructions (Orasure Technologies, Bethlehem, PA)
Rats were either sham-lesioned or lesioned with the selective dopaminergic neurotoxin 6-OHDA, as previously described (Cenci et al., 1998; Cenci et al., 2002). Briefly, rats were anesthetized with isofluorane (initial 5%, maintenance 1.75%), and then placed in a Kopf stereotaxic apparatus. Burr holes were drilled through the skull and 6-OHDA hydrochloride (2 μg/μl) stereotaxically injected into two separate sites of the right ascending medial forebrain bundle. We injected a relatively low dose of 6-OHDA (3 μg/μl) to yield ~50% decline in dopaminergic measures, as previously reported by Visanji et al. (2006). The coordinates for the position of these two sites were as follows relative to the Bregma and dural surface: (1) anteroposterior, - 4.4; lateral, 1.2; ventral, 7.8; tooth bar at -2.4; (2) anteroposterior, -4.0; lateral, 0.75; ventral, 8.0; tooth bar at +3.4. Buprenorphine (0.03 mg/kg) was injected subcutaneously immediately after surgery.
To assess the effect of nicotine treatment on motor deficits that arise with nigrostriatal damage, amphetamine-induced ipsilateral turning was determined as previously described (Cenci et al., 1998; Cenci et al., 1999; Cenci et al., 2002). Rats were tested for rotational behavior in an apparatus that consists of a cylindrical chamber connected to a computer with appropriate software (ROTOMAX, AccuScan Instruments Inc. Columbus, Ohio, USA). The rats were placed in the chamber for 30 min for acclimatization, after which amphetamine (4.0 mg/kg ip) was administered (Visanji et al., 2006). Circling behavior was assessed between 85 to 120 min after amphetamine administration when effects of nicotine on circling had returned to control levels. Testing was repeated one week later, and the data pooled.
The rats were subsequently killed, the brains removed and immediately frozen in isopentane on dry-ice and stored at -80°C. Brain sections (8 μm) were then cut on a cryostat (Leica Microsystems, Inc., Deerfield, IL) at -15°C, thaw mounted onto Superfrost Plus slides, air-dried and stored at -80°C until use for the autoradiographic studies described below.
Adult female squirrel monkeys (Saimiri sciureus) weighing between 0.6 and 0.8 kg were purchased from Worldwide Primates (Miami, FL) and quarantined for one month according to California State regulations. All animals were housed separately in a room maintained at 27 ± 3°C and humidity >30%, with a 13:11-h light/dark cycle. Food consisting of monkey chow, fruits and vegetables was given once daily, and water ad libidum. All procedures and treatments conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at the Parkinson's Institute.
Animals were randomly divided into four treatment groups: control (n = 3), nicotine-treated (n = 4), MPTP-lesioned (n = 4), and nicotine plus MPTP-lesioned (n = 4). Animals in the MPTP and nicotine plus MPTP groups received one injection of 2.0 mg/kg MPTP subcutaneously while the unlesioned groups (control and nicotine only) were given subcutaneous saline injections.
One month after MPTP lesioning, all animals were given commercially available Tang in the drinking water for ~10 days. Nicotine (free base) was then added to the drinking solution of the nicotine-treated groups starting at a concentration of 10 μg/ml and gradually increased to a final concentration of 650 μg/ml over 3 weeks. Animals were maintained on vehicle or nicotine for 2 months. Nicotine was withdrawn 24 hours before the animals were killed to minimize residual effects of nicotine in the binding assays.
Blood samples were taken after ~1 month on the final dose of nicotine. Blood from the femoral vein was drawn under ketamine anesthesia (15-20 mg/kg, im) and assayed for plasma cotinine as described above.
Animals were euthanized by anesthetic overdose. They were first given an intraperitoneal injection of 1.5 ml euthanasia solution (390 mg sodium pentobarbital and 50 mg phenytoin sodium/ml), followed by an intravenous injection of 2.2 ml/kg of the same solution. The brain was removed, rinsed in cold saline and sectioned along the midline as described (Quik et al., 2006b). From one half of the brain, 5 mg tissue samples were collected from medial and lateral caudate regions and ventral and dorsal putamen regions to measure striatal dopamine levels. The other half of the brain was subsequently dissected into 6 mm-thick blocks using stainless steel blades and a squirrel monkey brain mold. These blocks were then snap-frozen in isopentane on dry-ice and stored at -80°C. Frozen tissue blocks containing the striatum (level A15-A14) were then sectioned (20 μm thick) using a cryostat (Leica Microsystems, Inc., Deerfield, IL) and used for autoradiography.
