• nicotine;
  • nicotinic;
  • non-human primates;
  • MPTP;
  • Parkinson's disease;
  • striatum


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The present studies were done to investigate the effect of long-term nicotine treatment against nigrostriatal damage in non-human primates. Monkeys were administered nicotine in drinking water for 6 months to provide chronic but intermittent delivery as with smoking. Plasma nicotine levels ranged from 10 to 15 ng/mL, which were within the range in cigarette smokers. Animals were then lesioned with low doses of the dopaminergic neurotoxin MPTP for several months while nicotine was continued. The results showed that levels of striatal tyrosine hydroxylase, dopamine transporter, vesicular monoamine transporter, dopamine and nicotinic receptors were greater in nicotine-treated MPTP-lesioned primates than in lesioned animals not receiving nicotine. Nicotine had no effect in unlesioned animals. Monoamine oxidase activity was similar in unlesioned and lesioned animals treated with or without nicotine, suggesting that nicotine did not exert its effects through changes in MPTP or dopamine metabolism. MPTP-induced cell loss in the substantia nigra was unaffected by nicotine treatment, indicating that nicotine acts at the striatal level to restore/maintain dopaminergic function. These data further support the possibility that nicotine contributes to the lower incidence of Parkinson's disease in smokers.

Abbreviations used





dorsal putamen


glial cell line-derived neurotrophic factor


lateral caudate


medial caudate




tyrosine hydroxylase


ventral putamen

Tobacco use is generally known for its detrimental health-related effects. However, a large series of epidemiological studies over the past 50 years have shown an inverse relationship between smoking and Parkinson's disease, a progressive neurodegenerative movement disorder for which only symptomatic therapy is currently available (Lang and Obeso 2004; Olanow 2004; Samii et al. 2004). This decreased incidence (∼ 50%) of Parkinson's disease with tobacco use is reproducible, dose related and does not appear to be explained by selective mortality (Morens et al. 1995; Checkoway and Nelson 1999; Allam et al. 2004). The critical question is what agent in smoke is responsible, as its identification may provide a means to protect against the dopaminergic nigrostriatal damage that characterizes Parkinson's disease. However, tobacco contains numerous chemicals (∼ 4000), any one of which has the potential to alter biological processes and yield an apparent neuroprotective effect. One possibility, which is the focus of considerable research, is that nicotine in tobacco products may play a role.

Extensive evidence shows that nicotine exposure protects against toxic insults in cell culture systems including dopaminergic nigral neurons (O'Neill et al. 2002; Quik 2004). In vivo work to evaluate nicotine's protective potential against nigrostriatal damage has, however, yielded conflicting results, with protection observed in some studies (Janson et al. 1988, 1992; Carr and Rowell 1990; Shahi et al. 1991; Costa et al. 2001; Parain et al. 2001, 2003; Ryan et al. 2001), but not others (Behmand and Harik 1992; Janson et al. 1992; Hadjiconstantinou et al. 1994). These inconsistencies may be explained by inadequate nicotine dosing (too low or too high) and/or inappropriate nicotine treatment regimens, with more reproducible protection observed with frequent, intermittent rather than single dosing (Costa et al. 2001; Parain et al. 2001, 2003). Lesion size may also be an important factor, with the most robust protection observed with small chronic lesions (Costa et al. 2001). Coincidently, the factors that maximize the neuroprotective effects of nicotine in rodent studies resemble those encountered in humans who smoke and do not have clinically overt Parkinson's disease, that is frequent nicotine dosing during the day (as with smoking) and a slow progressive lesion.

Because of the importance of determining whether there is a link between nicotine and the reduced incidence of Parkinson's disease, we recently performed a study in non-human primates with nigrostriatal damage. This model offers the advantage of similar behavioral deficits to those in Parkinson's disease (Langston et al. 2000; Quik 2004). Moreover, nicotine is metabolized with a similar time course to that in humans (Schoedel et al. 2003; Hukkanen et al. 2005). Nicotine was administered over a 6-month period before MPTP treatment, and the animals were subsequently lesioned with MPTP over several months while nicotine treatment was continued. Nicotine administration significantly normalized aberrant striatal dopaminergic activity, including nicotine- and potassium-evoked dopamine release, dopamine turnover and synaptic plasticity in MPTP-treated non-human primates (Quik et al. 2006).

