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Is Nicotine Protective Against Parkinson´s Disease? An Experimental Analysis José-Rubén García-Montes1, Alejandra Boronat-García1, Ana-María López-Colomé1, José Bargas2, Magdalena Guerra-Crespo1 and René Drucker-Colín*,1 1
Departamento de Neuropatología Molecular, 2Departamento de Neurociencia Cognitiva, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, D.F., México Abstract: Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) and its projections. Reports show a lower incidence of PD in smokers compared to nonsmokers. Nicotine reduce motor symptoms of patients already diagnosed with PD. However, the mechanisms underlying the effects of nicotine in the dopamine (DA) depleted striatum remain elusive. This study evaluates the effects of chronic nicotine administration on PD motor symptoms in an attempt to mimic the chronic self-administration of nicotine in smokers. To achieve this, we used the 6-OHDA hemiparkinson rat model evaluating the amphetamine/apomorphine induced circling behavior, in rats whose daily water intake included nicotine. We found that chronic nicotine reduced amphetamine (AMPH) induced circling behavior by 40%, whereas apomorphine (APO) increased this behavior by 230%. High-performance liquid chromatography (HPLC) revealed that AMPH produced a 50% decrease of DA release in the intact hemisphere, while on the striatum of the lesioned side, receptor binding assays showed an increased affinity to D1 receptors and a concurrent decrease in D2 receptors. c-Fos activity showed through double labeling, that cell types involved in nicotine action were low threshold (LTS) and fast spiking (FS) inter-neurons, which increased in the DA-depleted striatum. We also observed an increase in the activity of D1 medium spiny neurons (D1 MSN), a striatal population with a major role in motor control. Our results show that chronic nicotine does not specifically protect against degeneration, but rather modifies DA receptor dynamics, suggesting that it could be used as a therapeutic element in PD pathology.
Keywords: Basal ganglia, chronic nicotine, dopamine receptor, inter-neurons, Parkinson´s disease, striatum. INTRODUCTION Parkinson´s disease, affects 1-3% of the population over 60 years of age [1]. The disorder is characterized by the gradual loss of DAergic neurons of the SNc, which results in a reduction of DAergic input into the striatum; as a consequence, a series of primary motor symptoms appear: tremor, rigidity, bradykinesia and postural instability [2]. To treat PD symptoms some pharmacological approaches have been employed, however, as the disease progresses, dyskinetic movements become the main complication of the most effective treatment for PD. Interestingly, epidemiological studies in open populations revealed that smokers have 50% less incidence of PD than nonsmokers [35]. Likewise, clinical studies have shown that nicotine, a major tobacco component, is involved in some improvements of motor and cognitive symptoms in PD patients [6, 7]. Although controversial, there is also evidence showing that nicotine prevents cellular damage with specific administration protocols [8, 9]. Moreover, some studies indicate that nicotine administration in rat and monkey PD models, chronically treated with L-DOPA, has antidyskinetic effects [10, 11]. It has been proposed that functional changes in motor nuclei, containing nicotinic
receptors (NR), could be part of the mechanism by which nicotine is improving motor symptoms [12]. The mammalian striatum contains two main neuronal populations of projection GABAergic neurons (Medium Spiny Neurons, MSN), and inter-neurons broadly divided into cholinergic and GABAergic neurons, which regulates the MSN [13, 14]. Interestingly, in the DA-depleted striatum, LTS but not FS GABAergic inter-neurons or cholinergic inter-neurons, shift their firing pattern from a tonic to an oscillatory mode and produce spontaneous repetitive giant GABAergic currents in one-half of the MSNs [13]. Remarkably, every FS and a third of LTS inter-neurons increase their firing rate in the presence of nicotine [15]. This finding suggests that NR are involved in the activation of striatal inter-neurons. It is noteworthy that the selective inhibition of FS inter-neurons in striatum has been shown to induce dyskinetic movements [16, 17]. Taken together, these studies suggest that the administration of nicotine seems to prevent the onset of PD. Thus, the aim of this study was to evaluate the effects induced by the chronic exposure to nicotine on the motor behavior of hemiparkinsonian rats, as well as to address the question of neuroprotection, DA receptor sensitivity and the activity of striatal neuronal populations. MATERIALS AND METHODS
*Address correspondence to this author at the Departamento de Neuropatología Molecular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Apdo. Postal 70-600, 04360, D.F. México, Tel/Fax: (52-55) 5550-0064; E-mail:
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Animals A total of one hundred ninety male Wistar rats, weighting 60-80 grs were used. Animals were individually housed © 2012 Bentham Science Publishers
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under standard colony conditions: 12:12 light/dark cycle (lights on at 7:00 a.m.), controlled room temperature (22 ± 1 ºC) and ad libitum food access. All studies were conducted in accordance with principles and procedures described by the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and according also to the local committee of the National Autonomous University of Mexico.
