Introduction

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Catecholaminergic systems are critical in mediating the physiological effects of nicotine. The self-administering and reinforcing properties of nicotine seem to arise from direct action on the mesocorticolimbic dopamine system. This system, with cell bodies in VTA, plays a key role in mediating the reinforcing effects of natural rewards as well as of drugs of abuse, including nicotine, through increased dopaminergic transmission in the nucleus accumbens. The nigrostriatal dopaminergic system, with cell bodies in the SN, innervates the caudate nucleus and putamen of the corpus striatum. Activation of striatal neuronal acetylcholine receptors (nAChRs) triggers release of dopamine. These receptors are also involved in mediating nicotine's effects on locomotion.

Abnormalities of dopaminergic neurotransmission are associated with a number of neuropsychiatric disorders, including schizophrenia and Tourette's syndrome. Nicotine binding in caudate nucleus and putamen is significantly reduced in Parkinson's disease, Alzheimer's disease, and dementia with Lewy bodies (Court et al., 2000). Nicotinic compounds have therapeutic potential for treatment of these disorders (Rusted et al., 2000).

In addition to triggering release of catecholamines, nicotine also promotes their biosynthesis. Several studies reveal that injections of high concentrations (1-4 mg/kg, free base) of nicotine increase activity of adrenal TH, the rate-limiting enzyme in the catecholamine biosynthetic pathway (Slotkin et al., 1976; Fossom et al., 1991). This is reflected by a rise in TH protein resulting from increased mRNA levels (Fossom et al., 1991; Hofle et al., 1991; Hiremagalur and Sabban, 1995). In the brain, TH is elevated by lower concentrations of nicotine. Nicotine injections (0.8 mg/kg, free base) raised TH activity in SN and VTA as well as in noradrenergic cell bodies of the locus coeruleus (Smith et al., 1991; Mitchell et al., 1993). A single injection of nicotine led to a large increase in TH mRNA in locus coeruleus even 6 days later (Mitchell et al., 1993). We have shown previously that injections of nicotine (0.35 and 1.75 mg/kg) doubled TH mRNA levels in VTA and SN (Serova et al., 1999a). It is unclear what is the lowest effective dose of nicotine needed to stimulate TH gene expression in dopaminergic locations.

Although TH is the rate-limiting step in biosynthesis of dopamine, as well as of other catecholamines, it is also dependent on levels of its essential cofactor, tetrahydrobiopterin (BH4) (Nagatsu and Ichinose, 1999). Intraventricular administration of BH4 increases the biosynthesis of dopamine in nigrostriatal neurons (Kettler et al., 1974). Moreover, patients with Parkinson's disease and dementia not only have reduced levels of dopamine but also reduced levels of BH4. These observations suggest that nicotine's effect on the dopaminergic system may be influenced by regulation of BH4 as well as of TH. The effect of nicotine on biosynthesis of BH4 has not been examined previously.

Nicotine mediates its actions through nAChRs, which exist as a variety of subtypes. Diverse nAChRs are formed as pentamers from different combinations of -type and -type subunits (for review, see Leonard and Bertrand, 2001). Receptors containing 42 and 7 subunits are, respectively, the major high- and low-affinity nAChRs in the brain. Both types of receptors are present in the SN and VTA (Tsuneki et al., 2000). Previously, we found that the 7 nAChR subtype is required for the nicotine-elicited elevation of TH mRNA in PC12 cells. Moreover, 7 receptor agonists were at least as effective as nicotine in triggering these effects (Gueorguiev et al., 2000). The 7 subunits can form a homopentameric receptor, which is reported to desensitize rapidly compared with other nAChR subtypes. Their high Ca²⁺ conductance could enable 7 nAChRs to take part in synaptic mechanisms in which calcium acts as a second messenger (Pugh and Berg, 1994). Whether these receptors play a role in regulation of gene expression of dopamine biosynthetic enzymes in vivo is unclear. Information regarding their role in the rewarding properties of nicotine and the withdrawal syndrome are contradictory. Studies by Schilstrom et al. (1998), Nomikos et al. (1999), and Panagis et al. (2000) indicate involvement of 7 nAChRs in nicotine-induced dopamine release in the nucleus accumbens, and in locomotion, whereas studies by Grottick et al. (2000) suggest a negligible role for 7 nAChRs in nicotine-induced hyperlocomotion and reward.