The present experiments were done to understand the molecular changes associated with the functional improvements described above. To approach this, we measured dopaminergic nerve terminal markers, including tyrosine hydroxylase (TH), the dopamine transporter, vesicular monoamine transporter, dopamine levels and nicotinic receptors, in striatum of unlesioned and lesioned monkeys treated with and without nicotine.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Animals and drug treatment

Twenty-six adult female squirrel monkeys (Saimiri sciureus) weighing from 0.5 to 0.8 kg were purchased from Osage Research Primates (Osage Beach, MO, USA) or the University of South Alabama (Mobile, AL, USA). The animals' record, coupled with their general appearance (dentition, fur, other), indicated that they were in late adulthood (10–12 years, lifespan ∼15–18 years), an age more relevant to Parkinson's disease. All animals were housed separately under a 13–11-h light–dark cycle, with free access to water. Monkey food chow and fruit/vegetables was provided once daily, as was standard for these animals. After quarantine, the monkeys were assigned randomly to the different treatment groups. To quantitate baseline motor activity, the monkeys were monitored in their home cage for a 6-h period for 10 days using a computerized webcam monitoring system (Togasaki et al. 2005). All animals were then given drinking solution containing 1% saccharin to mask nicotine's bitter taste. Because the study was chronic, the lowest dose of saccharin at which the monkeys tolerated the nicotine was used. The saccharin solution was given to all animals for 1 week. The controls were then continued on saccharin while nicotine (free base) was added to the saccharin solution of the treated group, with the nicotine saccharin solution adjusted to pH 7.0 throughout the course of the study. Nicotine dosing was started at 25 µg/mL for 1 week and 50 µg/mL for 1 week, with the concentration increased by 50-µg increments/week over ∼ 3–4 months, up to 650 µg/mL. This final nicotine dose was selected because the plasma nicotine levels fell within the range found in smokers (Table 1). Because the animals were relatively old and exhibited poor dentition, it was necessary to soften the dried food pellets with fluid. The food pellets of all animals were therefore moistened with ∼ 25 mL of either saccharin or nicotine–saccharin solution (for treated animals) to ensure adequate nicotine intake. All animals were routinely weighed to ensure that there was no weight loss, and consequently appropriate food intake.

Table 1.   Chronic nicotine treatment results in plasma nicotine and metabolite levels similar to those in smokers
TreatmentLesionNo. of primatesPlasma nicotine (ng/mL)Plasma cotinine (ng/mL)
  1. Animals were given nicotine in their drinking water, which also contained 1% saccharin to mask nicotine's bitter taste. Nicotine was started at a dose of 25 µg/mL and gradually increased to 650 µg/mL, a concentration at which the levels of plasma nicotine and cotinine (the primary nicotine metabolite) were similar to those in smokers. Nicotine was given for 6 months before MPTP, which included 3 months at the final nicotine dose. MPTP was then administered using a multiple low-dose regimen of 1.5 mg/kg, every 6–8 weeks for a total of three doses. Nicotine was included in the drinking water throughout MPTP treatment, until the animals were killed ∼ 2 months after the final MPTP injection. Nicotine and cotinine were measured using HPLC. Values are mean ± SEM.

ControlSaline70.6 ± 0.50 ± 0
NicotineSaline610.9 ± 1.7383.3 ± 94.6
ControlMPTP70.1 ± 0.10 ± 0
NicotineMPTP614.2 ± 1.9552.4 ± 56.9

The animals were maintained on the final nicotine dose for 3 months and subsequently injected with MPTP (1.5 mg/kg s.c.) to produce a lesion. The animals' weights were monitored to ensure adequate food and fluid intake. When necessary, animals were gavaged or syringe fed with Ensure© nutrition drink, also containing nicotine in the case of nicotine-treated animals only. MPTP administration was repeated at ∼ 2-month intervals for a total of three doses. Activity monitoring was repeated before treatment and at bimonthly intervals during the MPTP treatments. The animals were also rated for parkinsonism (Langston et al. 2000), before and 3 weeks after each MPTP or saline administration. They were killed ∼ 2 months after the last MPTP injection, or ∼ 6 months after initiation of the lesioning process with nicotine treatment present throughout the entire period. Animals were killed according to the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association. Animals were first given an intraperitoneal injection of solution containing 390 mg/mL sodium pentobarbital and 50 mg/mL phenytoin sodium, followed by 2.2 mL/kg of the same solution intravenously. All procedures used in this study 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.

Measurement of plasma nicotine and metabolites

Plasma nicotine and metabolite levels were measured throughout the course of the nicotine treatment regimen. Blood (1–2 mL) was drawn from the femoral vein under ketamine sedation (15–20 mg/kg i.m.) at ∼ 15:00 hours. Blood samples were centrifuged at 1000 × g for 12 min, and the plasma (∼ 600 μL) stored at − 80°C. Samples were analyzed using a modified solid-phase extraction followed by HPLC analyses, as described previously (Schoedel et al. 2003).

Tissue preparation

The brain was rapidly removed and sectioned sagitally along the midline. One half was placed in a brain mold and sliced into 6-mm thick coronal sections. The blocks were quickly frozen on glass slides in isopentane on dry ice, and used for immunohistochemical and autoradiographic studies. The midbrain was dissected from the other half and fixed for substantia nigra dopaminergic cell counting. The anterior portion of this half of the brain was sliced into 2-mm sections in the brain mold, and the caudate and putamen dissected for determination of dopamine/metabolite levels, western analyses and monoamine oxidase activity.