Striatal DA Release Measurement by HPLC with Electrochemical Detection (HPLC-ED)
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Chronic Administration of Nicotine We administered nicotine in drinking water throughout 27 weeks in order to reach blood concentration levels of nicotine similar to that of smokers [18]. Since rats present natural aversion to nicotine, the concentration was gradually increased, starting at 15 mg/L, and adding 3 mg/L weekly until a final concentration of 30 mg/L was reached. Two bottles were placed in consistent positions for 8 weeks [19]. Animals had access to water or nicotine solution at two schedules: 8-12 and 16-20 hours Animals were divided in two groups: 6-OHDA (n=95) or 6-OHDA plus nicotine treatment group (6-OHDA+nicotine) (n=95). Rats were housed for 12 hrs in metabolic cages to recover approximately 5-10 mL of urine for cotinine measurement. Cotinine levels were measured by HPLC with UV detection following a method described elsewhere [20].
Twenty l of sample were injected for analysis using a Gilson 734 autoinjector (Gilson Inc, Middleton USA). Electrochemical detection was performed using BAS 4C detector (EDC) prototype 650mV vs Ag/AgCl, glassy carbon electrode. The Gilson pump provided a flow rate of 80 l min-1 during 7 min through the column and guard columns (microprobe 100 x 1 mm, ODS C18-3 m). Rotational Behavior Rotational behavior was assessed based on the method described previously [23]. Animals were given AMPH (4 mg kg-1) or APO (1 mg kg-1) by i.p. injection. We recorded the number of left or right turns using a homemade computerized system, through 90 min for AMPH and 30 min for APO administration [24]. In order to evaluate the participation of NRs in this behavior, we used three NR antagonists: methyllycaconitine citrate hydrate (MC) (Sigma-Aldrich, No. M168), mecamylamine hydrochloride (MLA) (Sigma-Aldrich, No. M9020) and dihydro-betaerythroidine dihydrated (DHBE) (Sigma-Aldrich, No. D149). Immunofluorescence
6-OHDA Nigro-Striatal Lesion After nicotine administration protocol, two groups, 6OHDA and 6-OHDA+nicotine were infused with 6-OHDA into the left SNc. Coordinates were determined according to the rat brain atlas of Paxinos & Watson, 4.7 mm anteriorposterior (AP), 1.6 mm medial-lateral (ML), 8.2 mm ventral (V) (Paxinos & Watson, 1998). Briefly, rats were anesthetized by intraperitoneal (i.p.) route with ketamine/xylazine (87 and 13 mg kg-1, respectively). Each infusion consisted of 40 g of 6-OHDA (Sigma-Aldrich, St. Louis, MO) stabilized with 32 g l-1 of l-ascorbate in a total volume of 0.5 l of saline solution at 0.125 L min-1 [21]. Implantation of Guide Cannula for Microdialysis The guide cannula used for measuring in vivo DA levels during free movement microdyalisis was implanted stereotaxically in the dorsal striatum posterior to the 6OHDA infusion as previously described [22]. The following coordinates for dorsal striatum were used: 0.0 mm AP, 3.0 mm ML, 4.2 V. Cannulas (CMA/12 Guide Cannula, CMA Microdialysis, and Stockholm, Sweden) were secured to the head of the animal with dental cement and animals were allowed to recover for 4 days before the beginning of the behavioral experiments. The dialysis probe (CMA 12 elite 14/02 PAES, CMA Microdialysis AB, Stockholm, Sweden) allowed continuous perfusion (2 l min-1) with Krebs-Ringer solution (118 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 2.5 mM CaCl2*2H2O, 19 mM NaHCO3, 3.3 mM Glucose, pH 7.4 at 4 ºC) for microdialysis with a microinfusion pump (CMA400 Syringe Pump, CMA Microdialysis AB, Stockholm, Sweden).
