Introduction

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Cholinergic systems play an important role in cognitive functions, including attention, learning, and memory (Levin and Simon, 1998; Rezvani and Levin, 2001). Animal studies have shown that nicotinic acetylcholine receptor (nAChR) mechanisms are involved in attentional function (Blondel et al., 2000; Grottick and Higgins, 2000). Indeed, nicotine administration and cigarette smoking have been shown to improve attentiveness (Levin, 1992). There are several lines of evidence indicating that nicotinic acetylcholine mechanisms are responsible for cognitive impairments associated with attention deficit hyperactivity disorder (ADHD). Nicotine skin patches alleviate clinical ADHD symptoms as measured by the standard Clinical Global Impressions Scale in adults with ADHD (Conners et al., 1996; Levin et al., 1996, 2000, 2001; Levin and Rezvani, 2000). Moreover, a novel nAChR agonist, (S)-3-methyl-5-(1-methyl-2-pyrrolidinyl)isoxazole (ABT-418), is effective in the alleviation of the inattentive symptoms in adults with ADHD (Wilens et al., 1999).

Neuronal nAChRs are allosteric membrane proteins that are composed of five subunits according to the combination of  (2-9) and  (2-4) subunits. Neuronal subunits have been categorized into two major subtypes of nAChR pentamers in the brain on the basis of their high affinity for either nicotine or -bungarotoxin. Of the two, the former are considered to form 42 nAChR and the latter 7 nAChR, respectively (Lukas et al., 1999; Cordero-Erausquin et al., 2000). The 42 and 7 nAChR mechanisms are critical for nicotinic involvement in cognitive functions such as attention and working memory in rats (Felix and Levin, 1997; Levin et al., 1999; Bancroft and Levin, 2000; Blondel et al., 2000; Grottick and Higgins, 2000). However, the roles of the nAChR subtypes implicated in cognitive functions of patients with ADHD remain elusive.

Spontaneously hypertensive rats (SHR) are the most common animal model of ADHD (Wultz et al., 1990; Sagvolden et al., 1992, 1993). Recently, we have proposed juvenile stroke-prone spontaneously hypertensive rats (SHRSP), a substrain of SHR, as an animal model of a developmental disorder resembling ADHD (Ueno et al., 2002). We demonstrated that SHRSP exhibit behavioral abnormalities such as inattention, hyperactivity, and impulsivity similar to ADHD symptoms. Of note is the fact that male, but not female, SHRSP show impairment of cognitive function assessed by spontaneous alternation behavior. These behavioral symptoms were ameliorated by the psychostimulant methylphenidate, a first-choice drug for the treatment of ADHD. Central cholinergic dysfunction and concomitant cognitive dysfunction have been noted in SHRSP (Kang et al., 1990; Togashi et al., 1994, 1996; Minami et al., 1997; Nakamura and Shirane, 1999; Shirane and Nakamura, 2000). Moreover, the dysfunction of nicotinic systems, such as decreases in high-affinity (i.e., 42 sensitivity to [³H]nicotine) and low-affinity (i.e., 7 sensitivity to ¹²⁵I--bungarotoxin) nAChR binding sites, was also found in the brain regions of SHRSP (Yamada et al., 1987; Ferrari et al., 1999). However, the effects of nicotine and the role of nAChR subtypes associated with cholinergic dysfunction in SHRSP are still unknown.

The present study aimed to elucidate the effects of nicotine on spontaneous alternation behavior in juvenile SHRSP, an animal model of ADHD. Particular attention has focused on the characterization of nAChR subtypes involved in the effects of nicotine on spontaneous alternation behavior in SHRSP, using 42 and 7 nAChR antagonists.

	Materials and Methods

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Animals. Juvenile male SHRSP and a genetic control, Wistar-Kyoto rats (WKY), were used at 6 weeks of age. These animals have been inbred in our laboratory (current generation, F53). They were housed in a room with a maintained temperature (22 ± 2°C), relative humidity (55 ± 10%), and 12-h light/dark cycle (lights on at 6:00 AM), and allowed free access to food and water. All experimental procedures conformed to the Guidelines for the Care and Use of Laboratory Animals of the Animal Research Committee at Hokkaido University School of Medicine.