Nicotinic receptor binding and quantitation

Binding of 125I-labeled epibatidine to striatal sections (20 µm) was done as detailed previously (Davila-Garcia et al. 1997; Quik et al. 2000). Thawed sections were preincubated for 30 min, followed by 40 min incubation in standard buffer containing 125I-labeled epibatidine (0.015 nm, 2200 Ci/mmol; Perkin Elmer Life and Analytical Sciences, Boston, MA, USA). Sections were then washed with buffer and cold deionized H2O. After air-drying, slides were exposed to Kodak MR (Perkin Elmer Life Sciences, Boston, MA, USA) film for 2–5 days, together with 125I-standards. Nicotine (100 µm) was used to determine non-specific binding.

The optical density values from the autoradiograms were determined using an ImageQuant system (Molecular Dynamics, Sunnyvale, CA, USA), and were assessed by subtracting background from tissue values. These values were converted to nCi/mg tissue using standard curves generated from 125I standards. The optical density readings were within the linear range of the film. The receptor binding values (fmol/mg tissue) for the appropriate brain regions from each animal were averaged from two to four independent experiments.

TH immunocytochemistry

Fresh-frozen tissue sections (20 µm) were fixed in 4% paraformaldehyde. They were then preincubated in 4% normal goat serum for 40 min at room temperature (25°C), followed by overnight incubation at 4°C with a 1 : 600 dilution of a rabbit polyclonal antibody to TH (Pel Freez, Rogers, AR, USA). Control sections were incubated in the presence of non-immune IgG to ensure specificity. Antibody dilution was in 0.1 m phosphate-buffered saline, containing 1% bovine serum albumin and 0.1% Triton X-100. Sections were rinsed in phosphate-buffered saline and bound immunoglobulins visualized using the avidin–biotin immunoperoxidase reaction with the Vectastain ABC-peroxidase kit using 3,3′-diaminobenzidine as the chromagen (Vector, Burlingame, CA, USA). The sections were then dehydrated and coverslipped.

Stereological cell counting

The substantia nigra was immersion fixed in 4% paraformaldehyde for 3 days and 10% formalin for 10 days at 4°C. After cryoprotection, 40-µm horizontal sections were cut using the entire substantia nigra and every 12th section processed for TH immunohistochemistry (McCormack et al. 2004). Stereological analysis of the substantia nigra was then done using the delineations described previously (McCormack et al. 2004). Neurons were sampled using a 100 × oil immersion lens with a high numerical aperture (1.4), with the nucleolus used as the sampling unit.

Western analyses

Striatal samples (5–20 µg protein) were sonicated in lysis buffer (5 mm EDTA, 0.1 mm phenylmethylsulfonyl fluoride, 25 mm Tris, pH 7.2) and processed for sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The blots were subsequently exposed to one of the following primary antibodies: 1 : 3000 anti-TH, 1 : 10 000 anti-dopamine transporter, 1 : 4000 anti-vesicular monoamine transporter 2, or 1 : 10 000 anti-β-tubulin (to confirm equal loading). Secondary anti-mouse, anti-rat or anti-rabbit IgG conjugates were applied as appropriate. The blots were incubated with a chemiluminescent substrate and exposed to Hyperfilm ECLTM (Pierce, Rockford, IL, USA). Optical densities were determined using the ImageQuant program (Molecular Dynamics) and expressed as arbitrary units. Protein was determined using the bicinchononic acid assay (Pierce, Rockford, IL, USA).

Dopamine determination

Striatal tissue samples (5 mg) were sonicated in 0.25 mL 0.4 N perchloric acid (McCormack et al. 2004). The homogenates were centrifuged at 15 000 g for 12 min at 4°C, and the supernatants used for dopamine measurements by HPLC coupled to electrochemical detection. The pellets were resuspended in 250 µL 0.5 N NaOH, sonicated for 2–3 s, and assayed for protein using bovine serum albumin as standard.

Monoamine oxidase activity

Striatal tissue (∼ 5 mg) was homogenized in 50 mm sodium phosphate buffer, pH 7.4 (Quik and Di Monte 2001). A 30-µL aliquot was preincubated in buffer (150 µL) for 7 min, followed by a 20 min incubation period with 20 µL [14C]phenylethylamine (final concentration 0.25 mm). The reaction was terminated with 150 µL 2 m citric acid. Blanks were determined in the presence of 10 mm pargyline, or with no tissue. The products were extracted into 1.0 mL of a 1 : 1 solution of toluene and ethyl acetate. A 0.6-mL aliquot of the organic phase was counted using a liquid scintillation counter.

