Biomedical Research 34 (4) 189-195, 2013

Improvement of spontaneous alternation behavior deficit by activation of α4β2 nicotinic acetylcholine receptor signaling in the GM3- deficient mice

1 1 2 1 2 1 Kimie NIIMI , Chieko NISHIOKA , Tomomi MIYAMOTO , Eiki TAKAHASHI , Ichiro MIYOSHI , Chitoshi ITAKURA , 3, 4 and Tadashi YAMASHITA 1 Riken Brain Science Institute, Saitama 351-0198, Japan; 2 Graduate School of Medical Sciences, Nagoya City University, Nagoya 467- 8601, Japan; 3 Graduate School of Veterinary Medicine, Azabu University, Sagamihara 252-5201, Japan; and 4 World Class University Program, Kyungpook National University School of Medicine, Daegu, South Korea (Received 13 May 2013; and accepted 17 June 2013)

ABSTRACT We have reported that in ganglioside GM3-deficient (GM3−/−) mice, spontaneous alternation be- havior assessed by a Y-maze task was significantly lower, and total arm entries were significantly higher than in wild-type mice. The objective of the present study was to examine the role of nico- tinic acetylcholine receptor (nAChR) signaling in impairment of spontaneous alternation behavior of GM3−/− mice. Nicotine treatment (0.3, 1.0 mg/kg, s.c.) dose dependently improved the sponta- neous alternation deficit without affecting total arm entries −/−in GM3 mice. The nicotine-induced (1.0 mg/kg, s.c.) improvement was significantly abolished by the nAChR antagonist mecamyla- mine (1.0 mg/kg, i.p.). The α4β2 nAChR antagonist dihydro-β-erythroidine (2.5, 10.0 mg/kg, i.p.) dose dependently counteracted the nicotine-induced improvement of spontaneous alternation in GM3−/− mice, whereas the α7 nAChR antagonist methyllycaconitine (2.5, 10.0 mg/kg, i.p.) did not. In addition, the α4β2 nAChR agonist RJR-2403 (5.0, 10.0 mg/kg, s.c.) dose dependently and significantly improved the spontaneous alternation deficit, whereas the α7 nAChR agonist PNU120596 (0.3, 1.0, 3.0 mg/kg, i.p.) did not. These findings revealed that nicotine improved spontaneous alternation behavior of GM3−/− mice via the activation of α4β2, but not α7, nAChR. Thus, the ganglioside GM3 might be responsible for α4β2 nAChR signaling in the spontaneous alternation behavior.