Herein, we examine nicotine's effect on TH and GTPCH gene expression in the cell bodies of the nigrostriatal and mesocorticolimbic dopaminergic systems. The potential involvement of 7 nAChRs is evaluated, as well as the ability of specific nAChR agonists to elicit the effects of nicotine in central and peripheral catecholaminergic regions.

	Materials and Methods

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Animals, Drug Treatment, and Doses. All animal experiments were approved by the Animal Care and Use Committee. Adult, pathogen-free, male Sprague-Dawley rats (250 to 300 g) were purchased from Taconic Farms (Germantown, NY) and housed four per cage. They were maintained under controlled conditions on a 12-h light/dark cycle at 23 ± 2°C. Animals were given food and water ad libitum.

Isolation of RNA and Northern Blots. The levels of mRNAs for TH or GTPCH were determined by Northern blot analyses as described previously (Serova et al., 1999b). Briefly, the brain punches from each animal were homogenized in RNA-Stat 60 (Tel-Test, Friends Woods, TX). Total RNA was then isolated and fractionated on 1.2% agarose gels. The RNA was transferred to Gene-Screen Plus membranes (PerkinElmer Life Sciences, Boston, MA), and hybridizations were subsequently performed with rat [³²P]UTP-labeled GTPCH cRNA or with cDNA probes labeled with [³²P]-dCTP for rat TH and 18S rRNA as a control. Hybridization with cDNA probes was performed at 42°C with RNA probe at 68°C in ULTRAHyb solution (Ambion, Austin, TX). After washing, the blots were exposed to BioMax film (Eastman Kodak, Rochester, NY) within the linear range of the signal. Autoradiograms were scanned and analyzed by using Image-Pro Analysis software (Media Cybernetics, Silver Spring, MD). The values for TH and GTPCH mRNA were normalized to levels of 18S rRNA.

Determination of Plasma Cotinine Concentrations. Plasma was freshly prepared and kept at 70°C until the assay. Levels of cotinine, a principal blood and urinary nicotine metabolite, were measured by Double Antibody Nicotine Metabolite ¹²⁵I RIA kit (EURO/DPC Ltd., Gwynedd, UK) according to the manufacturer's protocol. The standard curve was determined using cotinine, concentrations that ranged from 100 to 15,000 ng/ml. The interassay coefficients of variation were less than 5%. Plasma (0.025 ml) from saline- or nicotine-treated rats was combined with 0.1 ml of ¹²⁵I-cotinine, and 0.1 ml of nicotine metabolite antiserum was added. Tubes were incubated at room temperature for 30 min. After centrifugation and precipitation, the ¹²⁵I-labeled precipitates were counted in a gamma counter.

Statistical Analysis. The data were analyzed by one-way analysis of variance followed by Fisher's least significant difference test for comparison of the means (for more than two experimental groups) or Student's t test (for two experimental groups). Comparison between changes in TH and GTPCH mRNAs was determined by correlation analysis. Levels of p < 0.05 were considered significant.

	Results

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Effect of Different Doses of Nicotine Injections on TH and GTPCH Gene Expression. Injections of 0.35 and 1.75 mg/kg nicotine were previously shown to elevate rat TH mRNA levels in VTA and SN (Serova et al., 1999a). Therefore, we studied whether lower concentrations of nicotine are also effective in triggering elevated mRNA levels. Rats were injected with several concentrations of nicotine (0.087, 0.175, and 0.35 mg/kg in saline), five times at 12-h intervals. Animals were euthanized 3 h after the last injection and the levels of cotinine, the major nicotine metabolite, were determined in plasma. The concentration of cotinine was 26 ± 3.2 ng/ml in animals treated with 0.087 or 0.175 mg/kg nicotine and was elevated to 44 ± 7.1 ng/ml with 0.35 mg/kg nicotine. With injections of the highest dose of nicotine (0.35 mg/kg), plasma cotinine was 320 ± 46 ng/ml.

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Effect of different doses of nicotine injections on TH mRNA levels in VTA and SN. Rats were injected with the indicated concentrations of nicotine (n) in saline, as described under Materials and Methods. RNA from brain tissue punches obtained from individual animals was analyzed separately by Northern blot. Representative Northern blots and summary data are shown for TH mRNA levels. Data are expressed as mean ± S.E., with levels of TH mRNA in the saline-treated control group taken as 1. *, p < 0.05 versus control; , p < 0.05 versus 0.087 mg/kg nicotine.