Data analysis

Results are expressed as mean ± SEM for the indicated number of animals. Statistical analyses were done with GraphPad Prism (GraphPad, San Diego, CA, USA) using two-way anova followed by Bonferroni post hoc test; p≤ 0.05 was considered significant.


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Nicotine treatment and MPTP lesioning

Nicotine was administered in the drinking water to provide a long-term pulsatile mode of delivery. Oral administration also offers the advantage that it is less stressful than injection, does not involve surgical manipulation, and is a method of treatment readily amenable to therapeutics in humans. The concentration of nicotine in the drinking water was gradually increased over a 3-month period to 650 µg/mL, and then maintained at this level for a further 3 months before MPTP. This final dose yielded plasma nicotine levels between 10 and 15 ng/mL (Table 1), which fall within the range of those in smokers (5–50 ng/mL) (Hukkanen et al. 2005). There were no obvious adverse effects of nicotine treatment on the animals. Fluid consumption was evaluated throughout the course of nicotine dosing regimen and was not significantly affected (p = 0.152), as shown previously (Matta et al. 2006). The animals were weighed routinely throughout the course of the study, at a minimum of once weekly. There were no significant weight changes throughout the course of the study. Body weights were similar (p = 0.152) among the four groups (controls 659 ± 35 g, n = 7; nicotine 568 ± 41 g, n = 6; MPTP lesioned 634 ± 23 g, n = 7; nicotine plus MPTP 668 ± 24 g, n = 6). Although the weights of the nicotine only group were somewhat lower than those of the other groups, this was not significant and most likely due to inherent variability between outbred animals. In addition, the body weights of the animals in the nicotine plus MPTP group were similar to those of controls.

Because Parkinson's disease is a chronic neurodegenerative disorder (Lang and Obeso 2004; Olanow 2004; Samii et al. 2004), a relatively long-term nigrostriatal lesioning paradigm was selected. Animals were given three low doses (1.5 mg/kg) of MPTP, a neurotoxin that selectively destroys the nigrostriatal dopaminergic system (Langston 1996). Toxin was administered at 6–8-week intervals for a total of three doses. Because our goal was to determine whether nicotine treatment attenuates nigrostriatal damage in analogy to smoking in humans, these studies focused only on the preclinical or asymptomatic stage of the disease, that is nigrostriatal damage with no detectable parkinsonism. Thus, animals were not parkinsonian with behavioral scores as follows (based on a 20-point rating scale, with 0 being normal): controls 0.7 ± 0.2 (n = 7), nicotine 1.0 ± 0.3 (n = 6), MPTP lesioned 1.6 ± 0.2 (n = 7) and nicotine plus MPTP 1.2 ± 0.1 (n = 6). Motor activity (pixel changes × 106), also evaluated using a computerized monitoring system (Togasaki et al. 2005), was similar (p = 0.704) between the groups (controls 39.9 ± 8.1, n = 7; nicotine 27.9 ± 5.5, n = 6; MPTP lesioned 39.1 ± 7.7, n = 7; nicotine plus MPTP 37.8 ± 6.3, n = 6). This also indicates that chronic nicotine treatment did not affect baseline motor activity.

Nicotine treatment increases nicotinic receptors in cortex and striatum from control and MPTP-lesioned animals

Receptor studies were done using 125I-labeled epibatidine, a radioligand that labels most CNS nicotinic receptors, that is those expressing α2–α6 subunits (Perry and Kellar 1995; Davila-Garcia et al. 1997). An increase was observed in 125I-labeled epibatidine binding (Figs 1a and b) in cortex from both unlesioned and lesioned monkeys, with a significant main effect of nicotine (F = 36.3; d.f. = 1,22; p < 0.001). There was no effect of MPTP on 125I-labeled epibatidine-binding sites in cortex (F = 0.03; d.f. = 1,22; p = 0.88) as the toxin does not damage non-dopaminergic terminals.


Figure 1.  Chronic nicotine treatment increases nicotinic receptors in control and lesioned primate brain. Animals were given nicotine in drinking water as detailed in Table 1 and subsequently administered MPTP, with nicotine treatment continued until the animals were killed. (a) Autoradiograms of coronal brain sections depicting binding of 125I-labeled epibatidine, a radioligand that measures nicotinic receptors. A schematic of the different striatal areas and cortex (CX) is provided on the left, showing medial caudate (MC), lateral caudate (LC), ventral putamen (VP) and dorsal putamen (DP). The colored bar denotes increasing image intensity in the sequence blue, yellow, red and black. The autoradiograms are representative of at least three independent experiments. Con, control. (b) Quantitative analyses showed that nicotine (Nic) treatment increased cortical nicotinic receptors. (c, d) Striatal nicotinic receptors were decreased with nigrostriatal damage (MPTP) and increased with nicotine treatment. Values are mean ± SEM (n = 5–7 animals). **p < 0.01, ***p < 0.001 versus saline control; †††p < 0.001 versus MPTP alone (two-way anova followed by Bonferroni post hoc test).