Spontaneous Alternation Behavior. Spontaneous alternation behavior requiring attention (Katz and Schmaltz, 1980) and working memory (Sarter et al., 1988) in a Y-maze was assessed by the methods of Sarter et al. (1988). Each arm of the Y-maze was 45 cm long, 10 cm wide, and 35 cm high, and both arms were positioned at equal angles. Each rat was placed at the end of an arm and allowed to enter the maze freely for an 8-min test session without reinforcers such as food, water, or electric shock. An arm entry was defined as the entry of all four paws into one arm. The sequence of arm entries was recorded with a video camera. The alternation behavior (actual alternations) was defined as the consecutive entry into three arms, i.e., the combination of three different arms, with stepwise combinations in the sequence. The maximum number of alternations was thus the total number of arms entered minus 2, and the percentage of alternation behavior was calculated as (actual alternations/maximum alternations) × 100.

Drugs. ()-Nicotine hydrogen tartrate and mecamylamine hydrochloride (MEC) were purchased from Sigma-Aldrich (St. Louis, MO). Hexamethonium dichloride (HEX), methyllycaconitine citrate (MLA), and dihydro--erythroidine hydrobromide (DHE), and RJR-2403 hemigalactarate were purchased from Sigma/RBI (St. Louis, MO). All drugs calculated as compounds with the base were dissolved in physiological saline solution. Nicotine or RJR-2403 was administered subcutaneously 20 min prior to the spontaneous alternation performance. MEC, HEX, MLA, or DHE was injected intraperitoneally 10 min before nicotine administration.

Statistical Analysis. All results are expressed as the means ± S.E.M. A two-tailed Student's t test was used to analyze differences between two group means (i.e., strain differences of WKY versus SHRSP). When more than two groups (i.e., drug effects) were compared, the significance of the differences among group means was evaluated by one-way analysis of variance, and further statistical analysis for post hoc comparison was done with Dunnett's multiple comparisons procedure (two-tailed). p < 0.05 was considered statistically significant. The statistical analysis program SPSS for Windows (SPSS, Inc., Chicago, IL) was used.

	Results

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Effects of Nicotine on Spontaneous Alternation Behavior in SHRSP. The acute effects of nicotine on spontaneous alternation behavior in SHRSP are shown in Fig. 1. SHRSP showed a significant decrease in alternation behavior (percentage) and a significant increase in total arm entries as compared with the normotensive genetic control WKY rats. Nicotine (0.1-1 mg/kg, s.c.) significantly ameliorated the lowered alternation (percentage) in a dose-dependent manner (Fig. 1A). However, nicotine did not affect total arm entries of SHRSP (Fig. 1B).

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Effects of nicotine on spontaneous alternation behavior (A) and total arm entries (B) in SHRSP. Nicotine (0.1-1 mg/kg, s.c.) was administered 20 min before the test. Data are expressed as the means ± S.E.M. (vertical bars). The numbers of animals used are shown in the columns. *, p < 0.05; **, p < 0.01 versus WKY; ##, p < 0.01; ###, p < 0.001 versus vehicle-treated SHRSP.

Effects of Centrally and Peripherally Acting nAChR Antagonists on Nicotine-Induced Improvement of Spontaneous Alternation in SHRSP. As shown in Fig. 2, the centrally acting nonselective nAChR antagonist MEC (1 mg/kg, i.p.) significantly abolished nicotine-induced (1 mg/kg, s.c.) improvement of spontaneous alternation in SHRSP (Fig. 2A). However, MEC did not affect total arm entries (Fig. 2B). In addition, the peripherally acting nonselective nAChR antagonist HEX (5 mg/kg, i.p.) had no effect on either alternation behavior (percentage) or total arm entries.