Gangliosides (i.e., [GSLs]) con- encoded by a single-copy gene, is a primary glyco­ taining one and more residues are present syltransferase for the synthesis of complex ganglio- in all mammalian cell plasma membranes and intra- sides. In previous studies (18, 24), mice with gene cellular membrane structures. They are especially disruption of GM3 synthase (GM3-KO) are viable abundant in central nervous tissues and considered without major abnormalities. Both males and fe- to have important roles in early development, cell males were fertile. The major brain differentiation and proliferation, and the stability of present in GM3+/+ mice (GM1a, GD1a, GD1b, and neuronal cells (8). The ganglioside GM3 synthase, GT1b) were absent in the GM3−/− mice (24). GM3−/− mice show significantly lower spontaneous alterna- tion in the Y maze test (18). Animal studies have Address correspondence to: Dr. Tadashi Yamashita, Department of Biochemistry, Graduate School of Veter- shown that nicotinic acetylcholine receptor (nAChR) inary Medicine, Azabu University, Sagamihara 252- mechanisms are involved in attentional function (3, 5201, Japan 7). Because GM3 is concentrated in plasma mem- Tel & Fax: +81-42-769-1623 brane domains that is specialized for cell sig- E-mail: [email protected] naling, there would be a possibility that spontaneous 190 K. Niimi et al. alternation in GM3-KO mice would be derived from behavioral testing room at least 1 h before testing. nAChR signaling dysfunction. The experiments were performed at 35 lux. Before Neuronal nAChRs are composed of five subunits behavioral testing, the mice were placed in one of according to the combination of α (α2-α9) and β the compartments and allowed to move freely on (β2-β4) subunits. Neuronal subunits have been cate- the one of the arms for 10 min. Each mouse per- gorized into two major subtypes of nAChR pentam- formed one trial. An arm entry was defined as three ers in the brain on the basis of their high affinity for legs entering one of the arms, and the sequence of either nicotine or α-bungarotoxin. Of the two, the entries was recorded manually using videotapes. An former are considered to form α4β2 nAChR and the alteration was defined as entry into all three arms latter α7 nAChR, respectively (4, 15). The α4β2 and with consecutive choices. The percentage of sponta- α7 nAChR mechanisms are critical for nicotinic in- neous alteration was calculated as (actual alteration/ volvement in cognitive functions such as attention maximum alteration) × 100. (2, 3, 6, 7, 13). The present study aimed to elucidate the effects Drugs. (−)-Nicotine hydrogen tartrate (nicotine) and of nAChR signaling on spontaneous alternation be- mecamylamine hydrochloride (MEC) were purchased havior in GM3−/− mice. Particular attention has fo- from Sigma-Aldrich (Saint Louis, MO, USA). cused on the characterization of nAChR subtypes Dihydro-β-erythroidine hydrobromide (DHβE), involved in the effects of nicotine on spontaneous methyllycaconitine citrate (MLA), PNU120596, and alternation behavior in GM3−/− mice, using α4β2 and RJR-2403 fumarate (RJR-2403) were purchased α7 nAChR antagonists. from Tocris Bioscience (Bristol, United Kingdom). Nicotine, MEC, DhβE, MLA or RJR-2403 were dissolved in saline solution. PNU120596 was dis- MATERIALS AND METHODS solved in 100 mM in DMSO. Control mice were not Animals. All procedures involving animals were ap- treated with nAChR related drugs, and received an proved by the Animal Experiments Committee of equivalent volume of vehicle. The doses used in this RIKEN and Hokkaido University. All animals were study were within the range reported to produce cared for humanely in accordance with institutional spontaneous alternations in various rodent models (9, guidelines for animal experimentation. The wild- 21). Nicotine, RJR-2403 or PNU120596 was admin- type GM3+/+ and GM3−/− mice with C57BL/6NCrSlc istered subcutaneously (s.c.) 20 min prior to the (Japan SLC, Inc., Shizuoka, Japan) genetic back- spontaneous alternation performance. MEC, DhβE ground (backcross generations; n = 16) were de- or MLA was injected intraperitoneally (i.p.) 10 min rived from a cross between +/− mice and genotyped before nicotine administration. by PCR using tail DNA (24). The mice were weaned at postnatal weeks 3 to 4 and were given Statistical analysis. The data are presented as the free access to water and food pellets (CRF-1; Orien- mean ± standard error of the mean (SEM). Statisti- tal Yeast Co., Ltd., Tokyo, Japan). The mice were cal analyses were conducted using Excel Statistics individually housed until use in a microisolation 2006 (SSRI, Tokyo, Japan). The student’s t-test ana- cage (MBS7115RHMV; Allentown, NJ, USA) with lyzed differences between two groups. When more bedding (TEK-FRESH; Harlan laboratories, Madi- than two groups were compared, the data were ana- son, WI, USA), under a 12:12-h light:dark cycle lyzed using analysis of variance (ANOVA), and fur- (dark period, 20 : 00 to 08 : 00) at 23 ± 1°C and ther statistical post hoc Tukey’s test between groups 55 ± 5% humidity. All behavioral analyses were was performed. The differences between groups conducted by a well-trained experimenter who was were considered significant at P < 0.05. blinded to the genotypes. Separate groups of juve- nile (aged 6 weeks) male mice were used for the RESULTS behavior tests. Effects of nicotine on spontaneous alternation be- Measurement of spontaneous alternation behavior in havior in GM3−/− mice the Y maze test. Spontaneous alternation behavior as The acute effects of nicotine on spontaneous alter- index of attention was evaluated using the Y maze nation behavior in GM3−/− mice are shown in Fig. 1. test (19). The Y-maze test was performed using the GM3−/− mice showed a significant decrease in alter- reported procedure (17) and was conducted between nation behavior (t(18) = 3.62, P < 0.01) and a signif- 09 : 00 and 16 : 00. The mice were moved into the icant increase in total arm entries (t(18) = 3.34, Acetylcholine receptor and GM3 191