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An increase in nicotinic receptors was observed in caudate and putamen of unlesioned primates after nicotine treatment (Figs 1c and d), consistent with previous studies in rodents (Marks et al. 1983; Schwartz and Kellar 1983; Gentry and Lukas 2002). MPTP treatment alone decreased nicotinic receptors because these receptors are present on dopaminergic terminals destroyed by the toxin (Quik et al. 2001). This decline was not observed in lesioned monkeys treated with nicotine, whose levels of striatal nicotinic receptors were similar to those in unlesioned monkeys not receiving nicotine. Quantitative analyses (Figs 1c and d) using two-way anova yielded a significant main effect of both MPTP lesioning (p < 0.001) and nicotine treatment (p < 0.001) in all striatal areas. Nicotine treatment to lesioned animals thus leads to striatal nicotinic receptor levels similar to those in unlesioned animals not receiving nicotine.

Increased levels of dopaminergic markers in striatum of nicotine-treated lesioned animals compared with lesioned animals not receiving nicotine

We next assessed effects of nicotine on markers of striatal dopaminergic neurons using immunocytochemistry and/or western analyses. Nicotine treatment alone did not affect the TH signal, whereas MPTP lesioning alone led to a decline in striatal immunoreactivity (Fig. 2a). In contrast, a more robust TH immunoreactivity was observed in striatum of lesioned animals treated with nicotine compared with that in monkeys that received MPTP but not nicotine (Fig. 2a). Because immunocytochemical results are difficult to quantify, we used western analyses to evaluate changes in TH protein levels (Figs 2b and c). Two-way anova yielded a significant main effect of MPTP (F = 50.8; d.f. = 1,21; p < 0.001), consistent with dopamine nerve terminal loss, with a decrease in TH levels in lesioned animals compared with those in unlesioned animals. Although there was no significant main effect of nicotine treatment (F = 3.25; d.f. = 1,21; p > 0.05), there was a significant interaction (F = 5.90; d.f. = 1,21; p < 0.05). Thus, nicotine treatment alone did not change TH protein (Fig. 2c, Table 2). However, it differentially affected TH levels in MPTP-treated animals, with less depletion in lesioned primates receiving nicotine (Fig. 2c). The western data shown are for ventral putamen, and similar results were obtained in the other striatal areas (Fig. 3a).


Figure 2.  Increase in striatal TH in lesioned primates treated with nicotine. (a) Representative sections depicting enhanced TH immunoreactivity in striatal sections in a nicotine-treated primate after MPTP lesioning (Nic + MPTP). (b) Western blot depicting changes in TH, the rate-controlling enzyme in dopamine synthesis. Nicotine (Nic) treatment had no effect compared with control (Con), whereas MPTP lesioning resulted in a decrease. The MPTP-induced decline in TH was less severe in animals that also received nicotine (Nic + MPTP). Each band represents the signal from an individual animal with the designated treatment. As a control, western blots were probed with the cellular marker β-tubulin (tubulin); similar band intensities were observed for all conditions. The blot shown is for the ventral putamen, with similar results in the other areas (see Fig. 3a), and is representative of three independent experiments. (c) Quantitative analyses of western blots as described above for ventral putamen. Nicotine treatment did not alter TH protein in unlesioned animals. In contrast, it significantly increased TH levels in MPTP-lesioned primates. Values are mean ± SEM (n = 5–7 animals), with the values for each animal representing the average from three independent experiments. ***p < 0.001 versus saline; †p < 0.05 versus MPTP alone (two-way anova followed by Bonferroni post hoc test).

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Table 2.   Effect of chronic nicotine dosing on levels of TH, dopamine transporter, vesicular monoamine transporter and dopamine in unlesioned monkeys
RegionTreatmentTH (% control)Dopamine transporter (% control)Vesicular monoamine transporter (% control)Dopamine (% control)
  1. Animals were given saccharin only (control) or saccharin plus nicotine in the drinking water using a dosing regimen as described in Materials and Methods. Values are mean ± SEM (n = 5–7 animals).