Rats were transcardially perfused after one hour and forty-five minutes of agonist administration with 0.1 M phosphate buffer solution (PBS) at 4 ºC followed by 4% paraformaldehyde (PFA) at 4 °C. Brains were extracted and post-fixed with 0.1% PFA at 4 ºC, and then placed in 30% sucrose solution. Brains were cut coronally into 30 m sections with a cryostat and the free-floating sections were rinsed in PBS. Thereafter, slices were blocked with 0.1 M PBS/3% horse serum /0.3% Triton X-100 1 hr at room temperature. Following a rinse in PBS three times for five minutes, sections were incubated with primary antibody for c-Fos (1:250, Santa Cruz Biotechnology, Santa Cruz, CA. No. L0302), mouse parvalbumin as a marker for FS interneurons (1:2000, Chemicon-Millipore, Billerica MA, USA, No. MAB1572), rabbit somatostatin to identify LTS interneurons (1:250, Chemicon-Millipore, Billerica MA, USA, No. AB5494), and rabbit P substance to identify D1 MSN cells at 4 ºC for 48 hrs (1:250, Chemicon-Millipore, Billerica MA, USA, No. AB1566). Sections were then washed three times in 0.1% PBS and then incubated with DyLight secondary antibodies (Jackson immuno Research, Pennsylvania USA) for 2 hours at room temperature: 488coupled donkey rabbit (No. 711-486-152), 649-coupled donkey rabbit (No. 711-496-152), 649-coupled donkey mouse (No. 715-496-151), 649-coupled donkey mouse, (No. 715-486-151), 649-coupled donkey goat, (No. 705496-147) and Alexa 488-coupled donkey goat (Invitrogen, Oregon, USA, No. A11055). Nuclei were stained with DAPI (1:10 000) diluted in PBS. Finally, sections were mounted on slides, and cover-slipped in fluorescence mounting medium (Dako, No. S3023).
Is Nicotine Protective Against Parkinson´s Disease? An Experimental Analysis
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Tyrosine Hydroxylase (TH) Immunohistochemistry
Aldrich, No. D054). Incubations were stopped at 60 min by centrifugation using an aerofuge (2 psi x 2 min) and supernatants were removed and washed twice with Tris-HCl (0.05 M, at pH 7.4 and 4 ºC). Radioactivity was measured in a scintillation spectrometer. Bmax were calculated for every condition using the following formula: Bmax: B = (Bmax*C)/(Kd+C), where B is the specific union site to the ligand concentration C; Kd is equal to the slope and Bmax is calculated by extrapolation of a nonlinear regression. Bmax and Kd were analyzed with Graph Prism4 Version 5.0 software.
As described above, rats were intracardially perfused, sectioned and subsequently blocked with 0.1 M PBS/2.5% bovine serum (BSA)/0.1 % Triton X-100 overnight at 4 ºC, followed by incubation with primary antibody for TH at 4 ºC for 48 hrs (1:1000, Chemicon-Millipore, Billerica MA, No. AB152). After 3 rinses with PBS, sections were incubated with secondary biotinylated antibody for 2 hrs (1:250, Chemicon-Millipore, Billerica MA USA, No. AB1542) followed by a 60-minutes incubation with avidinbiotinylated horseradish peroxidase (Elite Kit Vector Labs) at room temperature. Peroxidase was revealed with 3,3'diaminobenzidine (DAB)/H2O2. Cell Quantification for TH The number of TH-positive neurons was estimated in 10 animals per group. Five coronal sections of 40 m were sampled to represent a fourth part of the total SNc. We selected the 1, 5, 9, 13, 17 sections, beginning at bregma as zero. A stereomicroscope (Leica EZ4D) was used for picture acquisition and an image-analysis program was used for cell counting (ImageJ) [25]. Immunofluorescence Quantification of c-Fos A total of 45 rats were used and divided in 6 groups, 6OHDA treated, 6-OHDA+Nicotine, only Nicotine, Non Lesioned, SHAM, Sham+Nicotine. Photographs were obtained in a “z-stack” with a Leica microscope (DM6000 B, 40x magnification), using the LASAF software (Leica Application suite Advanced Fluorescence, 2.1.0 version). Counts were performed using the image analysis software ImageJ [25]. Areas counted in