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Effect of different dose of nicotine injections on GTPCH mRNA levels in VTA and SN. Rats were injected with the indicated concentrations of nicotine (n) in saline, as described in the legend to Fig. 1. After hybridization with TH, the filters were reprobed with RNA probe for GTPCH. Representative Northern blots and summary data are shown. Data are expressed as mean ± S.E, with the level GTPCH mRNA in saline-treated control group taken as 1. *, p < 0.05 versus control; , p < 0.05 versus 0.087 mg/kg nicotine.

Role of 7 nAChRs in Regulation of TH and GTPCH mRNA Levels. To examine which nicotine subtype is involved in the observed changes in TH and GTPCH mRNAs, MLA, a specific antagonist of the low-affinity 7 nAChR was used. It was injected (i.p.) 20 min before each injection of nicotine (0.175 mg/kg) or saline. MLA by itself did not significantly change TH and GTPCH mRNA levels in either VTA or SN. However, MLA prevented the effect of the nicotine on TH and GTPCH mRNA levels in both these areas (Figs. 3 and 4). These findings suggest that activation of 7 nAChRs is required for nicotine-elicited elevation of TH and GTPCH mRNA levels in these dopaminergic locations.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Antagonist of 7 nAChRs abolished the nicotine-triggered rise in TH mRNA levels in VTA and SN. Methyllycaconitine (MLA or m, 4.2 mg/kg) was injected intraperitoneally 15 min before each of five s.c. injections of 0.175 mg/kg nicotine (Nic or n) or saline (Sal or s). RNA from individual animals was analyzed by Northern blots. Representative Northern blots and summary data are shown for TH mRNA levels. Data are expressed as mean ± S.E. TH mRNA level in saline-treated control group was taken as 1. *, p < 0.05 versus control; , p < 0.05 versus nicotine.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Antagonist of 7 nAChRs abolished the nicotine-triggered rise in GTPCH mRNA levels in VTA and SN. The same filters from experiment described in Fig. 3 were reprobed with RNA probe for GTPCH. Representative Northern blots and summary data are shown for GTPCH mRNA levels. Data are expressed as mean ± S.E, with levels of GTPCH mRNA in saline-treated control group taken as 1. *, p < 0.05 versus control; , p < 0.05 versus nicotine.

View larger version (36K): [in this window] [in a new window]   Fig. 5.   Agonists of 7 nAChRs elevate TH mRNA levels in VTA and SN. DMXB (D) and 3CA. DMXB (d) at 1, 3, and 10 mg/kg; 3CA at 0.3 and 1 mg/kg; 1.75 mg/kg nicotine (N); or saline (S) was injected i.p. five times, 12 h apart. RNA from individual animals was analyzed by Northern blots. Representative Northern blots and summary data are shown for TH mRNA levels. Data are expressed as mean ± S.E. TH mRNA level in control saline-treated group was taken as 1. *, p < 0.05 versus control.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   7 nAChRs agonists DMXB and 3CA do not alter TH mRNA levels in superior cervical ganglia and adrenal medulla. Rats were treated with DMXB at 1, 3, and 10 mg/kg; 3CA at 0.3 and 1 mg/kg; 1.75 mg/kg nicotine (N); or saline (S) by i.p. injection five times, 12 h apart. RNA was isolated from superior cervical ganglia and adrenal medullae of individual rats and analyzed by Northern blots. Summary data for TH mRNA levels are shown. Data are expressed as mean ± S.E, with levels of TH mRNA in saline-treated control group taken as 1. *, p < 0.05 versus control.

	Discussion

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This study reveals, for the first time, that even low concentrations of nicotine are sufficient to elevate TH and GTPCH mRNA levels in central dopaminergic cell bodies of the mesocorticolimbic and nigrostriatal dopamine systems. Interestingly, the VTA is more sensitive than the SN to lower concentrations of nicotine. Thus, injections of 0.087 mg/kg nicotine significantly increased TH and GTPCH mRNA levels in VTA, but not in SN, of the same animals. This selective sensitivity to nicotine could be important for its rewarding properties. In the VTA, the lowest dose of nicotine was most effective. In contrast to their effect in the brain, these concentrations of nicotine are lower than required to increase gene expression of catecholamine-biosynthetic enzymes in adrenal medulla (Slotkin et al., 1976; Fossom et al., 1991; Hiremagalur and Sabban, 1995; Serova et al., 1999a). This might be partially due to the fact that nicotine administration results in higher effective concentrations in the brain compared with its concentration in periphery (for review, see Benowitz, 1990) or to lessened sensitivity to nicotine in the adrenal medulla.