Medial caudateControl100.0 ± 4.1100.0 ± 13.1100.0 ± 6.4100.0 ± 3.2
Nicotine93.2 ± 7.7102.1 ± 25.985.8 ± 12.7114.4 ± 8.4
Lateral caudateControl100.0 ± 10.3100.0 ± 14.4100.0 ± 19.2100.0 ± 6.2
Nicotine99.7 ± 5.989.2 ± 12.094.8 ± 20.8101.8 ± 9.5
Ventral putamenControl100.0 ± 5.0100.0 ± 5.8100.0 ± 21.7100.0 ± 10.3
Nicotine95.3 ± 9.096.2 ± 13.3124.4 ± 20.9110.0 ± 11.4
Dorsal putamenControl100.0 ± 6.3100.0 ± 6.3100.0 ± 9.0100.0 ± 11.5
Nicotine106.9 ± 7.3116.8 ± 9.9121.1 ± 15.8116.1 ± 10.3

Figure 3.  Increase in striatal dopaminergic protein markers in lesioned primates treated with nicotine compared with lesioned animals not receiving nicotine. (a) Quantitative analyses of western blots for TH. Two-way anova yielded a significant main effect of nicotine (F = 8.5; d.f. = 1,44; **p < 0.01). (b) Similar analyses for the dopamine transporter, a protein responsible for the reuptake of dopamine into the nerve terminal, yielded a significant main effect of nicotine (F = 5.55; d.f. = 1,44; *p < 0.05). (c) Analyses of the results for the vesicular monoamine transporter, a protein critical for dopamine storage in the nerve terminal, also yielded a significant main effect of nicotine (F = 5.86; d.f. = 1,44; p < 0.05). Nicotine had no effect on any of these measures in unlesioned animals (see Table 2), suggesting that nicotine administration selectively affects compromised dopaminergic terminals. Values are mean ± SEM (n = 5–7 animals).

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To determine whether the effect of nicotine extended to other striatal dopamine nerve terminal markers, we next measured levels of dopamine transporter and vesicular monoamine transporter using western analyses (Figs 3b and c). Nicotine treatment alone did not change either transporter in any striatal region in unlesioned animals (Table 2). MPTP treatment, on the other hand, led to significant decreases in both these measures (Figs 3b and c). Interestingly, both transporters were reduced to a smaller extent in nicotine-treated lesioned animals compared with animals treated with MPTP but not nicotine (Figs 3b, c). Thus, the pattern of change in the dopamine transporter and vesicular monoamine transporter in nicotine-treated lesioned animals was similar to that for TH, with higher levels compared with those in lesioned animals not receiving nicotine.

We next investigated the effect of chronic nicotine treatment on striatal dopamine levels (Fig. 4). Again, no change was observed with nicotine treatment alone (Table 2), whereas MPTP lesioning decreased dopamine levels by 70–95%. Notably, dopamine levels were greater in nicotine-treated lesioned primates than in MPTP-lesioned animals not receiving nicotine.


Figure 4.  Increase in striatal dopamine levels in lesioned primates treated with nicotine compared with lesioned animals not receiving nicotine. Dopamine levels were measured in the different striatal areas using HPLC. Two-way anova yielded a significant main effect of nicotine (F = 16.2; d.f. = 1,43; ***p < 0.001). Nicotine treatment did not induce any significant change in dopamine levels in unlesioned animals. Values are mean ± SEM (n = 5–7 animals).

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Nicotine treatment does not alter the number of TH-positive nigral neurons in MPTP-lesioned animals

Stereological cell counting was done based on both TH immunoreactivity and neuromelanin content (McCormack et al. 2004). The total number of substantia nigra dopaminergic neurons (unilateral) was similar in control and nicotine-treated animals (Table 3). MPTP treatment significantly reduced cell numbers to 49 056 ± 1008 (n = 3), with similar values in MPTP-lesioned animals treated with nicotine (46 959 ± 1812, n = 3). Analyses of these data showed a significant main effect of MPTP lesioning (F = 132.6; d.f. = 1,9; p < 0.001), with no effect of nicotine administration (F = 3.469; d.f. = 1,9; p = 0.095), nor an interaction between MPTP and nicotine treatments (F = 0.011; d.f. = 1,9; p = 0.917). These results suggest that nicotine does not prevent nigral neuronal cell loss, but acts selectively at the striatal dopaminergic nerve terminal level.

Table 3.   Effect of chronic nicotine treatment on total number of substantia nigra neurons
TreatmentLesionNo. of animalsNo. of nigral dopaminergic neurons
  1. Animals were given saccharin only (control) or saccharin plus nicotine in the drinking water using a dosing regimen as described in Materials and Methods. Nicotine was given for 6 months before MPTP, which included 3 months at the final nicotine dose. MPTP was then administered using a multiple low-dose regimen of 1.5 mg/kg, every 6–8 weeks for a total of three doses, with nicotine included throughout MPTP treatment until the animals were killed. Values are mean ± SEM. ***p < 0.001 versus control/saline or nicotine/saline (two-way anova followed by Bonferroni post hoc test).