striatum and orthogonal views are exemplified in Fig. (6). Receptor Binding Analysis A total of 34 rats were sacrificed six days after APO injection. After decapitation, striatum from both sides of the brain were dissected in a Krebs-Ringer solution and shredding in a Tris-HCl buffer (0.05 M at pH 7.4). Homogenized tissues were disposed in ultracentrifuge tubes and placed inside a 50Ti rotor (Beckman) in an ultracentrifuge (XL-90 Beckman ultracentrifuge) and were spun at 28,000 revolutions per minute during 5 minutes at 4 ºC in order to precipitate total membranes. Supernatants were removed from each centrifuge tube and the process was repeated twice. In the third wash an aliquot was taken for protein measurement by Bradford method. Based on the protein concentration, pellets were re-suspended in a TrisHCl buffer (0.05 M Tris + 120 mM NaCl and 50 M ketanserine). For total binding assays, D1 DA receptors membranes were deposited in a micro-scintillation tubes at room temperature with different concentrations of [3H]SCH23390 (0.125, 0.5, 0.5, 1, 1.25, 2.5, 3.75, and 5,6 nM 80.5 Ci/mmol RBI) in a final volume of 175 L. For nonspecific binding measuring we incubated membranes with equal concentrations of [3H]SCH-23390 (Perkin Elmer, Waltham, Massachusetts, USA, No. 930025UC) using R(+)-SCH23390 1 M (R(+)-7-Chloro-8-hydroxy-3-methyl-1-phenyl2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride, Sigma-
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Data Analysis For data analysis we used the Graph Prism4 Version 5.0 and statistical test are described in every figure legend. All compiled data passed the normality test of Shapiro-Wilk. All data are represented as mean ± SEM. RESULTS Chronic Nicotine Consumption The mean consumption of nicotine was estimated to be 3.75 ± 0.7 mg Kg-1 day-1. This analysis revealed that rats consumed comparable amounts of liquid with nicotine (32.8 ± 5.2 mL day-1) or without nicotine (31 ± 4.67 mL day-1). Urinary cotinine level in the period of nicotine drinking was 2.4 x 103 ± 1.2 ng mL-1. Thus, our long-term nicotine administration is similar to the human smoking condition at least in relation to cotinine urine levels 3.5 x 103 ng mL1 [26]. Rotation with AMPH After Chronic Nicotine In an attempt to evaluate the effect of chronic oral nicotine consumption (85 days) in the hemi-Parkinson model, we measured rotational behavior induced by AMPH, comparing 6-OHDA+Nicotine versus 6-OHDA group. Fig. (1) shows 90 minutes of rotational behavior, 15 days after lesion. As can be observed the number of rotations was significantly reduced in chronic nicotine treated rats. By contrast, chronic subcutaneous (s.c.) injection of nicotine (2.5 mg Kg-1 day-1) prior to the lesion (28 days) produced no changes in rotational behavior (results not shown). Therefore, we focused our research in the chronic oral nicotine administration. Chronic Nicotine and Neuroprotection In view of the suggestion that nicotine can affect the survival of neurons in the SNc [27], we quantified the number of TH positive cells remaining on the lesioned side (144±46. cells). Fig. (2) reveals that there is no sign of neuroprotection since comparing 6-OHDA+Nic group (Mean 99. ±31. cells) versus 6-OHDA only group (Mean 144±46 cells), there were no significant statistical differences. Chronic Nicotine and DA Release in the Intact Striatum When we determined in the 6-OHDA lesioned rats the level of DA released in the striatum of the intact side in the chronic nicotine group, we found a significant decrease of
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Fig. (1). Chronic nicotine administration decrease motor asymmetry induced by AMPH on PD-lesioned rats. 15 days after the lesion, the number of turns of each group was measured every 3 minutes within a 90 min interval after injection of AMPH. 6-OHDA n=30; 6OHDA+Nic n=42; Sham (surgically simulated rats) n=5. * p