The study reveals that the 7 nAChR subtype is involved in the nicotine-triggered elevation of mRNA for TH and GTPCH. These findings are based both on administration of MLA, the specific 7 nAChR antagonist, and two 7 nAChR agonists. MLA antagonized the ability of nicotine to induce TH or GTPCH mRNAs in both SN and VTA. In this regard, an equivalent dose of MLA, administered similarly, was found to inhibit nicotine's effect on footshock stress-elicited mesoprefrontal DA metabolism and immobility response (George et al., 2000a,b). Because MLA was administered by intraperitoneal injections, the specific location of the 7 nAChRs involved is not yet clear. It remains to be determined whether administration of MLA directly to the region of the VTA would have a similar effect. However, 7 nAChRs have been found in the region of the VTA, specifically on glutamatergic inputs to the DA neurons of the VTA (Schilstrom et al., 1998; Panagis et al., 2000).

There are contradictory studies regarding the importance of 7 nAChRs in nicotine's effect on brain dopaminergic systems. Several studies indicate that 7 nAChRs of the VTA mediate the rewarding effects, withdrawal syndrome, and reinforcing actions of nicotine. Nicotine-induced dopamine output in the nucleus accumbens was blocked by pretreatment with MLA in the VTA, indicating a role of 7 nAChRs in this mechanism (Schilstrom et al., 1998). The 7 nACh receptors in the VTA are reportedly involved in mediating the reinforcing actions not only of nicotine but also of cocaine (Panagis et al., 2000). Injection of MLA into the VTA of rats treated chronically with nicotine reduced their locomotion (Nomikos et al., 1999). In contrast, a study by Grottick et al. (2000) showed that agonists of the 7 nAChRs, AR-R-17779 and DMAC [()-spiro[1-azabicyclo[2.2.2]octane-3,5'-oxazolidin]-2'-one and [4-[(1E,3E)-3-(5,6-dihydro-4H-[2,3']bipyridinyl-3-ylidene)-propenyl]phenyl]dimethyl-amine, respectively], failed to stimulate locomotion activity in both nicotine-nontolerant and sensitized rats, and MLA pretreatment did not reduce nicotine-triggered hyperlocomotion. The discrepancy between these studies on the involvement of 7 nAChRs in nicotine-triggered locomotion and reinforcing behavior may be due to differences in the way MLA was administered or to the strain of rats.

The involvement of the 7 nAChR was confirmed with the administration of 7 nAChR agonists. DMXB and 3CA were able to elevate TH mRNA levels in VTA and SN. In contrast to the greater sensitivity of VTA to low concentrations of nicotine, the SN was more sensitive than the VTA to low concentrations of DMXB. Thus, mechanisms other than 7 nAChR mediated might be involved in nicotine's action on TH in the VTA. The lower sensitivity of the VTA to the 7 nAChR agonists, and the absence of an effect in the periphery are encouraging. Thus, 7 nAChR agonists may be able to increase TH gene expression and dopamine synthesis, for example, in patients with Parkinson's disease or schizophrenia, without eliciting its addictive or pressor side effects.

The locus of the 7 nAChR subunit gene has been genetically linked to schizophrenia (for review, see Leonard and Bertrand, 2001). Schizophrenics display a very high incidence of smoking, whereas nicotine is proposed to normalize a sensory gating deficit found in schizophrenics. In this regard, DMXB normalized an auditory evoked-potential deficit in mice with absence of sensory inhibition (Stevens et al., 1998).

The 7 nAChRs are also implicated in modulating synaptic neurotransmission and in regulating neuronal growth, differentiation, survival, and memory (Meyer et al., 1997; Broide and Leslie, 1999; Jonnala and Buccafusco, 2001). Administration of DMXB enhanced a variety of cognitive behaviors in mice, monkeys, rats, and rabbits (Meyer et al., 1997; Kem, 2000). It is neuroprotective in cultured cells and in vivo to a variety of insults (Meyer et al., 1997, 1998; Li et al., 1999). Based on these findings, DMXB has shown promise as a possible therapeutic target for Alzheimer's disease (for review, see Kem, 2000).