Striatal dopamine was determined as previously described (Quik et al., 2006b). In brief, 5 mg tissue samples were placed in 0.25 ml of cold 0.4 M perchloric acid, sonicated and centrifuged at 14,000g for 12 min. Dopamine was then assessed in the supernatants using high pressure liquid chromatography with electrochemical detection (Coulochem II detector; ESA, Chelmsford, MA) (Quik et al., 2006b). Protein concentrations were determined using the method of Lowry (Lowry et al., 1951).
Binding of 125I-RTI-121 (2200 Ci/mmol; PerkinElmer Life and Analytical Science, Boston, MA) was used to evaluate the dopamine transporter. Sections were preincubated in 50 mM Tris-HCl, 120 nM NaCl and 5 nM KCl, twice for 15 min. The sections were then incubated in the same buffer but containing 0.025% BSA, 1 μM fluoxetine, and 50 pM 125I-RTI-121 for 2 hours.
For 125I-epibatidine binding (2200 Ci/mmol, Perkin Elmer Life and Analytical Science, Boston, MA. USA), slides were preincubated at 22°C for 30 min in buffer containing 50 mM Tris, pH 7.5, 120 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, and 1.0 mM MgCl2. They were then incubated for 40 min with 0.015-0.030 nM 125I-epibatidine in the presence or absence of α-conotoxinMII (α-CtxMII, 100 nM) to distinguish α3/α6β2* from α4β2* nAChRs.
125I-α-CtxMII binding (2200 Ci/mmol) and competition studies were performed as previous (Bordia et al., 2007). Competition studies in rats were done by using the α-CtxMII analog E11A (MII[E11A], 0.1 fM - 0.1 μM). MII[E11A] was added to pre-incubation (20 mM HEPES buffer, pH 7.5, containing 144 mM NaCl, 1.5 mM KCl, 2 mM CaCl2, 1 mM MgSO4, 0.1% bovine serum albumin and 1 mM phenylmethylsulfonyl fluoride) and incubation buffer (the same solution without phenylmethylsulfonyl fluoride). Sections were pre-incubated for 15 min and then incubated for 1 h with 0.5 nM 125I-α-CtxMII in buffer with 0.5% BSA, 5 mM EDTA, 5 mM EGTA, and 10 μg/ml each of aprotinin, leupeptin, and pepstatin A.
For all autoradiographic studies, washing of the sections was done as previously described (Quik et al., 2003; Bordia et al., 2007). After air-drying, slides were exposed to Kodak MR film for several days. 125I standards were included to allow for quantitation.
The ImageQuant (GE Healthcare, Little Chalfont, Buckinghamshire, UK) system was used to determine optical density measurements from the autoradiograms. Optical density readings were converted to fmoles/mg tissue using standard curves generated from 125I standards. The optical density readings were within the linear range of the film. Competition and statistical analysis (one-way ANOVA followed by a Newman-Keuls post-hoc test or two-way ANOVA followed by a Bonferroni post-hoc test) were performed using GraphPad Prism (GraphPad, San Diego, CA). Rat rotational behavioral data was analyzed by three-way ANOVA using SPSS (SPSS Inc., Chicago, IL). Data are the mean ± SEM. P ≤ 0.05 was defined as significant.
Although numerous studies have reported administration of nicotine in the drinking water (Pietila et al., 1996; Sparks and Pauly 1999; Pietila and Ahtee 2000; Brunzell et al., 2003; Lai et al., 2005), little work has been done to investigate such a dosing regimen in rats. Experiments were therefore initiated to determine an effective nicotine treatment regimen in rats (Table 1). Nicotine was provided in 1% saccharin to mask its bitter taste, with the dose gradually increased from 12.5 to 100 μg/ml to allow the rats to accommodate to the drug. The rats refused to drink the solution when the nicotine dose was >100 μg/ml. The rats were maintained at each final dose (12.5, 25, 50 and 100 μg/ml) for 2 weeks. Nicotine doses of 25 and 50 μg/ml yielded plasma cotinine levels (Table 1) similar to those in moderate to heavy smokers (Matta et al., 2007).