View larger version (37K): [in this window] [in a new window]   Fig. 2.   Effects of MEC and HEX on nicotine-induced improvement of spontaneous alternation (A) and total arm entries (B) in SHRSP. MEC (1 mg/kg, i.p.) or HEX (5 mg/kg, i.p.) and nicotine (1 mg/kg, s.c.) were administered 30 and 20 min before the test, respectively. Data are expressed as the means ± S.E.M. (vertical bars). The numbers of animals used are shown in the columns. *, p < 0.05; **, p < 0.01 versus WKY; ###, p < 0.001 versus vehicle-treated SHRSP; $, p < 0.05 versus ()-nicotine alone.

Effects of Subtype-Selective nAChR Antagonists on Nicotine-Induced Improvement of Spontaneous Alternation in SHRSP. The effects of subtype-selective nAChR antagonists on nicotine-induced (1 mg/kg, s.c.) improvement of spontaneous alternation in SHRSP are shown in Figs. 3 and 4. The selective 42 nAChR antagonist DHE (3 and 10 mg/kg, i.p.) dose dependently counteracted the nicotine-induced improvement of spontaneous alternation in SHRSP. Statistical significance was noted at a dose of 10 mg/kg (Fig. 3A). However, DHE did not affect total arm entries (Fig. 3B). In contrast, the selective 7 nAChR antagonist MLA (3 and 10 mg/kg, i.p.) failed to counteract the nicotine-induced improvement of spontaneous alternation in SHRSP (Fig. 4A). Moreover, MLA did not affect total arm entries (Fig. 4B).

View larger version (34K): [in this window] [in a new window]   Fig. 3.   Effects of DHE on nicotine-induced improvement of spontaneous alternation (A) and total arm entries (B) in SHRSP. DHE (3-10 mg/kg, i.p.) and nicotine (1 mg/kg, s.c.) were administered 30 and 20 min before the test, respectively. Data are expressed as the means ± S.E.M. (vertical bars). The numbers of animals used are shown in the columns. *, p < 0.05; **, p < 0.01 versus WKY; ###, p < 0.001 versus vehicle-treated SHRSP; $, p < 0.01 versus ()-nicotine alone.

View larger version (33K): [in this window] [in a new window]   Fig. 4.   Effects of MLA on nicotine-induced improvement of spontaneous alternation (A) and total arm entries (B) in SHRSP. MLA (3-10 mg/kg, i.p.) and nicotine (1 mg/kg, s.c.) were administered 30 and 20 min before the test, respectively. Data are expressed as the means ± S.E.M. (vertical bars). The numbers of animals used are shown in the columns. *, p < 0.05; **, p < 0.01 versus WKY; ###, p < 0.001 versus vehicle-treated SHRSP.

Effects of a Selective 42 nAChR Agonist on Spontaneous Alternation Behavior in SHRSP. The effect of RJR-2403, a selective 42 nAChR agonist, was examined. RJR-2403 (1 and 10 mg/kg, s.c.) dose dependently and significantly improved the lowered alternation (percentage) in SHRSP (Fig. 5A). However, RJR-2403 did not affect total arm entries (Fig. 5B).

View larger version (34K): [in this window] [in a new window]   Fig. 5.   Effects of RJR-2403 on spontaneous alternation behavior (A) and total arm entries (B) in SHRSP. RJR-2403 (1-10 mg/kg, s.c.) was administered 20 min before the test. Data are expressed as the means ± S.E.M. (vertical bars). The numbers of animals used are shown in the columns. *, p < 0.05; **, p < 0.01 versus WKY; #, p < 0.05; ##, p < 0.01 versus vehicle-treated SHRSP.

	Discussion

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We demonstrated here that nicotine improved the spontaneous alternation deficit in juvenile SHRSP. The nicotine-induced effect was counteracted by an 42, but not an 7, nAChR antagonist. This is the first report showing that 42 nAChR activation improves cognitive dysfunctions, such as attentional disorder and/or spatial working memory impairment, in an animal model of ADHD.