In contrast to DMXB, 3CA has been less well studied. It was found to be even more effective than DMXB in eliciting a delayed but sustained rise in intracellular calcium in PC12 cells, and triggered marked elevations in TH and DBH mRNA levels (Gueorguiev et al., 2000). This 3-cinnamylidene anabaseine compound with mixed agonist/antagonist properties binds 7 nAChRs efficaciously, activates 7 nAChRs in a Xenopus oocyte expression system, and has low inhibitory activity. The concentrations of 3CA used in this study were also found to improve passive avoidance behavior in nucleus basalis-lesioned rats (Meyer et al., 1998).

Although the drugs used in the present study target 7, it should be noted that DMXB has partial agonist and antagonist properties on 5-hydroxytryptamine₃ receptors (Machu et al., 2001) and is also an antagonist of 42 receptors (Kem, 2000). Therefore, further studies are required to ascertain whether these actions are also involved in the DMXB-triggered changes in TH gene expression in the dopaminergic neurons observed in this study. In this regard, studies by Tsuneki et al. (2000) demonstrated that in mice nigral dopaminergic neurons, nicotine can elicit Ca²⁺ mobilization via activation of two distinct nAChRs, those containing 2 or 7 subunits. Therefore, nAChRs, other than the 7 subtype may also contribute to nicotine-triggered elevation of TH and GTPCH gene expression in the VTA and SN. However, the involvement of the 7 nAChR subtype is supported by the ability of MLA to block the nicotine-triggered increase in TH and GTPCH mRNA levels.

The results of this study indicate that low concentrations of nicotine induced not only TH but also GTPCH gene expression in the SN and VTA and required the 7 nAChR subtype. The nicotine-triggered changes in TH and GTPCH mRNA levels were significantly correlated. These findings suggest that there may be a common mechanism for regulation of expression of these two genes by nicotine. Previous studies have found that in TH and GTPCH mRNA levels are often, but not always concomitantly regulated by physiological treatments, such as stress or administration of estradiol (Serova et al., 1999b; Serova and Sabban, 2002). Both genes are responsive in cell culture to increases in cAMP and glucocorticoids (Zhu et al., 1994; Serova et al., 1997).

Although BH4 was not measured in this study, intracellular concentrations of BH4 are determined mainly by its de novo synthesis, with GTPCH as the rate-limiting enzyme (Nagatsu and Ichinose, 1999). In this regard, nicotine administration at a dose of 0.5 mg/kg was found to significantly increase biopterin levels in the striatum and the hypothalamus (Tsai and Lee, 1995). It has been shown that dopamine neurons contain exceedingly low levels of BH4 (Hirayama and Kapatos, 1998). Moreover, the number of TH molecules in brain dopaminergic nerve terminals is highly dependent on the intracellular concentration of BH4 (Sumi-Ichinose et al., 2001). This suggests that nicotine may activate biosynthesis of dopamine (and other catecholamines) not only by way of increased gene expression of TH but also through elevation of its essential cofactor.

The nicotine-elicited elevation of GTPCH mRNA levels found in this study, and the likely subsequent increase of BH4, may lead not only to activation of dopamine biosynthesis in cell bodies but also to protection of dopaminergic neurons. Recently, it has been shown that BH4, within dopaminergic neurons, is necessary and sufficient for maintaining lower reactive oxygen species (Nakamura et al., 2001). These multiple functions of BH4 may play a crucial role in biochemical and behavior recovery in 6-hydroxydopamine-lesioned rats (Parkinson's animal model) with administration of adenovirus-associated vectors expressing TH, aromatic-L-amino acid decarboxylase, and GTPCH. These rats display greater dopamine production and improvement in rotation behavior than rats that carry only TH and aromatic-L-amino acid decarboxylase viral vector (Shen et al., 2000). The findings of this study indicate that the neuroprotective effects of nicotine may also involve its regulation of biopterin biosynthesis.

Thus, our results reveal that nicotine use of 7 nAChRs may regulate brain dopaminergic systems by alterations in gene expression of TH and GTPCH. Agonists of 7 nAChRs have therapeutic potential for increasing dopamine biosynthesis in the central nervous system without side effects on the sympathoadrenal system.