The rats were killed after 2 weeks on the final nicotine dose, and the two primary striatal nAChR subtypes measured as an index of nicotine effectiveness in the CNS. The α6β2* nAChR subtype, measured using 125I-α-CtxMII, was not altered in rats receiving 12.5 μg/ml nicotine. However, there were significant decreases in 125I-α-CtxMII binding with the higher nicotine doses including 25 μg/ml (p < 0.01), 50 μg/ml (p < 0.001) and 100 μg/ml (p < 0.001) (Fig 1A). We next evaluated the effect of chronic nicotine dosing on α4β2* nAChRs by measuring 125I-epibatidine binding in the presence of α-CtxMII. α-CtxMII resistant 125I-epibatidine binding sites or α4β2* nAChR binding sites were significantly increased (p < 0.001) in rats treated with 12.5 μg/ml nicotine or higher, compared to control (Fig. 1B). Since nicotine at 50 μg/ml yielded a maximal decrease in α6β2* and increase in α4β2* nAChRs, this dose was used for further study.
Previous studies have shown that nicotine administered via injection or minipump protects against 6-OHDA-induced nigrostriatal damage in rats (Costa et al., 2001; Ryan et al., 2001; Visanji et al., 2006). The objective of the present experiments was to determine whether nicotine in drinking water is also neuroprotective against nigrostriatal damage. Rats were treated according to the nicotine (50 μg/ml) dosing and lesioning schedule depicted in Fig. 2A. Analyses of the data showed that there was a significant main effect of lesioning (p < 0.001), with no main effect of nicotine-treatment. However, there was a significant interaction between nicotine and lesioning (p < 0.01). These results indicate that the enhanced amphetamine-induced rotation is not observed in nicotine-treated 6-OHDA-lesioned rats as compared to 6-OHDA-lesioned rats only.
By contrast, there was no improvement in amphetamine-induced turning behavior in rats that were first lesioned and subsequently given nicotine in the drinking water (Fig. 2B and and3B).3B). There was a significant main effect of 6-OHDA lesioning (p < 0.001) with no main effect of nicotine treatment. There was also no significant interaction between nicotine and 6-OHDA lesioning, suggesting that nicotine post-treatment did not affect rotational behavior.
We assessed the striatal dopamine transporter as a measure of the effects of nicotine and 6-OHDA lesioning on striatal dopaminergic integrity. Nicotine treatment alone did not affect 125I-RTI-121 binding (control - 6.83 ± 0.33 fmol/mg tissue, n = 6; nicotine - 6.18 ± 0.26 fmol/mg/tissue, n = 6), as expected, while 6-OHDA lesioning significantly decreased transporter levels (Fig. 4A, B, p < 0.001). Two-way ANOVA followed by a Bonferroni post-hoc test showed that nicotine pre-treatment significantly attenuated the decreases in 125I-RTI-121 observed with 6-OHDA lesioning (p < 0.05) (Fig. 4A). By contrast, if the rats were first lesioned and nicotine subsequently administered 2 weeks later, there was no improvement in striatal dopamine transporter measurements (Fig. 4B). These data suggest that nicotine reduces ongoing nigrostriatal degeneration as assessed by measurement of the transporter, but does not restore damaged dopaminergic neurons. For these studies, we did not examine neuroprotection at the level of dopaminergic cell bodies in the substantia nigra, as previous studies had shown that nicotine does not improve tyrosine hydroxylase positive neurons in the nigra, despite improvements in dopaminergic measures in the striatum (Visanji et al., 2006).
We also measured nAChRs in striatum of these same groups of rats. Nicotine treatment alone decreased 125I-α-CtxMII binding or α6β2* nAChRs (control - 3.92 ± 0.26 fmol/mg tissue, n = 6; nicotine - 2.05 ± 0.10 fmol/mg/tissue, n = 6, p < 0.001), yet increased 125I-epibatidine binding to α4β2* nAChRs (control - 28.7 ± 1.3 fmol/mg tissue, n = 6; nicotine - 34.5 ± 1.2 fmol/mg/tissue, n = 6, p < 0.01) in agreement with the data in Fig. 1 and previous findings (Mugnaini et al., 2006; Perry et al., 2007). The effect of nicotine treatment on receptor levels was then compared to the corresponding intact hemisphere. If the rats were treated with nicotine before lesioning with the nicotine continued throughout (Fig. 4C and 4E), there was a significant increase in α6β2* nAChRs (p < 0.001) and α4β2* nAChRs (p < 0.001) compared to rats not pre-treated with nicotine using two-way ANOVA followed by a Bonferroni post-hoc test. However, α6β2* and α4β2* nAChRs levels were not enhanced when nicotine was administered two weeks after 6-OHDA lesioning (Fig. 4D and 4F). These findings further support the possibility that nicotine protects against nigrostriatal damage when given prior to the toxic insult but not when administered after nigrostriatal damage is complete.