Nicotine administration and cigarette smoking have been shown to improve attentiveness (Levin, 1992). It has been reported that nicotine improves cognitive dysfunctions, including attentional deficit, in patients with ADHD (Conners et al., 1996; Levin et al., 1996, 2000, 2001; Levin and Rezvani, 2000) as well as in animals (Blondel et al., 2000; Grottick and Higgins, 2000). Thus, nicotinic treatment has been proposed as a therapeutic approach for ADHD (Barkley, 1977; Wender et al., 1985; Pomerleau et al., 1995; Conners et al., 1996; Levin et al., 1996; Milberger et al., 1997). Notably, nicotine skin patches improve ADHD symptoms as measured by the standard Clinical Global Impressions Scale in adults with ADHD (Conners et al., 1996; Levin et al., 1996, 2000, 2001; Levin and Rezvani, 2000). The present study demonstrated that acute administration of nicotine (0.1-1 mg/kg, s.c.) dose dependently and significantly improved the lowered spontaneous alternation performance in juvenile SHRSP without affecting total arm entries. The nicotine-induced improvement of spontaneous alternation was significantly abolished by pretreatment with the centrally acting nonselective nAChR antagonist mecamylamine but not with peripherally acting hexamethonium. This indicated that nicotine-induced improvement was mediated via central nAChR. In contrast, nicotine did not affect spontaneous alternation performance, which may only be due to the ceiling effect in WKY. But nicotine elicited a significant increase in total arm entries (i.e., locomotor activity) in WKY at the subcutaneous doses of 0.3 and 1 mg/kg (data not shown). These findings indicated that nicotine was specifically effective in ameliorating cognitive dysfunctions, such as attentional disorder and/or spatial working memory impairment, in an animal model of ADHD. In addition, they further supported the idea that the SHRSP is an appropriate animal model to evaluate therapeutic efficiency for ADHD patients.

The 42 and 7 nAChR mechanisms are critical for nicotinic involvement in cognitive function, such as attention and working memory in rats (Felix and Levin, 1997; Levin et al., 1999; Bancroft and Levin, 2000; Blondel et al., 2000; Grottick and Higgins, 2000). However, it is still elusive which nAChR subtypes are implicated in spontaneous alternation deficit in SHRSP. The selective 42 nAChR antagonist DHE dose dependently and significantly counteracted the nicotine-induced improvement of spontaneous alternation performance in juvenile SHRSP, whereas the selective 7 nAChR antagonist MLA failed to affect it. In addition, the selective 42 nAChR agonist RJR-2403 dose dependently and significantly improved the spontaneous alternation deficit in SHRSP. These findings suggested that nicotine-induced improvement of spontaneous alternation behavior in SHRSP was mediated by the activation of 42, but not 7, nAChR. We have recently reported that juvenile SHRSP exhibited impairment of spontaneous alternation performance without impairment of hippocampal long-term potentiation, a cellular model of learning and memory. In addition, the impaired spontaneous alternation performance in SHRSP was ameliorated by a therapeutic agent for ADHD, methylphenidate (Fig. 6) (Ueno et al., 2002). It has been reported that nicotine improves attentional function via the activation of 42, but not 7, nAChR in the five-choice serial reaction time task, which assesses both selective and sustained attention in rats (Blondel et al., 2000; Grottick and Higgins, 2000). Moreover, it has also been reported that selective 7 nAChR activation improves learning and memory in fimbria-fornix lesioned rats (Levin et al., 1999) and enhances hippocampal long-term potentiation in rats (Hunter et al., 1994). Thus, it may be surmised that an 42 nAChR system is involved in attentional function, and an 7 nAChR system is involved in learning and memory function. This implies that juvenile SHRSP may exhibit impairment of spontaneous alternation behavior based on an attention deficit rather than learning and memory dysfunctions.

View larger version (29K): [in this window] [in a new window]   Fig. 6.   Effects of methylphenidate on spontaneous alternation behavior in SHRSP (A) and time course changes in hippocampal long-term potentiation in the dentate gyrus area in WKY and SHRSP (B). Methylphenidate (0.01-1 mg/kg, i.p.) was administered 20 min before the test. Data are expressed as the means ± S.E.M. (vertical bars). ***, p < 0.001 versus WKY; #, p < 0.05; ##, p < 0.01; ###, p < 0.001 versus vehicle-treated SHRSP.