Fig. 1 Effects of nicotine on spontaneous alternation behavior (A) and total arm entries (B) in GM3−/− mice. Nicotine (vehi- cle, 0.1, 0.3, 1.0 mg/kg) was administered 20 min before the test. Data are obtained from male mice (n = 10: each group) and expressed as the means ± SEM. **, P < 0.01 versus vehicle-treated GM3+/+ mice (t-test); #, P < 0.05, ##, P < 0.01 ver- sus vehicle-treated GM3−/− mice (Tukey’s test).

Fig. 2 Effects of MEC on nicotine-induced improvement of spontaneous alternation (A) and total arm entries (B) in GM3−/− mice. MEC (vehicle, 0.2, 1.0 mg/kg) and nicotine (1.0 mg/kg) were administered 30 and 20 min before the test, respectively. Data are obtained from male mice (n = 10: each group) and expressed as the means ± SEM. **, P < 0.01 versus vehicle- treated GM3+/+ mice (t-test); $$, P < 0.01 vehicle-treated GM3−/− mice (t-test); ##, P < 0.01 versus vehicle- and nicotine-treat- ed GM3−/− mice (Tukey’s test).

P < 0.01) as compared with wild-type mice. Nico- kg, P < 0.01] (Fig. 2A). However, MEC did not af- tine significantly ameliorated the lowered alternation fect total arm entries (Fig. 2B). in a dose-dependent manner [F(3,36) = 12.71, P < 0.01; 0.3 mg/kg, P < 0.05; 1.0 mg/kg, P < 0.01] Effects of subtype-selective nAChR antagonists on (Fig. 1A). However, nicotine did not affect total arm nicotine-induced improvement of spontaneous alter- entries of GM3−/− mice (Fig. 1B). nation in GM3−/− mice The effects of subtype-selective nAChR antagonists Effects of non-selective nAChR antagonist on nico- on the nicotine-induced improvement of spontane- tine-induced improvement of spontaneous alterna- ous alternation in GM3−/− mice are shown in Figs. 3 tion in GM3−/− mice and 4. The selective α4β2 nAChR antagonist DHβE As shown in Fig. 2, the non-selective nAChR antago- dose dependently counteracted the nicotine-induced nist MEC significantly abolished the nicotine-induced improvement of spontaneous alternation in GM3−/− (1.0 mg/kg) improvement of spontaneous alternation mice [F(2,27) = 15.45, P < 0.01]. Statistical signifi- in GM3−/− mice [F(2,27) = 18.95, P < 0.01; 1.0 mg/ cance was noted at a dose of 10.0 mg/kg (P < 0.01) 192 K. Niimi et al.

Fig. 3 Effects of DHβE on nicotine-induced improvement of spontaneous alternation (A) and total arm entries (B) in GM3−/− mice. DHβE (vehicle, 2.5, 10.0 mg/kg) and nicotine (1.0 mg/kg) were administered 30 and 20 min before the test, re- spectively. Data are obtained from male mice (n = 10: each group) and expressed as the means ± SEM. **, P < 0.01 versus vehicle-treated GM3+/+ mice (t-test); $$, P < 0.01 vehicle-treated GM3−/− mice (t-test); #, P < 0.05 versus vehicle- and nico- tine-treated GM3−/− mice (Tukey’s test).