Our previous work had shown that nicotine administered prior to MPTP improved striatal dopaminergic measures and nAChRs, suggesting that nicotine treatment protected against ongoing nigrostriatal damage (Bordia et al., 2006; Quik et al., 2006a; Quik et al., 2006b). Studies were next done to determine whether nicotine enhanced dopaminergic integrity/function when administered after nigrostriatal damage in monkeys, a parkinsonian animal model that more closely resembles Parkinson's disease.
For these experiments, nicotine was administered via the drinking water after MPTP lesioning as detailed in Methods. The monkeys were euthanized and the effect of nicotine assessed on nigrostriatal integrity. To approach this, we measured striatal dopamine levels and nAChRs. The results in Fig. 5A show that nicotine administered after nigrostriatal damage did not improve dopamine levels in monkey striatum. Results shown are for caudate with similar results in the putamen. These data contrast to our previous results (inset), which show that nicotine pre-treatment improves dopamine in striatum of lesioned monkeys (Quik et al., 2006b). We did not evaluate neuroprotection at the level of dopaminergic cell bodies in the substantia nigra, as our previous work in monkeys had shown that nicotine does not improve nigral tyrosine hydroxylase positive neurons despite improvements in dopaminergic measures in the striatum (Quik et al., 2006b).
With respect to nAChRs, nicotine treatment alone resulted in a small nonsignificant decline in striatal α3/α6β2* receptors, with a significant decrease after MPTP lesioning (p < 0.01), as expected (Fig 5B). However, in contrast to our previous results (see inset) with nicotine pre-treatment (Bordia et al., 2006), nicotine post-treatment did not improve striatal α3/α6β2* or α-CtxMII-sensitive nAChRs in lesioned monkeys as compared to non-nicotine treated lesioned monkeys. Results shown in Fig. 5B are for caudate with similar results in the putamen. Striatal α-CtxMII-resistant or α4β2* nAChRs were also assessed in lesioned monkeys given nicotine when nigrostriatal damage was complete. Nicotine treatment alone increased α-CtxMII-resistant or α4β2* nAChRs (main effect of nicotine, p < 0.05), while lesioning decreased these receptors (main effect of MPTP, p < 0.05), as expected (Bordia et al., 2006). However, the increase with nicotine treatment was of a similar magnitude in both MPTP-lesioned and unlesioned animals. This is in contrast to our previous data which had shown that there was a greater increase in α4β2* nAChR levels in lesioned monkeys pre-treated with nicotine as compared to unlesioned animals receiving nicotine (Bordia et al., 2006).
Thus nicotine administered after nigrostriatal damage does not significantly improve striatal dopamine levels or nAChRs.
Our previous data had shown that there are two major α6β2* nAChR populations expressed in rat striatum, the α6α4β2* and α6(nonα4)β2* subtypes, which are differentially regulated by nicotine treatment (Perez et al., 2008). Therefore, a question that arose was whether nicotine-mediated neuroprotection was associated with one or both of these α6β2* nAChR subtypes. To address this, we performed an 125I-α-CtxMII competition binding assay using varying concentrations of MII[E11A] in striatal slices of 6-OHDA-lesioned and/or nicotine treated rats and compared the results to the corresponding control groups. These studies were only performed using rat striatum as sections from monkey striatum were not available.