Nicotine administration has been shown to improve inattention in patients with ADHD (Conners et al., 1996; Levin et al., 1996, 2000, 2001; Levin and Rezvani, 2000). Nicotine skin patches may have advantages for the use of nicotine administration for certain therapeutic reasons. For instance, nicotine patches appear to be safer than cigarette smoking and have little likelihood of abuse, since nicotine is administered without the 4000 other compounds present in the tar and gaseous components of tobacco smoke (Pickworth et al., 1994). Thus, because nicotine skin patches are administered in a less hazardous form than cigarette smoking, they are thought to have therapeutic potential for treating attentional disorders in patients with ADHD. In the present study, nicotine clearly improved the spontaneous alternation deficit in juvenile SHRSP via the activation of 42 nAChR. Our finding coincides well with the article by Wilens et al. (1999) that the novel 42 nAChR agonist ABT-418 is effective in alleviating inattentive symptoms in patients with ADHD. Thus, the development of novel nicotinic ligands such as subtype-selective agonists for 42 nAChR may provide more potential therapeutic strategies with lower side effect profiles for ADHD patients than nicotine skin patches and cigarette smoking.

How nicotine improves the spontaneous alternation deficit in juvenile SHRSP is not fully understood yet. Nicotine is known to have a variety of actions on other neuronal systems, including the cholinergic, dopaminergic, noradrenergic, and serotonergic systems. It is possible that nicotine exerts its action on cognitive function by modulating these neuronal systems. Like methylphenidate and D-amphetamine, which are currently used in ADHD treatment, nicotine potentiates dopamine (DA) release (Marshall et al., 1997). It has also been reported that nicotine increases extracellular acetylcholine (ACh) levels via the activation of 42, but not 7, nAChR in the hippocampus and frontal cortex of the rat (Tani et al., 1998). Moreover, the 42 nAChR agonist ABT-418, but not the 7 nAChR agonist GTS-21, also increases the hippocampal extracellular ACh levels in rats (Tani et al., 1998). In addition, the selective 42 nAChR agonist RJR-2403 increases not only extracellular levels of ACh but also those of biogenic amines such as DA, norepinephrine, and serotonin in the hippocampus and frontal cortex of the rat (Summers et al., 1996). In SHRSP, central dopaminergic hypofunctions, such as lowered extracellular DA levels, have been found in the prefrontal cortex, nucleus accumbens, striatum, and amygdala (Nakamura et al., 2001). Moreover, SHRSP exhibit central cholinergic dysfunctions, such as lowered extracellular ACh levels, in the cerebrospinal fluid (Togashi et al., 1994) and prefrontal cortex (Shirane and Nakamura, 2000), decreased choline acetyltransferase activity in the brain (Nakamura and Shirane, 1999), impaired choline transport through the blood-brain barrier (Kang et al., 1990), and reduced numbers of cerebral high-affinity (i.e., 42) nAChR binding sites (Yamada et al., 1987). Thus, enhanced DA and ACh transmission by 42 stimulation might be a crucial mechanism for ameliorating effects of nicotine on the impaired spontaneous alternation performance in SHRSP. However, the precise mechanism of the action of nicotine on ADHD remains unsolved. Further studies using subtype-selective nAChR agonists promise to provide a better understanding of the nAChR mechanism of action of nicotine on cognitive functions and its therapeutic value for the treatment of ADHD.

In conclusion, our findings demonstrated that nicotine ameliorated spontaneous alternation impairment via the activation of 42 nAChR in juvenile SHRSP, an animal model of ADHD. Thus, the 42 nAChR mechanism might be involved in the spontaneous alternation deficit in SHRSP. These findings imply that selective 42 nAChR agonists may provide a useful therapeutic strategy for improving cognitive dysfunctions, such as inattention, in patients with ADHD.