Fig. 4 Effects of MLA on nicotine-induced improvement of spontaneous alternation (A) and total arm entries (B) in GM3−/− mice. MLA (vehicle, 2.5, 10.0 mg/kg) and nicotine (1.0 mg/kg) were administered 30 and 20 min before the test, respective- ly. Data are obtained from male mice (n = 10: each group) and expressed as the means ± SEM. **, P < 0.01 versus vehicle- treated GM3+/+ mice (t-test); $$, P < 0.01 versus vehicle-treated GM3−/− mice (t-test).

(Fig. 3A). However, DHβE did not affect total arm ternation in GM3−/− mice [F(3,36) = 11.87, P < 0.01; entries (Fig. 3B). On the other hand, the selective 5.0 mg/kg, P < 0.05; 10.0 mg/kg, P < 0.01] (Fig. 5A). α7 nAChR antagonist MLA failed to counteract the However, RJR-2403 did not affect total arm entries nicotine-induced improvement of spontaneous alter- (Fig. 5B). On the other hand, the selective α7 nation in GM3−/− mice (Fig. 4A). Moreover, MLA nAChR agonist PNU120596 failed to improve the did not affect total arm entries (Fig. 4B). lowered spontaneous alternation in GM3−/− mice (Fig. 6A). Moreover, PNU120596 did not affect to- Effects of subtype-selective nAChR agonists on tal arm entries (Fig. 6B). spontaneous alternation in GM3−/− mice The effect of a selective α4β2 nAChR agonist RJR- DISCUSSION 2403 and a selective α7 nAChR agonist PNU120596 were examined. RJR-2403 dose dependently and The α4β2 and α7 nAChR mechanisms are critical significantly improved the lowered spontaneous al- for nicotinic involvement in cognitive function, such Acetylcholine receptor and GM3 193

Fig. 5 Effects of RJR-2403 on spontaneous alternation behavior (A) and total arm entries (B) in GM3−/− mice. RJR-2403 (vehicle, 1.0, 5.0, 10.0 mg/kg) was administered 20 min before the test. Data are obtained from male mice (n = 10: each group) and expressed as the means ± SEM. **, P < 0.01 versus vehicle-treated GM3+/+ mice (t-test); #, P < 0.05, ##, P < 0.01 versus vehicle-treated GM3−/− mice (Tukey’s test).

Fig. 6 Effects of PNU120596 on spontaneous alternation behavior (A) and total arm entries (B) in GM3−/− mice. PNU120596 (vehicle, 0.3, 1.0, 3.0 mg/kg) was administered 20 min before the test. Data are obtained from male mice (n = 10: each group) and expressed as the means ± SEM. **, P < 0.01 versus vehicle-treated GM3+/+ mice (t-test). as attention (2, 3, 6, 7, 13). Both α4β2 and α7 and significantly improved the spontaneous alterna- nAChR agonists have been shown to improve per- tion deficit in GM3−/− mice. However, the α7 nAChR formance in cognitive tasks (14). However, it is not agonist PNU120596 did not improve the spontane- clear which nAChR subtypes are involved in spon- ous alternation deficit. These findings suggested that taneous alternation deficit in GM3−/− mice. The pres- the nicotine-induced improvement of spontaneous ent study demonstrated that acute administration of alternation behavior in GM3−/− mice was mediated nicotine dose dependently and significantly im- by the activation of α4β2, but not α7, nAChR. proved the lowered spontaneous alternation perfor- It has been reported that nicotine improves atten- mance in juvenile GM3−/− mice without affecting tion via the activation of α4β2 nAChR in the five- total arm entries. The selective α4β2 nAChR antag- choice serial reaction time task, which assesses both onist DHβE dose dependently and significantly selective and sustained attention (3, 7). The selec- counteracted the nicotine-induced improvement of tive α4β2 nAChR activation improved spontaneous spontaneous alternation performance in GM3−/− alternation behavior in juvenile stroke-prone sponta- mice, whereas the selective α7 nAChR antagonist neously hypertensive rats (SHRSP), an animal mod- MLA failed to affect it. 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