MII[E11A] inhibition of 125I-α-CtxMII binding to control rat striatal sections yielded a two-site inhibition curve (data best fit to a two-site model) with >10,000-fold difference in affinity between sites in intact and sham-lesioned rat striatum (Fig. 6A, Table 2). Nicotine treatment alone to rats also led to a biphasic curve but showed a decline in both the α6α4β2* and α6(nonα4)β2* nAChRs (Fig. 6B). Our previous results had shown that only α6α4β2* nAChRs were decreased with nicotine treatment (Perez et al., 2008). This difference may be due to the lower dose of nicotine used in the present compared to the previous study (50 μg/ml versus 100 μg/ml), as well as the longer nicotine dosing period currently used (~8 weeks versus 2 weeks). In rats lesioned with 6-OHDA, the two binding sites were no longer distinguished by MII[E11A], with a single IC50 similar to that of the α6(nonα4)β2* receptor subtype (Fig. 6A, Table 2), suggesting that the α6α4β2* receptor subtype was lost with 6-OHDA lesioning. In rats treated with nicotine when the lesion was complete, a monophasic inhibition curve similar to that with 6-OHDA lesioning alone was obtained (Fig. 6D). By contrast, nicotine pre-treatment yielded a two-site curve, although receptor levels of both the α6α4β2* and α6(nonα4)β2* populations were decreased (Fig. 6C). These combined data show that the α6α4β2* nAChR population is associated with nicotine-mediated neuroprotective effects.
The overall goal of the present studies is to investigate whether nicotine treatment improves striatal dopaminergic markers when administered to rats and monkeys with existing nigrostriatal dopaminergic damage. To evaluate this possibility, we gave nicotine in the drinking water since this mode of administration offers the advantage that it can readily be done on a long-term basis, is not stressful to the animals and provides nicotine in a pulsatile fashion. Moreover, this mode of treatment has extensively been used in mice with reproducible effects on nicotine-mediated behaviors and molecular measures in the brain, including changes in nAChR levels (Pietila et al., 1996; Sparks and Pauly, 1999; Pietila and Ahtee, 2000; Brunzell et al., 2003; Lai et al., 2005). Administration of nicotine via the drinking water has also successfully been used in monkeys over the long term (Quik et al., 2006a; Quik et al., 2006b; Bordia et al., 2006). However, no nicotine dose ranging studies have as yet been reported in rats. The present results show that an effective nicotine range for rats is ~10 fold lower than for mice. Doses of 12.5 to 50 μg/ml nicotine in the drinking water yielded rat plasma cotinine levels similar to that in plasma of moderate to heavy smokers (Matta et al., 2007). By contrast, doses of 100 to 500 μg/ml nicotine are generally given to mice using this same route of administration (Pietila et al., 1996; Sparks and Pauly, 1999; Pietila and Ahtee, 2000; Brunzell et al., 2003; Lai et al., 2005), Importantly, these lower doses of nicotine to rats yielded changes in receptor expression similar to those observed with the higher doses in mice. Rat striatal α4β2* nAChRs, assessed using 125I-epibatidine binding, were increased with nicotine administered in the drinking water, while α6β2* nAChRs or 125I-α-CtxMII binding sites were significantly decreased (Nguyen et al., 2004; Mugnaini et al., 2006; Perry et al., 2007; Mao et al., 2008). Since we obtained maximal receptor changes with the 50 μg/ml nicotine dose, this was used to evaluate neuroprotective and neurorestorative effects of nicotine against nigrostriatal damage.
Previous studies in rats had shown that nicotine injection or minipump administration protected against nigrostriatal damage (Janson et al., 1988; Costa et al., 2001; Ryan et al., 2001; Abin-Carriquiry et al., 2002; Soto-Otero et al., 2002; Visanji et al., 2006); however, the effect of nicotine administered via the drinking water had not yet been tested. Rats were pre-treated with nicotine, the nigrostriatal system then lesioned, with the nicotine continued until the rats were killed. 6-OHDA-induced behavioral changes were not present in lesioned rats pre-treated with nicotine. As well, declines in the dopamine transporter and nAChRs were partially attenuated compared to the intact hemisphere. Thus, nicotine given via the drinking water effectively improves striatal measures in parkinsonian rats, as it had in lesioned monkeys (Bordia et al., 2006; Quik et al., 2006a; Quik et al., 2006b). Interestingly, only a partial increase of the striatal dopamine transporter and nAChRs are associated with an almost complete return of normal motor function. This may suggest that only a small enhancement of striatal integrity results in significant improvements in Parkinson's disease motor symptoms. Such findings are in agreement with previous work which shows that Parkinson's disease motor symptoms only develop with ~70% or greater losses in striatal dopaminergic measures (Singh et al., 2007). In addition to dopaminergic neuroprotection, it is also possible that the improvements in rat motor behavior are due to alternate molecular mechanisms linked with nicotine treatment.