ControlSaline462 932 ± 1109
NicotineSaline360 581 ± 290
ControlMPTP349 056 ± 1008***
NicotineMPTP346 959 ± 1812***

Nicotine treatment does not affect monoamine oxidase activity in monkey striatum or cortex

To rule out the possibility that the changes in nicotine-induced effects may be due, at least in part, to alteration in MPTP metabolism via monoamine oxidase, we measured enzymatic activity in cortical and striatal homogenates from unlesioned and lesioned animals treated with or without nicotine. The results (Table 4) showed no significant change in monoamine oxidase activity after these treatments.

Table 4.   Effect of nicotine dosing and MPTP lesioning on monoamine oxidase B activity in monkey brain
RegionMonoamine amine oxidase B activity (nmol/h/mg tissue)
ControlNicotineMPTPMPTP + nicotine
  1. Animals were given nicotine in drinking water as detailed in Table 1 and subsequently administered MPTP, with nicotine treatment continued until the animals were killed. Values are mean ± SEM (n = 5–7 animals).

Cortex0.79 ± 0.230.71 ± 0.121.04 ± 0.220.95 ± 0.18
Medial caudate1.24 ± 0.121.41 ± 0.301.35 ± 0.221.76 ± 0.23
Lateral caudate1.05 ± 0.111.52 ± 0.271.30 ± 0.151.44 ± 0.08
Ventral putamen1.52 ± 0.191.73 ± 0.101.45 ± 0.191.40 ± 0.15
Dorsal putamen2.07 ± 0.392.53 ± 0.362.97 ± 0.621.65 ± 0.25


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The present data show that nicotine treatment to lesioned primates results in greater levels of several key dopaminergic nerve terminal measures in striatum. Striatal TH expression was shown to be higher in nicotine-treated lesioned animals using western analyses and immunocytochemistry. Similar results were obtained for the dopamine transporter, a membrane protein critical for dopamine reuptake. Importantly, the level of vesicular monoamine transporter was also increased in MPTP-lesioned animals treated with nicotine. This latter transporter is reported to be a very reliable marker of striatal dopaminergic nerve terminal integrity because it does not appear to be affected by drug treatments, unlike TH and the dopamine transporter (Vander Borght et al. 1995; Wilson and Kish 1996; Kemmerer et al. 2003). Striatal dopamine levels were also increased in MPTP-lesioned animals treated with nicotine. These results are consistent with the possibility that nicotine promotes the survival and/or regeneration of striatal dopaminergic terminals in animals with no overt parkinsonism. Studies to determine whether similar improvements are evident in monkeys with more severe nigrostriatal damage and consequent behavioral deficits remain to be done.

Such an interpretation is also supported by the results of recent studies (Quik et al. 2006), which show that chronic nicotine treatment of monkeys with nigrostriatal damage normalizes excessive dopaminergic activity in the striatum. This includes both nicotine- and potassium-evoked dopamine release, as well as dopamine turnover. In addition, chronic nicotine dosing to MPTP-lesioned monkeys restores corticostriatal synaptic plasticity, as measured using long-term depression, that arises with nigrostriatal damage (Quik et al. 2006).

The magnitude of the increase in dopaminergic markers in striatum of lesioned primates receiving nicotine is in agreement with that observed in other experimental models (O'Neill et al. 2002; Quik 2004). Work in cultured mesencephalic cells showed that nicotine exposure resulted in ∼ 20% protection against MPTP-induced toxicity. Nicotine treatment resulted in a similar protective effect against nigrostriatal damage in rodents, in which protection has been observed. Because Parkinson's disease develops only when there is a 70–80% deficit in the striatal dopaminergic system (Olanow 2004; Samii et al. 2004), a 20% reversal may delay the onset of symptoms by many years.

Nicotine administration and smoking are well known to increase CNS nicotinic receptors in rodents (Wonnacott et al. 1990; Wonnacott 1997; Gentry and Lukas 2002) and humans (Benwell et al. 1988; Breese et al. 1997; Perry et al. 1999). Various modes of administration are available in experimental animals, including its inclusion in the drinking water, a method that has been successfully used in rodents. This approach has the advantage that administration is long term and yet pulsatile because the animals drink intermittently during the course of the day, a mode that resembles smoking behavior. The present data demonstrating enhanced CNS nicotinic receptors with this method of nicotine treatment indicate that we were achieving biologically active levels of nicotine in monkey brain. Robust increases (∼ 50%) were observed in both cortex and striatum in control animals administered nicotine. Although MPTP lesioning decreased nicotinic receptors in striatum, concomitant nicotine treatment yielded nicotinic receptor levels similar to control values. Because nicotinic receptor stimulation modulates dopaminergic transmission (Wonnacott 1997), control receptor levels may allow for a more efficient regulation of striatal function.