The question next arose whether nicotine exerted its beneficial effects by preventing ongoing nigrostriatal damage, restoring damaged neurons, or through both these mechanisms. To address this, experiments were done in which nicotine was administered in the drinking water 2-3 weeks after the dopaminergic lesion when nigrostriatal damage is essentially complete. The present data show that under these conditions, turning behavior was not reduced with nicotine treatment nor was there an improvement in markers of striatal integrity, including the dopamine transporter and nAChRs. Thus, nicotine administered after the nigrostriatal insult is complete does not appear to be neurorestorative, at least in unilateral 6-OHDA-lesioned rats. Because the monkey model offers the advantage that it more closely resembles Parkinson's disease, we also did experiments in which lesioned monkeys were given nicotine administered at a time when the effects of lesioning are maximal. Again, no improvement in striatal measures was observed using such a paradigm. These combined results in rats and monkeys suggest that nicotine exposure primarily protects against ensuing nigrostriatal damage but does not restore dopaminergic neurons once they are destroyed. These data are important as they suggest that nicotine would be most useful in early Parkinson's disease management, at which time it could most effectively attenuate the neurodegenerative process. This could have considerable functional ramifications since the rat studies show that small improvements (25%) in dopaminergic integrity result in normal motor function.
As mentioned in the introduction, the two major nAChR populations in the rodent and monkey striatum are the α4β2* and α6β2* subtypes, with the latter consisting primarily of the α6α4β2* and α6(nonα4)β2* nAChRs (Gotti et al., 2007; Grady et al., 2007; Quik et al., 2007a). Since these two α6β2* subtypes are differentially affected by nigrostriatal damage (Bordia et al., 2007) and by nicotine treatment (Perez et al., 2008), we asked whether one or other of these subtypes may be more closely linked to neuroprotection versus neurorestoration. To address this, 125I-α-CtxMII competition studies with MII[E11A], an analog that can differentiate between the α6α4β2* and α6(nonα4)β2* subtypes were performed (Bordia et al., 2007). We focused on the rat because of the more ready availability of multiple brain sections from this species. The results show that the α6α4β2* subtype is expressed only under conditions in which nicotine-mediated protection is observed, that is, with the nicotine pre-treatment regimens. Thus, this latter subtype may be related to neuroprotection and an important target for the development of protective strategies against Parkinson's disease.
The question arises how an interaction at α6α4β2* nAChR confers a protective action against nigrostriatal damage. Since expression studies with this and other α6β2* nAChRs have proved difficult to date, information is lacking concerning the relevant signaling pathways. On the other hand, it is well known that other nAChR subtypes exert their effect by modulating a diversity of downstream pathways, depending on the nAChR subtype involved. This may include alterations in cellular kinases such as phosphatidylinositol 3-kinase (PI3K), Akt, mitogen-activated protein kinase (MAPK) and jnk kinase, caspases 3, 8 and 9, nitric oxide synthase, the cell survival protein Bcl-2, and other signalling molecules (Dajas-Bailador and Wonnacott, 2004; Quik et al., 2007b; Picciotto and Zoli, 2008). These in turn may modulate brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor-2 (FGF-2) levels in brain dopaminergic regions. Alterations in the levels of these trophic factors could potentially mediate nicotine's neuroprotective action against nigrostriatal damage (Belluardo et al., 1999b; Belluardo et al., 1999a; Belluardo et al., 2004; Zhou et al., 2004; Massey et al., 2006). Nicotine has also been shown to modulate immune responsiveness in both the peripheral and central nervous system (Shytle et al., 2004; Gahring and Rogers, 2005; Tracey, 2007; Vincler et al., 2006). This represents another mechanisms whereby nicotine may be neuroprotective since immune molecules have been associated with trophic effects (Gahring and Rogers, 2005; Tracey, 2007).
In summary, nicotine administered via the drinking water protects against nigrostriatal damage in 6-OHDA-lesioned rats when given prior to lesioning, similar to results in MPTP-lesioned monkeys. Thus protection against ongoing nigrostriatal damage is consistently observed independent of the animal model and neurotoxin used to induce nigrostriatal damage. However, nicotine given when nigrostriatal damage is complete does not restore dopaminergic integrity/function once damage has occurred. Since Parkinson's disease is a progressive disorder, nicotine treatment at the early stages of the disease may be beneficial to prevent further degeneration.
This work is supported by NIH grants NS42091 and NS47162 to MQ, and MH53631 and GM48677 to JMM.
The asterisk denotes the possible presence of other subunits in the nAChR complex.