To determine whether nicotine treatment also altered MPTP-induced decreases in substantia nigra neurons, dopaminergic cells were counted using stereological techniques (McCormack et al. 2004). No differences were observed in the number of dopaminergic neurons in lesioned animals treated without or with nicotine treatment. These data were somewhat unexpected because nicotine treatment partially reversed the dopaminergic nerve terminal deficits, as assessed by enhanced protein levels of striatal TH, dopamine transporter and vesicular monoamine transporter. These findings may suggest that nicotine treatment specifically affects the capacity of striatal dopamine terminals to respond to injury, perhaps through sprouting. Another hypothesis is that the remaining dopaminergic neurons in the substantia nigra of nicotine-treated lesioned animals are more functional than those in lesioned-only animals. Studies that include measurement of dopaminergic enzymes and transport processes in substantia nigra are necessary to evaluate this possibility.

The present finding of an improvement in striatal dopaminergic markers with no change in nigral dopaminergic cell number resembles that following treatment of MPTP-treated rhesus monkeys with glial cell line-derived neurotrophic factor (GDNF), which is being tested in patients with Parkinson's disease (Nutt et al. 2003; Harvey et al. 2005; Patel et al. 2005). Intranigral, intracaudate and intracerebroventricular infusion of GDNF led to ∼ 20% improvements in dopaminergic measures, including TH and dopamine levels on the lesioned side, in hemi-parkinsonian monkeys, but no significant increase in nigral dopamine neurons (Gash et al. 1996). This preferential effect at terminals is of interest because terminals are also damaged to a much greater extent. In animal models and also in Parkinson's disease, there is an ∼ 80% decline in striatal dopamine levels but only ∼ 50% loss in nigral dopaminergic neurons (Lang and Obeso 2004; Samii et al. 2004). Nicotine therefore appears to be most effective in the region that is most vulnerable.

Mechanisms whereby nicotine may exerts its protective effect include stimulation of trophic factors, which are increased in rodents after chronic treatment (Belluardo et al. 2000). Another possibility is that nicotine administration leads to receptor desensitization with a resulting reduction in dopamine release and a decrease in toxic metabolites arising from dopamine auto-oxidation (Rosenberg 1988; Masserano et al. 1996; McLaughlin et al. 1998; Berman and Hastings 1999; Quik et al. 2006). Alternatively or in addition, nicotine-stimulated dopamine release may have competed with MPTP for the dopamine transporter, although such a mechanism would also have applied to the substantia nigra, in which the dopaminergic cell number was not changed. Moreover, the lack of effect of nicotine on nigral cell number suggests that striatal changes are unlikely to be a mere consequence of nicotine affecting MPTP biodisposition. This is a relevant point as previous studies had shown that MPTP is metabolized to its active metabolite MPP+ by monoamine amine oxidase B (Langston 1996). Moreover, monoamine oxidase activity is decreased in the brains of smokers (Fowler et al. 1996a,b). However, no change was seen in enzymic activity in unlesioned or lesioned animals treated with nicotine.

As mentioned earlier, the conflicting data concerning the neuroprotective potential of nicotine against nigrostriatal damage in rodents may be explained by methodological differences between studies (O'Neill et al. 2002; Quik 2004). In addition, the variable degree of protection may relate to inherent species differences in nicotine pharmacokinetics and pharmacodynamics. Rodents metabolize nicotine somewhat differently, and there is a more rapid time course in rodents than in primates (Schoedel et al. 2003; Hukkanen et al. 2005). There are also differences in nicotinic receptor subtype distribution between species. The nicotinic α6* subtype forms only 15–20% of the total nicotinic receptor population in rodent striatum, but a much larger proportion (∼ 50%) in monkey striatum (Quik 2004). Although other nicotinic receptors are also present in striatum, the α6* subtype may be particularly relevant to Parkinson's disease because of its more restricted localization to the basal ganglia and selective vulnerability to nigrostriatal damage (Quik et al. 2001). Although it is uncertain at present which receptor subtypes mediate neuroprotection against nigrostriatal damage, evidence suggests that α2*–α6* subtypes may be involved (O'Neill et al. 2002; Quik 2004). It is possible that the larger population of α6* receptors in primate striatum may be linked to a more robust neuroprotective effect compared with that in rodents.

In summary, the present results show that nicotine treatment to lesioned primates results in greater levels of a wide range of dopaminergic nerve terminal measures in striatum. These molecular changes may be responsible, at least in part, for the normalization of aberrant activity in striatum of nicotine-treated lesioned animals compared with lesioned animals not receiving nicotine (Quik et al. 2006). Although the present results have not allowed us to evaluate whether nicotine is neuroprotective or neurorestorative, they do support the idea that the reduced incidence of Parkinson's disease in smokers may relate to the nicotine in tobacco. The present findings also have implications for the proposed use of nicotine as a therapeutic agent for Parkinson's disease, because nicotine or selective neuronal nicotinic receptor agonists (Lloyd and Williams 2000) may counteract disease progression particularly if given during the early stages.


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This work was supported by National Institutes of Health grants U54 ES012077 and NS42091.


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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